Translation in proximity to forming autophagosomes during sustained autophagy

Domain: neuroscience
Published: 2026-04-10
Editorial outcome: major_revision

Abstract
Autophagy is an evolutionarily conserved catabolic process. In a process requiring a cascade of
over 35 autophagy-related genes (Atg), a cupped phagophore membrane expands to surround
cytoplasmic material, and seals itself to form an autophagosome, which finally fuses with
lysosomes. Large numbers of autophagosomes form during stress responses, while
simultaneously cells drastically reduce translation to conserve energy. Here, using proximity-
labeling and Fluorescence in situ Hybridization we demonstrate that multiple mRNAs encoding
proteins required for autophagy preferentially localize in proximity to forming autophagosomes.
Polysome fractionation and proteomics of nascent proteins in proximity to forming
autophagosomes provides evidence for the local translation of these mRNAs. Translation and the
ribosome-binding protein RACK1 were required for the localization of these mRNAs to forming
autophagosomes. Inhibition of translation or knockdown of RACK1 caused depletion of several
proteins required for autophagy and a reduction in the number of autophagosomes. Local
translation may enable a rapid, energy-efficient supply of proteins for autophagy to enable cells
to massively induce autophagy while conserving energy during cell stress.

Introduction
Cells have evolved to utilize the minimum energy necessary in many processes. Cells and
organisms are frequently confronted with nutrient limitations accentuating the importance of
energy conservation. One mechanism by which cells conserve energy is by trafficking mRNA to
sites where the encoded proteins are required1,2. A single mRNA can be translated on average
140 times, and up to 1000 times in an hour3. Therefore, localizing mRNA to the needed location
or subcellular compartment for translation economizes energy by transporting one mRNA
instead of 100’s of its protein products. Local mRNA translation also provides a rapid supply of
specific proteins that can enhance the speed and efficiency of processes1,2. RNA localization can
be accomplished through several mechanisms. Localized mRNSAs often contain cis-acting
elements within their 3′-UTR, which are recognized by RNA-binding proteins (RBPs) for
transport. For example, the 3’UTR of β-actin mRNA directs it to the leading edge of cells where
cytoskeleton is abundant as cells migrate4. mRNAs encoding secreted or membrane proteins are
recruited to the ER during translation by nascent peptides which bind the signal recognition
particle5,6. It was commonly understood that spatial control of mRNA translation usually
involved translation being repressed during transport7, however recent reports suggest that, like
the localization of mRNAs encoding secreted proteins to the ER, mRNA localization frequently
occurs while an mRNA is being translated8,9. Co-translational localization of mRNAs may
therefore be dictated in part by partially translated proteins emerging from ribosomes or
differentiated subpopulations of ribosomes10,11 in addition to factors binding 3’UTRs.
When subjected to stress, cells utilize myriad pathways to reduce energy consumption to
conserve resources and promote survival. Translation consumes large amounts of cellular energy
and when subjected to stress cells reduce translation by over 70% by inhibiting mTORC112. At
the same time that cells profoundly reduce translation, inhibition of mTORC1 strongly induces
autophagy, a catabolic process devoted to the degradation of intracellular components. In
autophagy an entire organelle is formed de novo within minutes, and during stress 100’s of
autophagosomes are formed in a cell. In this process, membrane protrudes from the endoplasmic
reticulum (ER) forming an omegasome13,14. A membrane structure called a phagophore rapidly
initiates from this and expands to engulf cytoplasmic substrates. The phagophore membrane
seals itself to form an autophagosome, thereby sequestering its substrates from the cytoplasm.
Subsequent fusion of autophagosomes with lysosomes degrades autophagosome contents.

The intensity of autophagy is tightly regulated by the opposing effect of mTORC1 and AMPK
kinases15. Nutrient starvation activates AMPK kinases, which inactivate mTOR and
phosphorylate ULK1 to promote the activity of the ULK1/ATG13 complex thus initiating
autophagy16. Thus, in most systems, inhibition of mTOR leads to induction of autophagy. Once
activated, the ULK1 complex triggers phagophore nucleation by phosphorylating the PI3KC3
complex (e.g., VPS34, Beclin1) and activating PI3P (phosphatidylinositol 3-phosphate)
production on ER-derived omegasomes17. Omegasomes further mature to form phagophores, in a
process guided by recruitment of DFCP1 (Double FYVE-Containing Protein 1) and WIPI2 (WD-
repeat domain Phosphoinositide-Interacting 2) to PI3P18,19. Therefore, autophagosomes are
birthed on regions of ER marked by DFCP1 and WIPI2.

The formation of autophagosomes engages over 35 Autophagy-related-genes (Atg) in a cascade
that results in the covalent modification of LC3 proteins with the lipid phosphatidylethanolamine
(PE), to form LC3-II which populates the forming autophagosome membrane. Autophagy
receptors like p62/sequestosome1 (SQSTM1) and NBR1 (Neighbor of BRCA1) bind LC3 and
substrates for autophagy, recruiting these for engulfment by forming autophagosomes17,20. p62/
SQSTM1, NBR1 and LC3-II are also degraded by lysosomal hydrolases together with engulfed
cargoes, which is consistent with the relatively short half-lives of several autophagy-related
proteins21,22. LC3 and its lipid modification are essential, with rare exceptions, for autophagy and
thus the accumulation of LC3-II and its subsequent turnover in autophagosomes correlates with
the number of autophagosomes and autophagy activity23.During autophagy, rapid, and
continuous turnover of LC3, autophagy receptors and other proteins required for autophagy risks
exhausting essential autophagy related proteins without a continuously renewed supply. Together
this evidence raises the possibility that continued translation in the vicinity of forming
autophagosomes may be necessary for a continuous supply of key autophagy-related proteins to
sustain autophagy during prolonged stress.

Forming autophagosomes emerge from smooth ER, which is devoid of ribosomes directly
attached to the ER24, but multiple authors have remarked that the regions surrounding
omegasomes and phagophores are densely populated with ribosomes using electron microscopy
(EM)25-29. Ribosomes can also localize to forming autophagosomes to be targeted for
degradation by autophagy, in a process called ribophagy30,31. Whether ribosomes may also be
recruited in proximity to forming autophagosomes for local translation of mRNA has not been
assessed to the best of our knowledge. Besides Atg9, the other core proteins required for
autophagy are neither transmembrane nor secreted proteins32, and thus the mRNA encoding them
should not be recruited to the ER by the signal recognition particle. Many mRNAs which do not
encode transmembrane or secreted proteins have been observed to localize to the ER for reasons
that remain unclear6,33-35.

Here, we demonstrate that mRNAs encoding many proteins required for autophagy preferentially
localize in proximity to forming autophagosomes and are translated there. Localization of these
mRNAs to the forming autophagosome is dependent on the ribosome-associated protein RACK1
and enables sustained autophagy in cells undergoing stress responses.

Results
mRNAs encoding several proteins required for autophagy are proximal to forming
autophagosomes
Torin1, an mTOR inhibitor, induced autophagy, as confirmed by an increased LC3-II/I ratio and
the accumulation of LC3-II when lysosomal degradation was inhibited with Bafilomycin A1
(BAF), a vacuolar H+ ATPase inhibitor (Supplementary Fig.1a). Torin1 also reduced global
translation as indicated by a loss of ribosome occupancy on mRNAs in polysome fractionation
experiments (Supplementary Fig.1b). To assess whether translation occurs in proximity to
phagophores, or forming autophagosomes, we first validated markers of these structures. When
autophagy is induced, DFCP1 and WIPI1, a marker of phagophores, co-localized
(Supplementary Fig.1c).

APEX2, an engineered ascorbate peroxidase, catalyzes the oxidation of biotin-phenol (BP) in a
reaction potentiated by a 1-minute pulse of H2O2. This generates short-lived biotin radicals that
covalently ligate to nearby molecules within a 20 nm radius36,37. Molecules in proximity to
APEX2-DFCP1 or -LC3B were biotinylated and labeled with Alexa488-streptavidin. These
formed discrete foci only in cells treated with H2O2, and these colocalized with DFCP1 or LC3B,
respectively, confirming the specificity of this proximity-based biotinylation (Fig.1a). p62, in
addition to being an autophagy receptor, also is recruited during the early steps of
autophagosome formation38,39 and a fraction of p62 puncta co-localized with biotinylation
mediated by APEX2-DFCP1 (Supplementary Fig.1d). In cells expressing APEX2-myc-DFCP1,
APEX2-biotinylated signals colocalized with myc-DFCP1(Supplementary Fig.1e). In addition,
ATG14 proteins were successfully pulled down by APEX2-DFCP1-based proximity labeling and
pulldown (Supplementary Fig.1f). These results further confirmed the specificity of APEX2
proximity labeling. Cellular RNAs retrieved by proximity-labeling of APEX2-DFCP1 were
detected in a manner dependent on activation of APEX2 by H2O2 (Supplementary Fig.1g). In
addition, the protein levels of APXE2-DFCP1 and endogenous DFCP1 in APEX2-DFCP1
expressing cells were similar with or without Torin1 ± BAF treatment (Supplementary Fig.1h).
More biotinylated proteins by APEX2-DFCP1 were observed in pulldown comparing to input
samples under Torin1 conditions, which validated successful proximity labeling-based pulldown
under Torin1 treatment (Supplementary Fig.1i). mRNAs proximity-labeled by APEX2-DFCP1
and LC3B were enriched with streptavidin coated beads and quantified by RT-qPCR.

Phagophores form from smooth ER which lacks ribosomes, but may be within proximity
labeling distance of some rough ER. mRNA encoding transmembrane EGFR (Epidermal Growth
Factor Receptor), minimal and insignificant enrichment in proximity to DFCP1 or LC3B (Fig.1b,
c). Similarly, mRNA encoding a nuclear protein, Histone H1.1 was not enriched at these sites
(Fig.1b, c). mRNAs encoding proteins participating in autophagosome formation including
ATG3, ATG12, VPS34, DFCP1 and p62 were enriched in proximity to DFCP1, even though
these proteins lack transmembrane domains (Fig.1b). Some of these mRNAs were also enriched
in proximity to APEX2-LC3B (Fig.1c). 18S rRNA was also found enriched near DFCP1
(Fig.1b), suggesting that mRNAs may be actively translated in proximity to the phagophore. To
confirm these Atg mRNAs are specifically recruited to regions containing DFCP1 and LC3B, we
used two independent cytoplasmic APEX2-fused proteins (APEX2-Perilipin2, APEX2-
Cytoplasmic-V5 tag). These constructs exhibited proximity labeling localizing as expected, but
this did not co-localize with the phagophore marker DFCP1 (Supplementary Fig.2a). In contrast,
APEX2-DFCP1 proximity-labeling co-localized with DFCP1-mCherry (Supplementary Fig.2a).
Cytoplasmic APEX2-enriched Beta-actin mRNA by RT-qPCR as expected (Supplementary
Fig.2b). The mRNAs encoding proteins involved in autophagy which were enriched in proximity
to DFCP1, were not enriched in proximity to a diffuse cytoplasmic protein (Cytoplasmic V5-tag)
or Perilipin (Supplementary Fig.2c). This demonstrates that the enrichment of these mRNAs in
proximity to DFCP1 is not due to random sampling of the cytoplasm or RNA-binding sites.

To expand this principle, we performed RNAseq on mRNAs in proximity to DFCP1, CytoV5
and Perilipin. Volcano plots for DFCP1, CytoV5 and Perilipin proximity labeling demonstrate
enrichment of some RNAs with each protein (Supplementary Fig.2d). Cytoplasmic APEX2-
enriched RNAs were depleted of nuclear RNAs such as XIST and NEAT1, and mRNAs that were
more abundant in the nucleus in prior literature using APEX2 proximity labeling, validating the
specificity of the CytoV5-APEX2 labeling (Supplementary Fig.2d-f). mRNAs encoding an
extensive list of proteins involved in autophagosome formation, cargo recruitment and
maturation were enriched in proximity to DFCP1 to a greater extent than CytoV5, or Perilipin
when normalized to a negative control without H2O2-induction of proximity-labeling (Fig.1d).
These included mRNAs encoding ULK2, FIP200, ATG2B, ATG10, ATG12, ATG4C, VPS34
(PIK3C3), UVRAG, and TMEM41B among others. Beyond the canonical proteins involved in
autophagy, mRNAs encoding additional proteins implicated in autophagy were also enriched in
proximity to DFCP1. These include autophagy receptors and adaptors (OPTN,
CALCOCO2/NDP52, TAX1BP1, CCPG1), mitophagy mediators (FUNDC1, BNIP3L/NIX), the
ER-phagy receptor SEC62, fusion-associated SNARE proteins (STX17, SNAP29, STX7),
trafficking regulators (SNX4), and mTOR-associated signaling components (LAMTOR3,
RICTOR).

Another set of mRNAs which encode proteins involved in autophagy processes were under-
represented in proximity to DFCP1. Several of these proteins localize to sites other than the
phagophore or are only involved in autophagy when selective autophagy of specific structures is
triggered. For example, PHB240 is a protein in the inner mitochondrial membrane involved in
mitophagy. TEX264 is an autophagy receptor for dsDNA breaks41. DNM2 is an endosomal
protein42. SNX17 is involved in recycling components of autophagolysosomes rather than their
formation43. LAMTOR 1, 2, 4 and 5 associate with the lysosome to anchor the RAG GTPases
there44. Intriguingly, mRNAs encoding ULK1, ATG13 and ATG101, which form a complex
involved in phagophore initiation45, were all under-enriched in proximity to DFCP1, suggesting
something unique about the localization of all mRNAs encoding this complex.

To confirm using an orthogonal method that mRNAs encoding proteins required for autophagy
localize in proximity to forming autophagosomes, we used single-molecule FISH (Fluorescence
In Situ Hybridization) probes for ATG3 or ATG12 mRNA conjugated to Quasar® 570 Dye.
ATG3 or ATG12 mRNA frequently co-localized with or were immediately adjacent to the
phagophore marker, WIPI1 (Fig.2 a, b, c, Supplementary Fig.2g). ATG3 mRNA also co-
localized with DFCP1 (Supplementary Fig.2h). In contrast, ACTB mRNA encoding β-actin co-
localized much less frequently with WIPI1 (Fig.2 a, b, c, Supplementary Fig.2g). These results
reinforce the proximity-labeling data (Fig.1b, c) demonstrating that mRNAs encoding several
proteins involved in autophagy preferentially localize in proximity to phagophores.

Recruitment of mRNAs encoding autophagy proteins to DFCP1 (Fig.2d) or LC3B
(Supplementary Fig.2i) locales required treatment with Torin1. This suggests that these mRNAs
were specifically recruited to phagophores or autophagosomes, which are induced by Torin1. To
test this, experiments were repeated in FIP200-/- cells. FIP200-/- cells fail to form ULK1
nucleation sites and block formation of phagophores in response to mTOR inhibition46. FIP200-/-
cells nonetheless do not impair non-canonical autophagy pathways and their production of
LC3B-II, meaning that untreated FIP200-/- cells do have LC3B-II, but this does not increase
when autophagy is induced. In agreement, LC3B-II was present in FIP200-/- cells, but was not
increased by Torin1 treatment (Supplementary Fig.2j). APEX2-DFCP1 enriched mRNAs
encoding autophagy-related proteins, but the enrichment of 5/6 of these was ablated in FIP200-/-
cells (Fig.2e), supporting evidence that these mRNAs localize to a site of autophagosome
genesis. The enrichment of DFCP1 mRNA in proximity to APEX2-DFCP1 was minimally
affected by FIP200-/- (Fig.2e). This is consistent with DFCP1 mRNA localizing in proximity to
APEX2-DFCP1 it encodes independent of phagophore formation. The requirement of FIP200 for
localization of mRNAs to DFCP1 also shows that this is not a site involved in non-canonical
autophagy processes47, like microautophagy, LC3-associated endosomes or LC3-dependent
packaging of extracellular vesicles48,49.

mRNAs encoding proteins required for autophagy are translated near phagophores.
To test for nascent protein synthesis near forming autophagosomes, APEX2-DFCP1 expressing
cells were briefly pulsed with a Click-iT amino acid analogue (AHA, L-azidohomoalaine) and
molecules in proximity to APEX2-DFCP1 were biotinylated and pulled down. AHA was then
detected on SDS-PAGE gels by coupling it to Alexa488. Nascently-translated proteins were
present in proximity to APEX2-DFCP1 (Fig.3a), and this was blocked when translation was
inhibited with cycloheximide (CHX, Fig.3a). More biotinylated proteins were observed in
pulldown comparing to input proteins of APEX2-DFCP1 cells treated with or without CHX,
indicating successful proximity labeling and pulldown with CHX treatment (Supplementary
Fig.1i).

If proteins are translated in proximity to DFCP1, then the mRNAs should be in translation-
competent ribosomes. We performed proximity labeling with APEX2-DFCP1, then polysomes
were fractionated into heavy and light polysome, and monosome fractions50,51 (Supplementary
Fig.2k, l). Heavy and light polysome fractions could be ablated by EDTA which disrupts
ribosome association with mRNA52 (Supplementary Fig.2l). From these fractions, we pulled-
down biotinylated RNAs proximal to DFCP1 and performed RT-qPCR.mRNAs encoding
proteins involved in autophagosome formation, like ATG2, ATG3, ATG4, ATG5, VPS34,
DFCP1, ATG12 and p62/SQSTM were found enriched in heavy polysome fractions in proximity
to APEX2-DFCP1, while β-actin, mitochondrial MTCO2, and nuclear H1.1 mRNAs were not
(Fig.3b). This evidence further suggests that mRNAs encoding several proteins essential for
autophagy are selectively and actively translated in proximity to phagophores.

To determine which mRNAs encoding essential autophagy proteins are actively translated in
proximity to phagophores, we gave a short pulse of SILAC heavy amino acids to cells, labeled
and pulled-down proteins in proximity to DFCP1 and then performed mePROD mass
spectrometry12. The significantly enriched proteins with heavy isotype labelling were mainly
involved in autophagy-related pathways, indicating that the APEX2-DFCP1 successfully
biotinylated proteins close to DFCP1 (Supplementary Fig.2m). Among proteins enriched in
proximity to DFCP1 versus control pulldown, a large number of ribosomal proteins from both
large (with the prefix "RPL") and small ribosomal subunits (with the prefix "RPS”) were
identified (Supplementary Fig.3a). VSP34, ATG5, ATG12, ATG3, p62/SQSTM, and ATG16L
were identified as nascent proteins in proximity to DFCP1, with significant heavy/light amino
acid (H/L) ratios (Fig.3c). In general, the amount of nascent protein synthesis (SILAC H/L
ratios) in proximity to DFCP1 correlated with the enrichment of mRNAs encoding proteins
involved in autophagy (APEX2-DFCP1-seq) as shown in (Fig.3d). Notably, ATG3, ATG5,
ATG12, and PIK3C3 were significantly enriched in the APEX2-DFCP1-seq data and also
exhibited strong labeling in the SILAC data, suggesting pronounced mRNA enrichment and
translation of these mRANAs in proximity to DFCP1. Weaker correlation of other mRNAs with
nascent peptide abundance may be attributable to differences in the translational rate or other
post-transcriptional regulation among these mRNAs.

A substantial number of mRNAs without known roles in autophagy were enriched in proximity
to DFCP1 in our RNAseq analysis (see Source Data file, sheet “AP2-DFCP1 RNAseq
enrichment”). GO-term analysis of these mRNAs showed an enrichment for proteins involved in
chromosome replication and cell cycle (Supplementary Fig.3b). Nascent peptides were not
identified for these mRNAs encoding proteins involved in cell division. This suggests a second
pool of mRNAs which are not translated, may localize in proximity to phagophores.

Translation is required for sustained autophagy.
If translation of proteins involved in autophagy occurs in proximity to phagophores (Fig.3), then
this translation may be required for autophagy to operate at full capacity. Previous attempts to
use CHX to study the impact of translation on autophagy, were confounded by the use of serum-
starvation to activate autophagy53. CHX stops the incorporation of amino acids into proteins,
which also increases the free amino acid pool, thereby re-activating mTOR and reducing
autophagy53. To avoid this confounding effect, mTOR inhibitors (INK128, Torin1) were used to
ensure complete inhibition of mTOR during CHX treatment which was confirmed by the
sustained dephosphorylation of eIF4EBP (Supplementary Fig.3c, d).

To identify proteins involved in autophagy that continue to be translated during sustained
autophagy, HeLa cells were treated with Torin1 and BAF, to negate the influence of protein
turnover caused by lysosomal degradation. The cellular protein levels of ATG3, ATG5, ATG12,
and ATG7 significantly decreased when translation was blocked with CHX (Fig.4a, b). ATG2
protein levels were also decreased by CHX but with the P value of 0.056 (Fig.4a, b).
Intriguingly, the mRNA encoding ATG16L1 was minimally, or not enriched in proximity to
DFCP1 (Fig.1b), and the levels of ATG16L1 were not significantly altered by CHX (Fig.4a, b).
Notably, the levels of several autophagy receptors including p62, NBR1, OPTN (Optineurin),
LC3A-II (autophagosome marker) and NEDP52 (CALCOCO2) were also depleted when
translation was inhibited (Fig.4c, d). These data suggest that translation of several proteins
involved in autophagy is required to maintain their levels and sustain autophagy.

To further evaluate the requirement of translation for sustained autophagy, HEK293T cells
expressing a GFP-mCherry-LC3B autophagy reporter were treated with mTOR inhibitor, with or
without CHX. Quenching of GFP, but not mCherry by the low pH of the lysosome enables
quantification of autophagosomes and autophagolysosomes respectively54. Inhibition of
translation reduced the numbers of both autophagosomes and autophagolysosomes (Fig.4e, f, g)
and this was not caused by the fluorescence intensity change of GFP and mCherry
(Supplementary Fig.3e, f).

RACK1 is required for localization of autophagy-related mRNAs to forming
autophagosomes.
A specific factor may be involved in recruiting mRNAs to forming autophagosomes for in situ
translation. Recruitment of mRNAs encoding autophagy proteins into the vicinity of DFCP1
(Fig.5a) and LC3B (Supplementary Fig.3g), was abrogated when translating ribosomes were
displaced from mRNAs with puromycin. Recruitment of 28S rRNA to DFCP1 was also
significantly inhibited by puromycin (Fig.5a). While EGFR mRNA was enriched in DFCP1
proximity labeling samples, but to a lesser extent than mRNAs encoding autophagy proteins.
This enrichment was likely due to EGFR presence at ER (Fig.5a). Interestingly, recruitment of
several of these mRNA to DFCP1 was also abolished by Homoharringtonine (Supplementary
Fig.3h), which arrests ribosomes at the start codon and disrupts polysomes55,56. This indicates
that autophagy-related mRNAs require ongoing translation, and potentially a ribosome-
associated factor for the recruitment to or the retention at DFCP1 locales. RACK1 (Receptor for
Activated C-Kinase 1, GNB2L1), which can bind the 40S small ribosomal subunit near the
mRNA exit tunnel57, was previously shown to bind to a complex of VPS15, ATG14L, and
Beclin1, placing it at the site of phagophore formation58. RACK1 is also known to control the
translation of mRNAs encoding proteins involved in autophagy59.

In cells treated with Torin1, RACK1 immunoprecipitated mRNAs including ATG3, ATG12,
VPS34, p62 and DFCP1, while GAPDH, and β-actin mRNAs were not significantly associated
with RACK1 (Fig.5b). By selectively associating with complexes containing these mRNAs,
RACK1 is a prime candidate for recruiting these mRNAs to phagophores. In agreement,
fluorescence microscopy for RACK1-GFP transfected cells demonstrated that it colocalizes with
or is immediately adjacent to many puncta of DFCP1 (Fig.5c) and co-localizes with both ATG12
mRNA and the phagophore marker WIPI2 (Supplementary Fig.3i). In proximity ligation assays
(PLA), RACK1 proteins were also found in the vicinity of DFCP1 proteins but not Histone H3
proteins (Supplementary Fig.3j). In addition, DFCP1 protein, ribosomal proteins (RPL7, RPL18,
RPS9) and ER protein (ATP2A2/SERCA2), were immunoprecipitated by RACK1 after Torin1
treatment (Supplementary Fig.3k). This confirms that RACK1 associates with phagophores and
mRNAs encoding autophagy proteins.

Depletion of RACK1 with siRNA was validated by Western blot and RT-qPCR (Supplementary
Fig.4a, b) and did not affect cellular levels of autophagy-related mRNAs (Supplementary
Fig.4c). More biotinylated proteins by APEX2-DFCP1 were observed in pulldown comparing to
input samples with or without RACK1 siRNAs transfection, indicating successful proximity
labeling and pulldown under RACK1 knockdown (Supplementary Fig.1i). RACK1 knockdown
abrogated the recruitment of mRNAs essential for autophagy, including ATG3, ATG12, VPS34,
DFCP1 and p62 into the vicinity of DFCP1 using proximity labeling pulldowns with APEX2-
DFCP1 (Fig.5d). Similarly, ATG3 or ATG12 mRNA co-localized less with the phagophore
marker WIPI1 by FISH when RACK1 levels were reduced (Fig.5e, Supplementary Fig.4d).
Therefore, RACK1 is necessary for recruitment of mRNA encoding proteins involved in
autophagy to phagophores. Together, these multiple lines of evidence support a model in which
mRNAs encoding proteins essential for autophagy like ATG3 are recruited to forming
autophagosomes in a process dependent on active translation and RACK160-62.

RACK1 regulates translation of autophagy-related mRNAs in proximity to phagophores.
RACK1 associates with ribosomes and selectively regulates the translation of subpopulations of
mRNAs59,63,64. Since RACK1 co-localizes with forming autophagosomes and recruits mRNAs to
this site, we hypothesized that RACK1 controls translation of these mRNAs in proximity to
forming autophagosomes. Consistent with the enrichment of RACK1 to phagophores (Fig.5c,
Supplementary Fig.3i, j), knockdown of RACK1 detectably diminished levels of translation in
cells, as detected by polysome profiling (Supplementary Fig.4e) and AHA nascent protein
labelling (Supplementary Fig.4f). However, RACK1 knockdown selectively had a more
profound effect on translation in proximity to APEX2-DFCP1, even when this was normalized to
account for its effect on global translation (input AHA-labelled proteins) and APEX2-DFCP1
pulldowns (Supplementary Fig.4f). RACK1 knockdown also reduced levels of ATG3, DFCP1
and VPS34 mRNAs in heavy polysome fractions in proximity to DFCP1 (Fig.6a). Cumulatively,
this data demonstrates that RACK1 localizes to forming autophagosomes and preferentially
controls translation of mRNAs encoding proteins required for autophagy there.

If the recruitment of mRNAs encoding proteins that are required for autophagy to the early
phagophore is critical for sustained autophagy, then depleting RACK1 should decrease the rate
of autophagy. Similar to cycloheximide treatment, RACK1 knockdown significantly decreased
the protein level of p62/SQSTM, LC3A/B-II, NBR1, ATG3, ATG5-ATG12, VPS34, and OPTN
required for autophagic degradation (Fig.6b-j). While DFCP1 and NDP52protein levels appeared
to decrease after RACK1 knockdown, this did not reach statistical significance (Fig.6f and 6j).
RACK1 knockdown significantly inhibited autophagosome and autolysosome formation, as
measured using the LC3B-GFP-mCherry system (Supplementary Fig.4g, and Fig.6k, l), and
again the reduction of autophagosomes and autophagolysosomes was not caused by a
fluorescence intensity change of GFP (Supplementary Fig.4h) and mCherry (Supplementary
Fig.4i).

RACK1 associates with an autophagosome biogenesis complex (Atg14L-Beclin 1-Vps34-
Vps15) upon its phosphorylation by AMPK at Thr5058. RACK1 also binds 40S ribosomes, and a
R36D/K38E mutant of RACK1 inhibits this65. Thr50 and R36D/K38E mutants of RACK1 were
generated and used to further dissect the mechanism by which RACK1 localizes mRNAs to
phagophores. APEX2-DFCP1 expressing cells were co-transfected with RACK1 siRNAs
targeting the 3’UTR of RACK1 and Flag-RACK1 mutants (Fig.6m). Wild-type RACK1 rescued
the recruitment of Atg mRNAs into proximity of DFCP1 (Fig.6n). Either RACK1 mutant
abrogated the enrichment of transcripts encoding autophagy proteins transcripts to DFCP1 (Fig.
6n) and inhibited the formation of autophagosomes (Fig.6o). This indicates that interrupting
RACK1 association with ribosomes or with phagophores impairs recruitment of these mRNAs to
forming autophagosomes and autophagy.

Cumulatively, our data demonstrates that several mRNAs essential for autophagy preferentially
localize in proximity to forming autophagosomes and were translated there in a RACK1-
dependent manner. Local translation of these mRNAs in a RACK1-dependent manner is critical
for sustaining levels of autophagy.

Methods
Cell culture HeLa (CCL2, ATCC) and HEK293T (CRL-3216, ASTCC) cells were cultured in
complete medium (Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum
and 1% penicillin-streptomycin) in the incubator at 37°C with 5% CO2.

In vitro APEX2 proximity labelling APEX2-DFCP1 plasmid, APEX2-DFCP1 and -LC3B
expressing Hela cells were generated by Dr. Christian Behrends’ lab37. APEX2 expressing cells
were seeded on 15 cm plates until they were 90% confluent. 24 h before collection, APEX2
expression was induced with doxycycline (40 µg/mL). On the day of collection, cells were
treated with Torin1 to induce autophagy. APEX2 labeling was then initiated by changing the
medium to fresh DMEM medium containing 500 mM (Biotin-Phenol/ biotin-tyramide) BP and
Torin1. The cells were incubated at 37°C under 5% CO2 for 30 min, followed by gentle agitation
for one min after the addition of 1 mM H2O2. The unlabeled controls were processed identically,
except that the H2O2 addition step was omitted.

The APEX2 reaction was quenched by replacing the medium with an equal volume of quenching
buffer (5 mM Trolox, 10 mM sodium ascorbate and 10 mM sodium azide in DPBS) and gently
agitated plate for 10 min. Cells were further washed with cold PBS for two times, scraped with
cell lifters, and transferred to 1.5-mL Eppendorf tubes. The cells were pelleted by centrifugation
at 300 g for three minutes at 4°C and stored at -80°C for further experiments.

Streptavidin purification of biotinylated RNAs TRIzol was added to APEX2 proximity
biotinylated or unlabeled (controls) cell pellets. The cellular RNAs were extracted following the
Tri-Reagent isolation protocol. Dynabeads™ MyOne™ Streptavidin C1 Beads were used to
enrich biotinylated RNAs. 10 µl beads were used for every 25 µg of total RNA. Beads were pre-
washed with 1×Binding and Washing (B & W) Buffer (5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA
and 1 M NaCl) three times, followed by two washes with Solution A (DEPC-treated 0.1 M
NaOH and DEPC-treated 0.05 M NaCl), and one wash with Solution B (DEPC-treated 0.1 M
NaCl). The beads were then suspended in 500 µL of 2 × B & W Buffer and incubated with above
RNAs, dissolved in 500 µL of water on a rotator for two hours at 4°C. After rotation, beads were
washed with 1× B & W Buffer on a magnet rack three times. The beads were resuspended in 7
µL RNase-free water for downstream RT-qPCR applications.

RT-qPCR on biotinylated RNAs 10 µL M-MuLV Reverse Transcriptase Reaction system was
used to syntheses cDNA from input RNAs (1 µg) or captured biotinylated RNAs on beads. After
reverse transcription, the cDNA was separated from the beads using a magnet. The cDNA was
diluted 10 times for qPCR. Custom primers (Table 2 below) were ordered from IDT. The
SYBR™ Green Master Mix was used for qPCR as follows: 95°C for 2 min, (95°C for 30 sec,
55°C (vary with primers) for 30 sec, 72°C for 45 sec) × 50 cycles, 72°C for 10 min.

APEX2-seq APEX2 biotinylated RNAs were pulled down by streptavidin beads and purified
with TRIzol before being sent to sequencing service provider Innomics for RNA sequencing
with DNBSEQ platform at reading length of PE 100bp. RNA-seq libraries were constructed
following the Standard DNBSEQ Eukaryotic Transcriptome Resequencing protocols.
The raw data from the DNBSEQ platform was processed using nf-core/rnaseq v3.14.0 (doi:
https://doi.org/10.5281/zenodo.1400710) of the nf-core collection of workflows86. Sequences
were aligned to the human GRCh38 genome and v45 GENCODE annotations using STAR
2.7.9a87 and quantified using Salmon 1.10.188, generating gene based read counts and TPM files.
Limma-Voom was used to quantify the adjusted P-value and fold change of genes between H2O2
groups and omit conditions from APEX2-DFCP1, -Cyto-V5, and -Perilipin groups. The dot plot
showing fold-change and adjusted P-value was generated.

Immunofluorescence Cells were seeded on cover slips. On the day of collection, cover slips
were fixed with 1% PFA (paraformaldehyde) for 10 min at RT and washed with PBS three
times. Fixed cells were permeabilized with permeabilization buffer (20 mM NH4Cl, and 0.2%
Triton in PBS) for 30 min at RT. Cells were subsequently blocked with 1% BSA for 1 h before
incubating with primary antibody overnight at 4°C. The next day, coverslips were washed with
PBS and incubated with fluorophore-tagged secondary antibody for 1 h at RT. Coverslips were
then washed with PBS and mounted on slides with VECTASHIELD mounting medium.

Polysome fractionation APEX2 expressing cells were processed through proximity labelling as
previously described. The cells were treated with 100 µg/µL of CHX for 5-10 min before
applying quenching buffer. The cells were scraped and pelleted at 400 g at 4°C. Cell Pellets was
suspended in 450 µL of the 1st lysis buffer (5 mM Tris-HCl pH 7.5, 1.5 mM KCl, 2.5 mM
MgCl2, 1× Protease Inhibitor, 20 U/mL RNAse Inhibitor, 1 mM DTT, and prepared in RNAse-
free conditions) and vortexed for 5 seconds. 50 µL of the 2nd lysis buffer (0.5% Triton X-100,
0.5% Sodium deoxycholate) were subsequently added to the cell suspension which was then
vortexed for 5 seconds. The cells in lysis buffer were incubated on ice for 15 min. Cell lysate
was collected by pelleting cell debris at 14,000 RPM at 4°C for 15 min. Absorbance at 260 nm
was used to normalize sample amounts in 500 µL. Ten percent of lysate samples (50 µL) was
kept in -80°C as input. 60% (w/v) sucrose was diluted in polysome buffer containing 20 mM
HEPES (pH 7.6), 0.1 M KCl, 5 mM MgCl2, 1 × Protease Inhibitor, 20 U/mL RNAse Inhibitor,
0.1 mM DTT, and prepared in RNAse-free conditions to make 5% (w/v) and 50% (w/v) sucrose
solutions. Sucrose gradients were prepared with the BioComp model 108 Gradient Master. Five
hundred µL were removed from the top of each sucrose gradient and 450 µL of cell lysate
samples were slowly loaded on top to prevent disrupting the gradient. The tubes were balanced
using the hypotonic lysis buffer before centrifugation at 39,000 RPM at 4°C for two hours with
an SW41Ti rotor. After centrifugation, sucrose gradients were fractionated into ribosome-free
sub-polysome fractions (sub), light polysome fractions (light) and heavy polysome fractions
(heavy) using a density gradient fractionation system by monitoring their absorbance at 254 nm.
The separated sucrose gradient fractions were stored at -80°C prior to further processing.

Streptavidin purification of biotinylated RNAs from different ribosome fractions 1.8 mL of
2 mL of sucrose samples (containing RNAs and proteins) from ribosome-free sub fractions, light
polysome fractions or heavy polysome fractions were incubated with 5-10 µL of MyOne™
Streptavidin C1 beads and rotated for two hours at 4°C. The leftover 0.2 mL of sucrose was used
as input. Biotinylated samples coated with Streptavidin C1 beads were then washed with 1× B
&W Buffer and washed with a magnet three times. TRIzol was then added to the input samples
and beads to purify biotinylated RNAs for RT-qPCR.

FISH staining Cells were seeded on glass coverslips and transfected with plasmid for 48 hr.
Cells were fixed in 4% PFA in PBS for 10 min at room temperature (RT) and washed with PBS,
the slides were subsequently permeabilized and blocked for 1 h at RT with (0.2% Triton X-100,
1% BSA, 100 µg/mL salmon sperm DNA and 250 µg/mL yeast extract RNA in PBS). Slides
were washed with Stellaris Wash Buffer A at RT. Stellaris fluorescent mRNA probes (ATG3,
ATG12, and β-actin) were placed in hybridization buffer (90% Stellaris Hybridization buffer,
10% formamide) and incubated with cells in the dark at 37°C overnight. Slides were washed
with Wash Buffer A, mounted with VECTASHIELD mounting medium with DAPI and sealed
with nail polish.

Streptavidin purification of APEX2 biotinylated nascent proteins APEX2-DFCP1
expressing cells cultured in methionine-free RPMI medium with Torin1 were treated with or
without AHA (50 µM) for 30 min before APEX2-DFCP1 proximity labeling. Cells were washed
two times with PBS, scraped with cell lifters, and transferred to 1.5-mL Eppendorf tubes and
pelleted. Cell pellets were lysed with 50-100 µL of the lysis buffer (0.5% sodium deoxycholate,
0.5% Triton X-100 in PBS) before centrifugation at 10,000 g for 15 min. Biotinylated proteins in
the supernatant were clicked to Alexa Fluor™ 488 Alkyne (5 µM) using the Click-iT® Protein
Reaction Buffer Kit. APEX2 biotinylated proteins were pulled down with Streptavidin C1 beads
for Western blot and detected with HRP-conjugated anti-Alexa488.

SILAC labeling and sample preparation for mass spectrometry APEX2-DFCP1 expressing
cells were incubated in Torin1 containing DMEM consisting of 13C6 15 N2 L-Lysine-2HCl and
13C6 15 N4 L-Arginine-HCl for 30min, before being biotinylated with BP and H2O2, which
were lysed with RIPA buffer and followed by streptavidin beads pulldown. For 100% labeling.
Cells were incubated in DMEM consisting of 13C6 15 N2 L-Lysine-2HCl and 13C6 15 N4 L-
Arginine-HCl and passaged for five doubling events, and the incorporation of heavy amino acids
was tested using mass spectrometry analysis.

Biotin-streptavidin pulldown samples were prepared for mass spectrometry analysis with an on-
bead digestion method. Briefly, a magnetic stand was used to remove liquid from the beads. And
beads were resuspended in 9 µL of 20mM Tris-HCl (pH 8) with 1.6 µL of 25mM DTT, then
incubated for 30 minutes at room temperature. After adding 1.2 µL of 50mM iodoacetamide and
incubate for 10 minutes, 10 µL of 100 ng/µL mass-spec grade trypsin was then added to achieve
a final concentration of 45 ng/µL. The mixture was then incubated on the rotator at 37 °C
overnight. The next day, the beads were removed with a magnetic stand, and additional 500ng of
trypsin was added for 4 hours incubation at 37 °C with gentle vortex. Digestion was stopped with
formic acid (F.A.) at a final concentration of 2%. For desalting, Sep-Pak tc18 1cc cartridges were
conditioned in 900 µL of 100% acetonitrile (ACN) followed by 300 µL of 50% ACN and 0.5%
acetic acid (HAcO). Cartridges were then equilibrated with 900 µL of 0.1% trifluoroacetic acid
(TFA). Samples were adjusted to a final concentration of 0.4% TFA, passed through the
cartridges, desalting with 900 µL of 0.1% TFA, and washing with 90 µL of 0.5% HAcO and
elution with 500 µL of 50% ACN, 0.5% HAcO.

The peak lists of all raw files were processed and analyzed with software MaxQuant (Version
1.6.17.0). against Swissprot protein database of Human (2012, July Release), including
commonly observed contaminants. Cysteine carbamidomethylation was selected as a fixed
modification; the methionine oxidation (+15.99492 Da), protein N-terminal acetylation. Enzyme
specificity was set to trypsin, not allowing for cleavage N-terminal to proline. Up to two missing
cleavages of trypsin were allowed. For SILAC labeled samples, Arg10 and Lys8 were chosen as
light and heavy labels with Multiplicity 2 for quantification. The precursor ion mass tolerances
were 7 ppm, and fragment ion mass tolerance was 0.8 Da for MS/MS spectra. Razor and unique
peptides were used for quantitation. FDR was set 0.01 on both protein and peptide level. For
LCMS Analysis was processed by following this protocol89 and generated data was recorded
using Xcalibur software (ThermoFisher Scientific).

Live cell imaging Cells were seeded on µ-Slide 8-well chambered polymer coverslips at a
density of 15,000 cells per well. A live imaging chamber maintained at 5% CO2 and 37°C on a
Quorum spinning disk confocal microscope (Leica DMI6000B inverted model) was used.
Multiple cells in the field of view were selected for imaging and phenol-free DMEM medium
containing INK128 (50 nM) with or without CHX (100 µg/mL) was added. Cells were imaged
every 30 min for up to six hours. DAPI signals (405 nm laser and DAPI emission filter set,
460/50 nm), GFP signals (490 nm laser and FITC emission filter set, 525/50 nm), and mCherry
signals (561 nm laser and Cy3 emission filter set, 605/52 nm) were used. A 63X oil immersion
objective/1.4 NA was used. Images were captured with a sCMOS camera: Photometrics Prime
BSI (1966x1966).

Imaris software analysis Colocalization of GFP and mCherry positive foci was analyzed by the
distance between these using Imaris software. If the distance was less than or equal to the sum of
half the diameter of GFP and mCherry foci these were defined as colocalized. Based on this, the
software counted the number of single mCherry positive dots per cell and the number of double
fluorescent dots (LC3-mCherry and LC3-GFP) per cell.

DNA plasmid transfection with Polyethylenimine (PEI) Cells were rinsed with PBS and fresh
complete medium was added. DNA was added to media without FBS and PEI (4 µL PEI to 1 µg
plasmid DNA) was added. The mixture was incubated at room temperature for 15-30 min before
adding it to the cell culture. Cells were used 48 hr later.

Immunoprecipitation (IP) Torin1 treated WT HeLa cells were lysed with NP40 lysis buffer
(50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP40, 1 Roche Complete Protease Inhibitor
Cocktail Tablet) for 20 min at 4°C and centrifuged at 5000 g for 10 min. Protein concentration
was measured with a BCA assay. Ten µL of protein G-Dynabeads were added the lysate and
rotated for 20 min at 4°C to reduce non-specific binding to beads. One thousand µg pre-cleared
lysate was then incubated with 4 μg RACK1 or mouse IgG antibody for 3 h at 4°C before
incubation with 25 μL wet volume of protein G-Dynabeads (1 hr, 4°C). Subsequently, beads
were collected by using a magnetic rack and washed with 1 mL wash buffer (50 mM Tris-base,
pH 7.5 and 150 mM NaCl, 0.1% NP-40) three times. Half of the samples were suspended in 1X
SDS-sample buffer and boiled at 99°C for 10 min and beads quickly removed using a magnet.
Supernatants were used for Western blot. The remaining samples were used for RNA extraction
and RT-qPCR.

RNA Immuno-Blot Assay 2 µL of APEX2 biotinylated RNAs and un-biotinylated RNAs were
pipetted onto a positively charged nylon membrane (Roche, Mannheim, Germany). RNA was
cross-linked to the membrane with UV light: 120 mJoule for 1min of both sides at 254 nm.
Biotinylated RNAs were detected with Stabilized Streptavidin-Horseradish Peroxidase
Conjugate and the Substrate Working Solution of Chemiluminescent Nucleic Acid Detection
Module Kit and imaged with CCD camera.

Proximity Ligation in situ Assay Duolink® In Situ Detection Reagents Orange kit (including
Ligase, Ligation buffer, DNA polymerase, and Amplification Orange buffer) was used. Cells
seeded on coverslips were fixed with 4% PFA and permeabilized before being blocked with
Duolink Blocking buffer for 60 min at 37°C. The coverslips were incubated with primary
antibodies diluted in Duolink® Antibody Diluent for 1 h at room temperature. Coverslips were
washed 2 times for 5 min in Wash Buffer A and then incubated with PLA probes MINUS and
PLUS probes corresponding to the primary antibodies (Duolink® In Situ PLA Probe Anti-Mouse
MINUS and Duolink® In Situ PLA Probe Anti-Rabbit PLUS) for 1 h at 37°C. Then, coverslips
were washed twice for 5 min with Wash Buffer A and then incubated with a DNA ligase diluted
in Ligation buffer for 30 min at 37°C. Coverslips were washed 2 times for 5 min with Wash
Buffer A and incubated with a DNA polymerase diluted in Amplification buffer for 100 min at
37°C. Finally, coverslips were washed 2 times for 10 min with Wash Buffer B and subsequent
0.01x Wash Buffer B for 1 minute before being mounted with Duolink® In Situ Mounting
Media with DAPI. Fluorescence was visualized with a Zeiss LSM800 confocal microscope.

siRNA transfection with Lipofectamine RNAiMAX APEX2-DFCP1 expressing cells were
transfected with siRNAs by Lipofectamine™ RNAimax as follows: Cells were plated one day
before transfection to be 50% confluent at the time of transfection. SiRNA- LipofectamineTM
RNAiMAX complexes were prepared in Opti-MEM and incubated for five minutes at room
temperature. Plates were gently rocked to promote mixing, and cells were harvested 72 hr later.

Lentiviral vector production and transduction HEK293T cells stably expressing APEX2-
DFCP1 were generated via lentiviral transduction. Lentivirus was produced through co-
transfection of WT HEK293T cells with the packaging plasmids psPAX2 and pMD2.G, along
with the APEX2-DFCP1 plasmid. The media was harvested 48 h and 72 h post-transfection and
filtered through a 0.45 µM PES membrane. Lentivirus was concentrated by ultracentrifuging
media at 20,800 rpm with a 32Ti rotor for 2 h. HEK293T cells were seeded in 6 well plates 24 h
before transduction with concentrated lentivirus promoted by 8 μg/mL of polybrene. After 48 h,
media was changed, and cells were selected with puromycin (2 μg/mL).

Statistics & Reproducibility Statistical analyses were performed using GraphPad Prism version
8. Data are presented as mean ± SEM / mean ± SD unless otherwise indicated. Sample sizes
were based on those generally used in the field for similar types of experiments. No data were
excluded from the analyses. Plates were seeded from a single batch of cells and randomized to
receive treatments. The experiments were not randomized.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript by Xue et al. sheds light on the interesting concept of localized translation at phagophores. The authors suggest that the translation of some autophagy-relevant mRNAs occurs in proximity to forming autophagosomes in a RACK1-dependent manner and that this allows for sustained autophagy during stress conditions. While the idea is intriguing, the presented data do not convincingly support these claims.\nCritical points\n1. The use of the APEX2-based proximity labeling coupled to qRT-PCR for selected transcripts throughout the manuscript raises major concerns due to the biased nature of the readout combined with a lack of specificity and adequate controls. Expanded below:\na) The use of qRT-PCR as a readout for the APEX2 is not robust enough to support the authors claims. The transcripts shown vary highly from figure to figure giving an unfortunate feeling of cherry picking. For instance: negative control transcripts vary inconsistently between selections of beta actin, MTCO2, H1.1, EGFR, MTCO2. Likewise, the panel of selected autophagy transcripts is also highly variable, for instance between Figures 1, 3 and 6. To get around this issue, the authors should employ an unbiased transcriptome-wide APEX-seq to enable robust conclusions. For examples on this see PMIDs: 31442426, 31230715. If the authors then wish to validate key findings from the seq by qpcr, that is fine, but a consistent set of positive and negative control transcripts selected from the seq should be used.\nb) The most important control in all APEX-related experiments is lacking. The authors should make use of an APEX2-fusion protein which does not localize to the phagophore. Preferably two independent negative control fusion proteins should be used in parallel to DFCP1 and LC3B in order claim true phagophore proximity of selected transcripts. Such a control would also be crucial for the APEX-seq suggested above.\nc) At least some of the key experiments should be repeated in an autophagy-deficient cell line. For instance, the LC3B-APEX qRT-PCR could be performed in a lipidation-deficient line (ATG5/7 KO) or with a lipidation deficient mutant LC3B mutant (G120A). Indeed, LC3B that cannot localize to phagophore should not demonstrate enrichment of these transcripts, and this would be essential to support the author’s claim of localized translation.\nd) It is unclear why sometimes both APEX-DFCP1 and APEX LC3B are used (Figs 1&6), while other experiments are done only with APEX-DFCP1 (eg. Fig 3, 4). The authors should be consistent or at least provide some explanation for these choices.\nd) The images shown in eg. Fig 1 show a large extent of background biotinylation beyond the localized puncta at phagophore sites. This makes it hard to understand how this system could be robust enough to detect localized transcripts – emphasizing the need for adequate controls discussed above.\n2. The authors claim localized translation in proximity to phagophores however the data do not support this claim for reasons specified below:\na) The AHA-click assay coupled to western blotting in Fig 4A is merely a control experiment showing that AHA-incorporation is occurring nearby DFCP1. This would be expected to happen nearby a majority of cytosolic proteins as ribosomes are spread throughout the entire cytoplasm. Therefore, the result does not convincingly tell us anything about localized translation.\nb) Showing presence of selected autophagy mRNAs in heavy polysome fractions in eg. Fig 3 is not enough to support the claim of localized translation. The authors should show the relevant nascent autophagy proteins, for instance by using the AHA-click assay coupled proteomics or at least to western blotting for a panel of autophagy proteins. In this case, the non-phagophore localized control is again crucial to claim specificity.\nc) The reduction in puncta in Figure 4B&C after 6h CHX treatment is not surprising given that this likely reflects a global translational shutdown which would affect a majority of proteins and processes.\nd) The rational for Figure 5 is unclear. How is it novel that translation is required to maintain levels of autophagy proteins? This is expected for a majority of proteins. This does not tell us anything about localized translation. Also, key information about timing and concentration is lacking, and there is no control for- or mention of how the proteasome-regulated half-lives of the proteins would expectantly contribute.\ne) If there are local ribosomes, it is difficult to understand why a large fraction of these would not simply be engulfed and degraded by autophagy. This would make the process highly inefficient and energy-wasting. The authors speculate about selective degradation of damaged ribosomes, but without any supportive data. To enhance the credibility of their claim, the authors should try to understand how ribosomes performing localized translation could be positioned to avoid their random engulfment.\n3. The link to RACK1 is not sufficiently examined. RACK1s implications for autophagy are clear (and previously known) but I am not convinced that this occurs via local translation. Therefore, the novelty here is highly questionable:\na) The use of depletion experiments of RACK1 is not sufficient to tell us about its ability to conduct localized translation at phagophores. Rather than RACK1 depletion, the authors should create relevant mutations to dissociate RACK1 from its binding to 1. autophagy factors and 2. the ribosome. For instance, Thr50 mutation would be important since AMPK-mediated Thr50 phosphorylation enhances RACKs direct binding early autophagy factors (PMID 26549445). Likewise, the RACK1-40S interface is well described and mutations can easily be made to disrupt this. The mutant should be used in key experiments including 6b, 6f, 7c.\nb) RACK is already known to positively regulate autophagy (PMIDs: 22562043, 26549445) and therefore Fig 7d and 7e do not provide any new information. The reduction in puncta upon transient knockdown (7e) is therefore expected and it is unclear how this could reflect a localized translation effect. The rationale for expecting an effect on total protein levels (7d) is unclear and in fact, these reductions may rather suggest broader (non-localized) translation effects.\nc) And why do the RACK1 experiments generally only address a subset of transcripts from those in Figure 3? Is this because RACK1 is only regulating translation of some of these?\nWhy are the transcripts in 7c not the same as the proteins in 7d? These figures should support each other. Why also, are the authors only showing 3 transcripts in 7c + MTCO2 as a control, what about all the others from 6b?\nd) If only some mRNAs are selectively translated near the phagophore in a RACK1-dependent manner, what would be the mechanism by which this occurs? The authors should investigate whether there are certain features pertaining to the translated mRNAs including sequence motifs or other means of post-transcriptional regulation, to at least touch upon this mechanism.\nAdditional comments\n4) It is unclear if there are any endogenous Ab stains throughout the manuscript. For instance, the authors mention that they are looking at endogenous RACK1 in Figure 6c. However, the figure shows RACK1-GFP? Also, the authors mention that they are showing endogenous DFCP1 and LC3B signals in Fig 1c. But in these cells they are expressing the APEX2-tagged constructs. So how can it be said that the puncta co-localize with endogenous proteins? It is understandable that not everything can be done with endogenous stains, but at least some key data should be confirmed with endogenous stains and the authors should be more careful in their experimental descriptions.\n5) Figures 1a,b,c do not present any novel information. They show well-known effects of drugs on LC3BII and polysome profiles. This type of data should – at most – be presented in the supplementary data.\n6) In general, the methods, legends and experimental details are poorly described and often lacking details regarding eg. timing and concentrations of treatments. Also, the handling of qRT-PCR data and methods of normalization are inadequately described and seem variable, sometimes displaying Ct values, sometimes displaying data “vs. omit” and sometimes “vs. omit and input”.\n7) While it is OK to perform qRTpcrs on the pooled sub, light and heavy polysome fractions, the quality of the full polysome profiles should be verified by protein extractions from individual non-pooled fractions in order to verify the presence of 40S, 60S, monosomes, polysomes in the expected fractions. It would additionally be useful to see the full profile for some of the mRNAs to understand how enrichment in some fractions may affect other fractions.\n8) For Figures 3b and c, why are these two graphs separated and why are different controls used in the two graphs? Fig 3c EGFR in fact looks to be 6-7 fold upregulated between “input H” vs. “pulldown H”. Wouldn’t this indicate a similar enrichment as seen for all autophagy transcripts in 3b?\n9) Figure 1d shows enrichment of 18S in the pulldown. Is this also the case for 28S and 5.8S? In later figures there are discrepancies between small and large subunit rRNAs. Surely the full ribosome would be required to be present for this translation task. The authors should test this more extensively and comment.\nMinor comments:\nIt would have been helpful with line nrs and page nrs.\nWestern blots should include MW markers\nThe authors should be careful to distinguish between MTOR and MTORC1\nThere are several mistakes with the references to wrong papers. See eg. #44 and others.", "reviewer_2": "In recent years, localized translation of mRNAs has emerged as a more pervasive phenomenon than previously thought, and it was shown to be associated with several eukaryotic organelles. In this work, Xue et al. study the impact of autophagosome-adjacent translation on autophagy. They show that mRNAs encoding autophagy-related (Atg) proteins tend to be enriched in proximity of forming autophagosomes. These mRNAs are preferentially translated there in comparison to other mRNAs. Autophagy-related proteins are usually continuously degraded during the process itself, making localized translation a convenient mechanism to ensure a continuous supply of these proteins during prolonged exposure to stress. Puromycin treatment, as well as depletion of the ribosome-associated protein RACK1, disrupts the localization of Atg mRNA to the autophagosomes.\nOverall, the manuscript is written clearly. The authors provide evidence that autophagy-related mRNAs tend to be preferentially localized in proximity to autophagosomes and preferentially translated there, showing that autophagosome are associated with localized translation. They also show that translation is required for sustained autophagy, which is in line with Atg proteins being short-lived proteins. However, the evidence speaking in favor of localized translation being required for sustained autophagy is not as strong (see Major comments).\nMajor comments\n- The manuscript adds autophagosomes to a growing list of eukaryotic compartments associated with localized translation. Besides the ER, the authors should cite other relevant work on targeting mRNA to specific organelles, such as mitochondria, centrosomes, peroxisomes, etc.\n- Fig. 1D – Are cytosolic transcripts such as beta-actin and GADPH mRNAs enriched in proximity to APEX2-DFCP1 or -LC3B? These transcripts are used as controls in most other experiments, so it seems relevant to to quantify them here to validate the specificity of this approach, especially since the cytosolic 18S transcripts were also found to be enriched.\n- Fig. 6A – Puromycin induces premature termination of translation, while CHX blocks translationelongation stabilizes polysomes. Does treatment with CHX change the enrichment of Atg mRNAs in proximity to APEX2-DFCP1 or -LC3B? The authors could also test the impact of inhibitors such as harringtonine and emetine. These tests would address whether active translation or association of mRNA with ribosomes are important for localization of Atg mRNAs near autophagosomes.\n- Figs. 6-7 – The observation that the ribosome-associated protein RACK1 is important for proper targeting of Atg mRNAs to autophagosomes and their localized translation is quite interesting. However, since knockdown of RACK1 also negatively impacts global translation, it makes it challenging to assess whether RACK1’s role at autophagosomes is specific. Could the authors perform comparative studies of other ribosome-associated co-factors to demonstrate specificity of the RACK1 depletion phenotype? Also, could the authors make use of RACK1 point mutants perturbing its association with ribosomes (e.g. the R36D/K38E mutant studied in Ref#45) or other protein modules to further investigate the impact of autophagy.\n- The resolution of several of the figure panels are not good (e.g. yellow writing in Figure 1, the microscopy data presented in Figure 6 is not good, in particular for panels 6d and 6e. The authors should take steps to improve the resolution.\n- Can the authors speculate on the targeting mechanisms of Atg mRNAs to autophagosomes?\nMinor comments\n- H2O2 is written with a zero instead on a O (H202) in many parts of the manuscript, as well as some figures, which should be corrected.\n- Line 200: “Previous attempts to use CHX…” please include references to papers in which this effect was confounded.\n- Several statement phrases in the introduction are also not accompanied by appropriate citations (e.g. phrases ending on line 72, 77), this should be rectified.\n- Figure 5d is not cited properly in the text, as is the case for Figures 6c and 6d later in the text.\n- The quantification graph in Figure 5c should include all proteins analyzed in 5a.", "reviewer_3": "In this study, Xue et al., based on proximity-biotinylation technology, revealed that mRNAs encoding several ATG proteins involved in autophagosome biogenesis are enriched at the site of the biogenesis and actively translated there. The authors showed that translation of these mRNAs is important for targeting them to the autophagosome formation site. Moreover, they identified RACK1 as responsible for this targeting and local translation of mRNAs. Collectively, the authors proposed that RACK1-dependent local translation of ATG mRNAs is important for sustained autophagosome formation during nutrient-limiting conditions. Thus, this study could provide a new and interesting insight into cellular mechanisms that support autophagosome biogenesis. However, the present version of the study is preliminary and requires further experiments for the authors to draw their conclusions as listed below.\nMajor comments:\n1. In the absence of RACK1, not only ATG mRNA targeting to the autophagosome formation site but their translation is also attenuated. Therefore, the authors cannot conclude that the local translation of ATG mRNAs is important for autophagosome formation; decreasing ATG protein levels should result in defective autophagosome formation irrespective of the site of their translation. To address this issue, the authors need to show that non-localized translation of an ATG protein causes defects in autophagosome formation even if it produces the same (or more) level of the protein as that control cells.\n2. Given the authors' result that translation is required for the localization of ATG mRNAs to the autophagosome formation site, it is still unclear RACK1, whose knockdown attenuates translation of these mRNAs, is directly involved in mRNA localization. The authors would need to obtain a RACK1 mutant specifically defective in mRNA recruitment to the autophagosome formation site.\n3. The authors should examine whether mRNA biotinylation and localization to the autophagosome formation site are abolished in cells lacking a complex of VPS15, ATG14L, and BECN1 (ATG14L knockout cells), which were reported to interact with RACK1.\n4. Fig. 1d: The authors should add mRNAs encoding autophagy-unrelated cytoplasmic proteins to this analysis. Autophagy-unrelated cytoplasmic proteins fused to APEX2 should also be checked whether they do not significantly biotinylate ATG mRNAs.\n5. Fig. 4b, c and 7e-g: The authors should confirm that the level of GFP-mCherry-LC3B decreased by CHX treatment or RACK1 knockdown was not the cause of a decrease in the dots.\n6. Fig. 7f: Unlike the effect of CHX treatment on autophagosome formation shown in Fig. 4c, that of RACK1 knockdown appeared immediately after Torin1 treatment. How would the authors explain this difference?\n7. The authors should examine whether ATG mRNAs are localized to the autophagosome formation site when autophagosome formation is induced by signals independent of TORC1 inactivation.\nMinor comments:\n1. Line 131: \"H2O2 labeling\" should be \"H2O2 treatment\".\n2. Line 176: \"involving\" should be \"involved\".\n3. Line 181: \"suggest\" should be \"suggests\".\n4. Lines 218-220: Data for LC3-II and p62 are not shown in Fig. 5a-c.\n5. Line 223: Fig. 5c should be Fig. 5d.\n6. Fig. 6b: Are the labels “scramble siRNA” and “RACK1 siRNA” in Input correct?\n7. Lines 241-256: Figure panels should be cited correctly.\n8. Line 253: Data for ATG12 mRNA are not shown in the figure.", "reviewer_4": "In this manuscript, using proximity labeling based on APEX2-DFCP1/-LC3B and polysome fractionation, the authors illustrate that mRNAs encoding proteins participating in autophagosome formation, including ATG3, ATG12, VPS34, DFCP1 and p62, preferentially localize in and are translated in proximity to forming autophagosomes. In addition, by combining methods such as quantification of nascent protein synthesis and siRNA, the authors verify that some of those mRNAs are translated there in a RACK1-dependent manner. The work gives an insight into cell autophagy that proximal translation may enable a rapid, energy-efficient supply of proteins for autophagy to enable cells to massively induce autophagy while conserving energy during cell stress. It is an interesting work. However, there are a few concerns that should be addressed.\n1. A control group of proximity labeling based on APEX2-DFCP1/-LC3B should be added when autophagy is not induced. Such as in the experiment of Fig.1c, Supplementary Fig.1b and Fig.3b, c. Cells treated without Torin 1 before being labelled with BP + H2O2 should be set as reference.\n2. The levels of APXE2-DFCP1 and -LC3B induced by doxycycline (40 μg/mL) in APEX2-DFCP1 and -LC3B expressing Hela cells should be detected and compare with endogenous levels of DFCP1 and LC3B.\n3. The bait protein levels in input and pulldown should be detected and displayed in all APEX2 proximity labeling experiments for confirming successful labeling and pulldown.\n4. The specific enrichment of each mRNA in the data were not very obvious, only a few folds. Especially the specific enrichment of each mRNA in Fig.3 was about 5 to 7 folds change, except for p62 mRNA which is about 35 folds, and the enrichment folds of EGFR mRNA, which is a negative control, are about 7. Can these data represent that those mRNA are really enriched?\n5. In the line 204 of manuscript, the authors have mentioned “mTOR inhibitors (INK128, Torin1) were used to… (Supplementary Fig. 1f)”,but there is no data processed by INK128.\n6. The figures and figure Legends should be described in more detail. Such as in Fig.1a, the treated concentrations for BAF, CHX and Torin1 should be provided. In Fig.1a and Fig.1b, the time of treatment should be noted. These problems also exist in other Figure legends, which should indicate the concentration and time of drug treatment.\n7. There are some writing problems. Such as line117, “Torin1 a small molecule mTOR inhibitor was used to ...” may be changed to “Torin1, a small molecule mTOR inhibitor, was used to ...” Line 223 Fig.5c should be Fig.5d."}, "author_response": "Response: In initial proximity labeling experiments, we included multiple control mRNAs including those that should localize to ER (EGFR), mitochondria (MTCO2, Fazal et al. Cell 2019) or be abundant and most likely to be randomly labeled in proximity to a cytoplasmic protein (B-actin and Histone mRNAs). After this initial validation of the DFCP1-proximity labeling system and its specificity, we routinely included at least one control mRNA and 3-8 mRNAs encoding proteins involved in autophagy. We could not always include every mRNA because of the limiting amount of cDNA. The mRNAs we included also changed somewhat as the project developed over several years. The scale of the enrichment above background can vary across experiments depending on the passage number of the cells and epigenetic silencing of the transduced APEX2-DFCP1.\nRNAseq from proximity labeling pulldowns is challenging. Indeed, I was invited to present this work at the RNA Society International meeting in Edinburgh in a session on RNA localization. I had conversations with numerous internationally renowned experts in RNA localization over the conference about RNAseq of proximity-labeling pulldowns. Many had tried, and none had succeeded in getting useable data. After extensive efforts at optimization, we have obtained RNAseq data from APEX2-DFCP1 as well as three different control proteins, one a diffuse cytosolic APEX2 fused to a V5 tag, or cytosolic APEX2 fused to Perilipin2, and another APEX2 fusion to an RNA-binding protein (NPM1). mRNAs encoding a wide range of autophagy proteins are enriched in proximity to DFCP1 more than in proximity to APEX2-cytoV5, APEX2-Perilipin2, or APEX2-NPM1 when this RNAseq data is normalized to samples not treated with H O (Fig.1f). 2 2\nThis shows in a comprehensive and unbiased manner that these mRNAs are enriched in proximity to phagophores.\nb) The most important control in all APEX-related experiments is lacking. The authors should make use of an APEX2-fusion protein which does not localize to the phagophore. Preferably two independent negative control fusion proteins should be used in parallel to DFCP1 and LC3B in order claim true phagophore proximity of selected transcripts. Such a control would also be crucial for the APEX-seq suggested above.\nResponse: The addition of these controls was addressed above in respect to RNAseq data. In RT-qPCR we have now included data using APEX2 fused to a diffuse cytoplasmic protein (CytoV5 tag), a nucleocytoplasmic RNA-binding protein NPM1, and cytoplasmic Perilipin2. No enrichment above our control mRNAs EGFR and Histone1 was observed of five mRNAs encoding proteins involved in autophagy (Fig.1e). This reinforces our data that many mRNAs encoding proteins involved in autophagy are enriched in proximity to phagophores.\nc) At least some of the key experiments should be repeated in an autophagy-deficient cell line. For instance, the LC3B-APEX qRT-PCR could be performed in a lipidation-deficient line (ATG5/7 KO) or with a lipidation deficient mutant LC3B mutant (G120A). Indeed, LC3B that cannot localize to phagophore should not demonstrate enrichment of these transcripts, and this would be essential to support the author’s claim of localized translation.\nResponse: FIP200 is required for phagophore formation but not for later stages of autophagy, or for non-canonical autophagy pathways. We therefore chose to address this comment more specifically by using FIP200-/- cells. In these cells no enrichment of mRNAs encoding proteins involved in autophagy was observed (Fig.2g, h). Recruitment of mRNAs to DFCP1 also required mTOR inhibition which drives creation of phagophores (Fig.2e, f). These new data demonstrate that mRNAs are recruited to the phagophores and excludes their recruitment to a site of non-canonical autophagy (DOI: 10.1126/sciadv.abo1274).\nThe LC3 mutant experiment the reviewer suggests is not so straightforward. While these LC3-mutants are not included in mature autophagosomes or autophagolysosomes, their localization to phagophores is much less clear. LC3-I must be recruited to an ATG5-12-16L1 complex, at the phagophore, to be converted to LC3-II. LC3-I can also be recruited to the phagophore by p62. The LC3 mutants likely accumulate at phagophores. Similarly, many mutations, like in ATG7 cause phagophores to accumulate and therefore could give counterintuitive results if one is looking to examine LC3 or DFCP1 at phagophores.\nd) It is unclear why sometimes both APEX2-DFCP1 and APEX2-LC3B are used (Figs 1&6), while other experiments are done only with APEX2-DFCP1 (eg. Fig 3, 4). The authors should be consistent or at least provide some explanation for these choices.\nResponse: As this project began, we used both APEX2-LC3B and -DFCP1. Results with the two constructs were similar overall, but enrichment was often lower with the LC3B construct. We eventually decided to use DFCP1 as it is more a precise marker of phagophores, whereas LC3B labels phagophores, maturing autophagosomes, mature autophagosomes and autophagolysosomes. We decided to keep the LC3B data in the paper for transparency and to provide more data to readers.\nd) The images shown in e.g. Fig 1 show a large extent of background biotinylation beyond the localized puncta at phagophore sites. This makes it hard to understand how this system could be robust enough to detect localized transcripts – emphasizing the need for adequate controls discussed above.\nResponse: We purposefully tend to leave fluorescence intensity in our microscopy images high enough to see some cytoplasmic labeling so one can better see the cell shape, and to show that images haven’t been over-processed. We have eliminated this background signal to avoid confusion about the specificity of the signal. We have now also included control cytoplasmic APEX2-fusion proteins. These exclude further that the labeling of autophagy mRNAs is non-specific cytoplasmic labeling.\n2. The authors claim localized translation in proximity to phagophores however the data do not support this claim for reasons specified below:\na) The AHA-click assay coupled to western blotting in Fig 4A is merely a control experiment showing that AHA-incorporation is occurring nearby DFCP1. This would be expected to happen nearby a majority of cytosolic proteins as ribosomes are spread throughout the entire cytoplasm. Therefore, the result does not convincingly tell us anything about localized translation.\nResponse: Figure 4A (now Fig.3a) shows that translation does occur within the 20-40 nm radius labeled by proximity labeling. As many papers in the field state that phagophores emerge from ribosome-free regions of the ER, the observation that translation occurs here is important to make. This is confirmed by fluorescent microscopy where we see punctae of AHA labeling co-localizing with phagophores (Fig.3d), demonstrating that nascent translation is prominent at this location. Evidently, translation may be more or less active in many other cellular locations. We are not attempting to claim that the amount of translation in proximity to phagophores somehow exceeds that in other locations, simply that it exists.\nb) Showing presence of selected autophagy mRNAs in heavy polysome fractions in eg. Fig 3 is not enough to support the claim of localized translation. The authors should show the relevant nascent autophagy proteins, for instance by using the AHA-click assay coupled proteomics or at least to western blotting for a panel of autophagy proteins. In this case, the non-phagophore localized control is again crucial to claim specificity.\nResponse: The Fig.3 has been revised. We used a pulse of SILAC labeling to identify nascent proteins in proximity to phagophores. To enable this we also used a recent method MeProd (DOI: 10.1016/j.molcel.2019.11.010) to enhance sensitivity and accuracy of MS/MS for low levels of SILAC proteins. This data demonstrates that many of the nascent proteins detected in proximity to DFCP1 are involved in autophagy and are encoded by the mRNAs we found in heavy polysomes in proximity to DFCP1, such as ATG3, p62/SQSTM, ATG5, ATG12 and VPS34 (Fig.3h, i). Collectively, this data shows that mRNAs encoding autophagy proteins localize to phagophores (proximity-labeling and FISH, Fig.1), are in heavy polysomes near phagophores (Fig.3f, g), where translation is occurring (Fig.4a-d), and that the nascent proteins in proximity to phagophores are overwhelmingly autophagy-related and encoded by these mRNAs.\nc) The reduction in puncta in Figure 4B&C after 6h CHX treatment is not surprising given that this likely reflects a global translational shutdown which would affect a majority of proteins and processes.\nResponse: As the reviewer surely appreciates, experimentally separating the functional impacts of local translation, without any effect on other cellular translation is virtually impossible. The functional impact of local translation is still debatable even for some of the most studied examples. In the case of autophagy, the literature is not even clear on the importance of translation in general for autophagy (e.g. https://doi.org/10.1016/j.bbrc.2014.01.180 or 10.15252/embr.201846072). The goal of Fig.4B and C (now Fig.4e-g) is to demonstrate in general that continued translation is required to sustain autophagy beginning within 30-60 min after CHX treatment, as this principle is not clear in the literature. We try to make a more direct case for the impact of local translation by knocking down RACK1 in Fig. 6l-n.\nd) The rational for Figure 5 is unclear. How is it novel that translation is required to maintain levels of autophagy proteins? This is expected for a majority of proteins. This does not tell us anything about localized translation. Also, key information about timing and concentration is lacking, and there is no control for- or mention of how the proteasome-regulated half-lives of the proteins would expectantly contribute.\nResponse: The Fig5 (now is Fig.4). We agree it is normal that proteins disappear over time. However, some proteins have half-lives of 30 minutes while others have half-lives of three weeks like SOD1 in the brain. The extent to which these proteins disappear is not known and helps to define the relative impact that continued translation could have. For example, this data unexpectedly showed that NBR1 almost entirely disappears without continued translation, while ATG16L1 is relatively stable (Fig.4a, b). This helps us and readers to understand, for example, that while a 50% decrease in ATG5 is unlikely to matter, given that only 5-10% of cellular levels of ATG5 are required to sustain autophagy, NBR1-mediated degradation may be particularly susceptible to changes in global or local translation.\nWe have added information on timing and concentration. Our focus is on translation and protein production, so we believe that defining which proteins are degraded by the proteasome is not critical.\ne) If there are local ribosomes, it is difficult to understand why a large fraction of these would not simply be engulfed and degraded by autophagy. This would make the process highly inefficient and energy-wasting. The authors speculate about selective degradation of damaged ribosomes, but without any supportive data. To enhance the credibility of their claim, the authors should try to understand how ribosomes performing localized translation could be positioned to avoid their random engulfment.\nResponse: During phagophore formation, a large number of the key proteins involved like FIP200, DFCP1, ATG5-12 and others avoid excessive degradation by autophagy, while LC3 and autophagy receptors are degraded. For this reason, we do not think it would be particularly surprising for ribosomes at this site to avoid degradation by autophagy too.\nSeveral papers have described degradation of ribosomes by autophagy and hypothesized that damaged ribosomes are selectively degraded (e.g. PMID: 18391941). We have revisited the text to ensure that it is clear we are not claiming our evidence shows degradation of ribosomes or damaged ribosomes, but that this has been claimed by others. We added data showing that knockdown of the ribophagy receptor NUFIP1, does not affect translation in proximity to DFCP1 using either the SDS-PAGE or microscopy methods to detect this (Fig.3b-e). This demonstrates that recruitment of mRNAs to phagophores for translation is independent of ribophagy but requires RACK1 (Fig.6).\n3. The link to RACK1 is not sufficiently examined. RACK1s implications for autophagy are clear (and previously known) but I am not convinced that this occurs via local translation. Therefore, the novelty here is highly questionable:\na) The use of depletion experiments of RACK1 is not sufficient to tell us about its ability to conduct localized translation at phagophores. Rather than RACK1 depletion, the authors should create relevant mutations to dissociate RACK1 from its binding to 1. autophagy factors and 2. the ribosome. For instance, Thr50 mutation would be important since AMPK-mediated Thr50 phosphorylation enhances RACKs direct binding early autophagy factors (PMID 26549445). Likewise, the RACK1-40S interface is well described and mutations can easily be made to disrupt this. The mutant should be used in key experiments including 6b, 6f, 7c.\nResponse: We have added experiments showing that recruitment of mRNAs encoding autophagy proteins to phagophores in RACK1 knockdown cells is rescued by wild-type RACK1, but not RACK1 Thr50 or RACK1 R36D/K38E which is required for its binding to ribosomes (Fig.6q, r). This shows, for the first time, that RACK1 binding to complexes involved in phagophore formation and its binding to ribosomes are required to recruit mRNAs to the phagophore site.\nb) RACK is already known to positively regulate autophagy (PMIDs: 22562043, 26549445) and therefore Fig 7d and 7e do not provide any new information. The reduction in puncta upon transient knockdown (7e) is therefore expected and it is unclear how this could reflect a localized translation effect.\nResponse: As we said above, it is extremely difficult to show a functional impact of local translation in the field in general, because of challenges like these of separating RACK1’s local effects on translation, from its effects on translation elsewhere in the cell or autophagosome formation. We have rewritten key parts of the paper to temper statements that our findings demonstrate that local translation is required for sustained autophagy. Our data in Fig.6s shows that formation of autophagosomes is disrupted by mutations in RACK1 which block its association with ribosomes. This data provides improved support for the hypothesis that local translation is important for autophagy.\nc) And why do the RACK1 experiments generally only address a subset of transcripts from those in Figure 3? Is this because RACK1 is only regulating translation of some of these?\nWhy are the transcripts in 7c not the same as the proteins in 7d? These figures should support each other. Why also, are the authors only showing 3 transcripts in 7c + MTCO2 as a control, what about all the others from 6b?\nResponse: Experiments in Fig.7c (now is Fig 6c) require proximity labeling pulldown from ribosome density gradient fractions. The yields are not high, and we only recovered enough RNA to do RT-qPCR for 4 mRNAs involved in autophagy (ATG3, ATG12, VPS34, DFCP1) and a negative control (MTCO2) (Fig.6c). This demonstrates the phenomena is not restricted to a single mRNA. We do not have antibodies that work well for some of the proteins, so we did not test the protein for every mRNA evaluated. We used antibodies for some multiple key proteins in autophagy (ATG3, LC3, ATG5-12). We also validated more autophagy related proteins in the new Fig. 6d-k.\nd) If only some mRNAs are selectively translated near the phagophore in a RACK1-dependent manner, what would be the mechanism by which this occurs? The authors should investigate whether there are certain features pertaining to the translated mRNAs including sequence motifs or other means of post-transcriptional regulation, to at least touch upon this mechanism.\nResponse: We agree it would be great to identify an RNA sequence or structure motif that enables recruitment of these mRNAs to phagophores. This would require several additional figures of experiments to identify and characterize. For example, Fazal first identified local translation in proximity to mitochondria (Cell 2019), and subsequently began to identify RNA features required for this (Qin Nat. Comm 2021). We feel this is more appropriate for a follow-up manuscript. We have nonetheless added data showing that the mRNA localization observed requires binding of ribosomes to RACK1 (Fig.6q, r) and the mRNA (puromycin, Fig.5a, Supplementary Fig.2f).\nAdditional comments\n4) It is unclear if there are any endogenous Ab stains throughout the manuscript. For instance, the authors mention that they are looking at endogenous RACK1 in Figure 6c. However, the figure shows RACK1-GFP? Also, the authors mention that they are showing endogenous DFCP1 and LC3B signals in Fig 1c. But in these cells, they are expressing the APEX2-tagged constructs. So how can it be said that the puncta co-localize with endogenous proteins? It is understandable that not everything can be done with endogenous stains, but at least some key data should be confirmed with endogenous stains and the authors should be more careful in their experimental descriptions.\nResponse: We have used antibodies for endogenous proteins in three places. In Fig.1a we show the proximity-labeling with APEX2-DFCP1 and APEX2-LC3 occurs in discrete spots as expected and these co-localize with DFCP1 and LC3B detected with antibodies. In Supp. 1d we also show that endogenous p62 which is recruited to phagophores (https://www.nature.com/articles/s41467-020-20185-1) also co-localizes with proximity-labeling from APEX2-DFCP1. Proximity-ligation assays in Fig.5e use antibodies recognizing endogenous RACK1 and DFCP1 to show that these endogenous proteins are in proximity to one another.\n5) Figures 1a, b, c do not present any novel information. They show well-known effects of drugs on LC3BII and polysome profiles. This type of data should – at most – be presented in the supplementary data.\nResponse: We have moved Fig.1a and Fig.1b to the supplementary data. For Fig.1c (now is Fig .1a), we keep it in the main figure to confirm the specificity of this proximity-based biotinylation.\n6) In general, the methods, legends and experimental details are poorly described and often lacking details regarding eg. timing and concentrations of treatments. Also, the handling of qRT-PCR data and methods of normalization are inadequately described and seem variable, sometimes displaying Ct values, sometimes displaying data “vs. omit” and sometimes “vs. omit and input”.\nResponse: We have revisited methods and figure legends to ensure critical details are included. We consistently normalize proximity-labeling experiments to the omit (without H O ). The 2 2\nexceptions to this are when we assess effects on translation in proximity to phagophores after RACK1 or NUFIP1 depletion, where the global translation in input is also likely affected.\n7) While it is OK to perform qRTpcrs on the pooled sub, light and heavy polysome fractions, the quality of the full polysome profiles should be verified by protein extractions from individual non-pooled fractions in order to verify the presence of 40S, 60S, monosomes, polysomes in the expected fractions. It would additionally be useful to see the full profile for some of the mRNAs to understand how enrichment in some fractions may affect other fractions.\nResponse: We have included A260 profiles of the ribosome fractionation as standard in the field to show the expected peaks. We also showed the expected effect of mTOR inhibition and EDTA on the A260 profiles as the most common way to validate that peaks are due to ribosomes identity (Fig.3f and Supplementary Fig.1b). We have now also added blots of small and large ribosome proteins which fractionate as expected (Supplementary Fig.2c).\n8) For Figures 3b and c, why are these two graphs separated and why are different controls used in the two graphs? Fig 3c EGFR in fact looks to be 6-7 fold upregulated between “input H” vs. “pulldown H”. Wouldn’t this indicate a similar enrichment as seen for all autophagy transcripts in 3b?\nResponse: These are always relative measures. There was a higher background level in this experiment and so more EGFR mRNA was identified. As EGFR mRNA will localize to ER not very far from the phagophore formation site this is not entirely unexpected if labeling of the DFCP1-APEX2 system was a bit too strong here. The important point is that p62 mRNA is much more highly enriched in proximity to phagophores vs. EGFR mRNA in this experiment. Because of the inconsistency in this data, we have removed it from the paper. Data with other negative and positive controls remains in all other figures.\n9) Figure 1d shows enrichment of 18S in the pulldown. Is this also the case for 28S and 5.8S? In later figures there are discrepancies between small and large subunit rRNAs. Surely the full ribosome would be required to be present for this translation task. The authors should test this more extensively and comment.\nResponse: Fig.1d (now is Fig.1b, c) shows 18S and Fig.5a shows 18S and 28S. We also show that heavy polysomes containing multiple autophagy mRNAs are located in proximity to DFCP1 (Fig.3f, g), translation occurs there (Fig.3a-e), and nascent proteins involved in autophagy are present there (Fig.3h, i). Together this shows with multiple independent approaches that active ribosomes are present in proximity to DFCP1.\nMinor comments:\nIt would have been helpful with line nrs and page nrs.\nWestern blots should include MW markers\nThe authors should be careful to distinguish between MTOR and MTORC1\nThere are several mistakes with the references to wrong papers. See eg. #44 and others.\nResponse: We have made these changes."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed many of my concerns. Still, I have remaining critical points that must be addressed, including the need for additional evidence, in order for the work to support the conclusions and claims of the authors. In addition, there are methodology descriptions and statistical analysis that are lacking at several places. Some initial questions also still remain unanswered.\nRegarding original point 1a)\nI do not find any methodological description of the APEX2-seq, only the heatmap which contains minimal information. Are these changes significant? How many replicates is this based on? Such standard information on the setup, inclusion of p-values etc. must be added as a minimum to support any claims.\nThere are several inconsistencies between 1b and 1f which substantiate the previous concern about the robustness of the data. For instance, p62, a key target in 1b and throughout the entire paper, seems even downregulated in proximity to DFCP1 in the sequencing? How do the authors reconcile such contradictory data?\nAlthough it is understandable that data was collected over years, with slightly changing focus on transcripts over time, this is an issue encountered by most researchers when publishing. Considering the additional concerns introduced from the sequencing, together with the previous inconsistencies that are still there, Figure 1 as a whole comes across as confusing and still requires consolidation by performing some repeats for each panel, to show consistency for a selected group of transcripts and controls.\nRegarding original point 1b)\nFig 1e is now added as an essential control to Fig 1b and the authors state “The mRNAs encoding proteins involved in autophagy which were enriched in proximity to DFCP1, were not enriched in proximity to a diffuse cytoplasmic protein (Cytoplasmic V5-tag) or Perilipin (Fig.1e). Similarly, these mRNAs were not enriched in proximity to NPM1 (Fig.1e).” If Fig 1e should function as a proper negative control (for those transcripts enriched at DFCP1, as stated) it should certainly be done on the same set of transcripts, not on new ones or omitting old ones. Considering the importance of this control, this data is key and must be provided. Fig 1e should also be depicted in the same way as 1b/c, with statistical significance calculated relative to inputs, to allow for comparison.\nRegarding original point 1c)\nIt seems that the FIP200 -/- cells were generated in this study (as stated in author contributions section). If this is the case, a description of their generation and validation is lacking. If they are previoulsy described, the authors should provide a reference. Also, the western in Fig 2g should include blotting for FIP200. In Fig 2h the results from the FIP200-/- cells should be depicted in comparison to WT (as in 2g), including a statistical comparison between the two cell types. A KO cell line cannot stand alone as a negative result without comparison to its WT counterpart from the same experiment. This is standard practice.\nRegarding original point 2a)\nEven if phagophores emerge from ribosome free regions of the ER, there are likely at any given moment, multiple ribosomes in proximity to a phagophore, especially in conditions of ribophagy induction by mTOR inhibition. The authors should comment this in the manuscript. This relates to the concern below about excluding an impact of ribophagy.\nRegarding original points 2c&e)\nThe previous comment: “To enhance the credibility of their claim, the authors should try to understand how ribosomes performing localized translation could be positioned to avoid their random engulfment” was not replied to. Ribosomes are not comparable in abundance, size or nature to the proteins mentioned by the authors that avoid degradation.\nThe role of NUFIP1 in ribophagy has been thoroughly refuted (PMID: 32612236) and it is widely acknowledged in the field that the original findings from (PMID: 29700228) are not reproducible. Therefore, the NUFIP knockdown experiments do not provide value to the manuscript and should be removed. This still leaves the general concern that the findings may be due to a bystander effect of ribophagy, which should be adressed by different means.\nRegarding original point 3b)\nThe authors rightly state that a functional impact of localized translation is debatable, also leaving the biological consequences of their own work questionable. Even more so when potential interference from ribophagy is not excluded (as described above). The authors state that they have rewritten key parts of the paper to “temper statements that our findings demonstrate that local translation is required for sustained autophagy”. Yet there is still even speculation on this in the abstract and elsewhere. Please temper further and point out all changes by indicating line number.\nRegarding original point 3c)\nThe lack of consistency is still problematic between 6c and 6d because without this consistency, the idea that localized translation of specific mRNA leads to local protein production is simply not supported. The corresponding antibodies for the four transcripts in 6c are widely used, available and suitable for western blotting (as shown extensively in the litterature). This includes DFCP1 that the authors even use themselves (Supp. Fig 1F). The experiments should be done to fullfil this.\nRegarding original point 4)\nThe previous question is not answered: “the authors mention that they are showing endogenous DFCP1 and LC3B signals in Fig 1c. But in these cells, they are expressing the APEX2-tagged constructs. So how can it be said that the puncta co-localize with endogenous proteins?” Again, please answer: How can the authors claim that the signals come from the endogenous proteins? The Abs surely recognize the APEX-tagged proteins, which the authors even show are present at several fold higher amounts?\nRegarding original point 6)\nThe authors explain why APEX-qpcr data is sometimes displayed relative to input or not. 1e should be displayed relative to input, according to this explanation. With statistics, as mentioned above.\nRegarding original point 9)\nThe previous question is not answered: “Figure 1d shows enrichment of 18S in the pulldown. Is this also the case for 28S and 5.8S? Please answer. The other findings mentioned here only indirectly adress this point.", "reviewer_2": "The authors have done an excellent job in addressing the main concerns that were outlined previously and have adequately responded to the criticisms. Below are a few minor points that could be addressed in a revised version to improve the paper.\n-In Figure 1F, the authors describe conducting RNA-seq of mRNAs purified from their proximity labeling reactions for APEX-tagged DFCP1, CytoV5 and Perilipin. However, they provided very little information about this experiment. It would seem strategically advantageous to give more information about these results, e.g. how many RNAs were identified in each purification, whether these had any functional signatures via GO-term enrichment analyses, etc. This data would enhance the paper without much additional work and could be added as a supplement, with some elements included in the main figure. It would also be a valuable resource to the community, who may be interested in consulting the data via resources such as GEO.\n-Similar to the comment above, in Figure 3h-I the authors present results of MS profiling datasets. It would again be to their benefit to give more information about these results (e.g. number of proteins identified in the different labeling assays, functional signatures, etc).\n-Line 67: ‘… autophagosomes are formed in a cell.’\n-Line 76: ‘Once activated, the ULK1 complex…’\n-Line 247: ‘… translation of several proteins involved in autophagy is required to…’", "reviewer_3": "The authors have addressed all the concerns I raised in the review of the original manuscript in a satisfactory manner.", "reviewer_4": "The authors have addressed my comments properly."}, "author_response": "Response: We have added the requested details on the APEX2seq sample preparation and RNAseq analysis to the Methods section.\nAre these changes significant? How many replicates is this based on? Such standard information on the setup, inclusion of p-values etc. must be added as a minimum to support any claims.\nResponse: The p-value of each RNA in the APEX2-seq datasets was determined by comparing the three replicates of H O labeling versus H O omission using an independent T-test (see Fig.1f of the Source data file). This confirms that many of the mRNA enrichments in proximity to DFCP1 are not random.\nThere are several inconsistencies between 1b and 1f which substantiate the previous concern about the robustness of the data. For instance, p62, a key target in 1b and throughout the entire paper, seems even downregulated in proximity to DFCP1 in the sequencing? How do the authors reconcile such contradictory data?\nResponse: We included >80 mRNAs from a comprehensive list of proteins involved in autophagy from a recent review from the Mizushima group (PMID: 36635405) in Fig.1f. When such a large number of mRNAs are tested, statistically, with a p=0.05 cutoff we will have several mRNAs that do not show their actual effects. Therefore, statistically, some inconsistencies must be expected. The overarching message of the APEX2-seq analysis is that a very large number of mRNAs encoding proteins involved in autophagy are enriched in proximity to DFCP1 compared to the three control APEX2 fusion proteins.\nThis is shown with multiple independent methods (FISH, RT-qPCR, RNAseq). Each method has inherent biases in the RNAs it will detect, and measure based on RNA structure, interactions, and RNA modifications. For example, FISH probes may be impacted by RNA structure or a RNA’s accessibility. Sequencing and RT-qPCR methods can be biased by changes in RNA purification or an enzyme’s ability to reverse-transcribe or ligate an RNA that is chemically modified (e.g biotinylated) or highly structured. This is well-established with the contrast between standard RNAseq and TGIRT RNAseq. In our experiments, the biotinylated RNAs pulled down for RNA-seq were isolated from streptavidin beads using TRIzol, whereas the RNA for RT-qPCR was directly reverse-transcribed while bound to the beads, different reverse-transcriptases and ligation steps were involved. Such minor variations/inconsistencies are to be expected when one notes the complexity and inherent biases of each method. Collectively, the data with many mRNAs and independent methods reinforces the conclusion that many mRNAs encoding proteins involved in autophagy are enriched in proximity to phagophores.\nAlthough it is understandable that data was collected over years, with slightly changing focus on transcripts over time, this is an issue encountered by most researchers when publishing. Considering the additional concerns introduced from the sequencing, together with the previous inconsistencies that are still there, Figure 1 as a whole comes across as confusing and still requires consolidation by performing some repeats for each panel, to show consistency for a selected group of transcripts and controls.\nResponse: Each panel of Figure 1 already includes data from a minimum of three repeats of the same experiment. Throughout the paper, we have shown the enrichment of these mRNAs in a minimum of three biological replicates in Fig.1, Fig. 5a, Fig. 5f, and Fig 6r confirming that these results are highly reproducible over 12 or more replicates of the experiment.\nRegarding original point 1b)\nFig 1e is now added as an essential control to Fig 1b and the authors state “The mRNAs encoding proteins involved in autophagy which were enriched in proximity to DFCP1, were not enriched in proximity to a diffuse cytoplasmic protein (Cytoplasmic V5-tag) or Perilipin (Fig.1e). Similarly, these mRNAs were not enriched in proximity to NPM1 (Fig.1e).” If Fig 1e should function as a proper negative control (for those transcripts enriched at DFCP1, as stated) it should certainly be done on the same set of transcripts, not on new ones or omitting old ones. Considering the importance of this control, this data is key and must be provided. Fig 1e should also be depicted in the same way as 1b/c, with statistical significance calculated relative to inputs, to allow for comparison.\nResponse: We encourage the reviewer to consider all data provided. Three of the six mRNAs included in Fig.1b were tested on the negative controls to confirm a pattern. One must also consider that APEX2-seq data displayed in Fig.1 further shows across over 80 mRNAs encoding proteins involved in autophagy that most are enriched in proximity to DFCP1 compared to all three control APEX2 fusions. These should compensate for minor changes in which mRNAs were tested in Fig.1b and e.\nWe tested effects on inputs in H₂O₂ treated and untreated groups repeatedly throughout the paper (Fig.1b, c, Fig.2e, f, h, Fig.3g, Fig.5a, f). As the only difference between these two samples is a 1 min H₂O₂ treatment, it is not surprising that consistently there was no change between these input samples. Therefore, the result would be identical in Fig.1e whether this was included or not. We considered it superfluous and unnecessary to analyze input samples in Fig.1e for this reason. The key control (H₂O₂ omit pulldown) is used for normalization instead.\nRegarding original point 1c)\nIt seems that the FIP200 -/- cells were generated in this study (as stated in author contributions section). If this is the case, a description of their generation and validation is lacking. If they are previously described, the authors should provide a reference. Also, the western in Fig 2g should include blotting for FIP200. In Fig 2h the results from the FIP200-/- cells should be depicted in comparison to WT (as in 2g), including a statistical comparison between the two cell types. A KO cell line cannot stand alone as a negative result without comparison to its WT counterpart from the same experiment. This is standard practice.\nResponse: A description of the generation of this cell line using CRISPR has been added to the methods. The FIP200 knockouts were validated by Sanger sequencing (shown below). Both alleles of FIP200 showed frameshifting mutations. Neither we, nor our collaborator (Ryan Russell) could manage to detect FIP200 by Western blot. We therefore showed that these FIP200 knockout cells exhibit the established phenotype: lack of induction of LC3-II, and accumulation of p62 in response to mTOR. While we understand the reviewer’s perspective about including the wild-type control, we believe that the fact that mRNA enrichment in proximity to DFCP1 was reproducible in 12 replicates across Fig.1, Fig. 5a, Fig. 5f, and Fig 6r makes the same point.\nRegarding original point 2a)\nEven if phagophores emerge from ribosome free regions of the ER, there are likely at any given moment, multiple ribosomes in proximity to a phagophore, especially in conditions of ribophagy induction by mTOR inhibition. The authors should comment this in the manuscript. This relates to the concern below about excluding an impact of ribophagy.\nResponse: We do not exclude that ribosomes could localize to phagophores for degradation. We have expanded our analysis of the potential impact of ribophagy in the Discussion now.\nAvailable literature on ribophagy, that the reviewer tells us is the most reliable below, states that ribophagy is a non-selective bystander process of autophagy. Therefore, ribophagy would NOT cause selective enrichment of mRNAs encoding proteins involved in autophagy as our evidence demonstrates using RT-qPCR, RNAseq and FISH.\nThe reviewer’s preferred reference on ribophagy from Heeseon et al. in Wade Harper’s lab (PMID: 32612236) finds only a 3-4% almost undetectable decrease in ribosomes and this only after 24 h of mTOR inhibition. Data from other publications on ribophagy (Lopez et al. Cell Reports 2023, Wyant et al. Science 2018) show that ribophagy is undetectable until at least 10-24 h of mTOR inhibition or 72 h of senescence induction. No published evidence shows any detectable ribophagy at the time points used in the experiments in our article of 2-4 h mTOR inhibition.\nFinally, no data suggests that ribosomes degraded by autophagy are actively translating. Indeed, current models suggest these ribosomes are defective. Our data demonstrates that mRNAs encoding autophagy proteins are in heavy polysomes in proximity to phagophores. Our pulse-chase labeling also shows selective accumulation of nascent ATG3, ATG5, p62, DFCP1 and VPS34 proteins in proximity to phagophores. These findings again contradict current models of ribophagy.\nIn sum, our data contradicts a role for ribophagy in three key areas: (i) selectivity for specific mRNAs, (ii) translation of these mRNAs (heavy polysomes, SILAC) and (iii) a time frame incompatible with detectable ribophagy. Nonetheless, we have modified the Discussion substantially to more fully analyze these issues and raise the possibility that ribophagy could account for the presence of some ribosomes at phagophores.\nRegarding original points 2c&e)\nThe previous comment: “To enhance the credibility of their claim, the authors should try to understand how ribosomes performing localized translation could be positioned to avoid their random engulfment” was not replied to. Ribosomes are not comparable in abundance, size or nature to the proteins mentioned by the authors that avoid degradation.\nResponse: There could be simple explanations. For example, phagophores form a cup-shaped structure which expand and engulf substrates in a specific direction, positioning ribosomes on the exterior surface of the phagophore or underneath this cup would allow them to escape degradation while producing relevant proteins in proximity to forming autophagosomes. We do not exclude that ribosomes translating mRNAs encoding autophagy proteins could be degraded at some point by autophagy, after translating autophagy proteins. If ribophagy is undetectable (Heeseon et al.) at the time frames of mTOR inhibition we use, it is not possible to study it and exclude it.\nThe role of NUFIP1 in ribophagy has been thoroughly refuted (PMID: 32612236) and it is widely acknowledged in the field that the original findings from (PMID: 29700228) are not reproducible. Therefore, the NUFIP knockdown experiments do not provide value to the manuscript and should be removed. This still leaves the general concern that the findings may be due to a bystander effect of ribophagy, which should be addressed by different means.\nResponse: While the reviewer is evidently an expert on ribophagy, and is privileged to knowledge from private discussions, the published peer-reviewed literature is not as definitive. For the time being, we have retained this data in the paper and whenever we mention NUFIP1 we state that its role in ribophagy is disputed. If the editor agrees with the reviewer, we can remove data on NUFIP1.\nAs we outlined above in response to original point 2a there are multiple reasons why ribophagy cannot account for our results. (i) Ribophagy as non-selective would not cause selective enrichment of mRNAs encoding proteins involved in autophagy, (ii) ribosomes targeted for ribophagy are poly-ubiquitinated and are believed to be defective and not translating, while our data shows these mRNAs are in heavy polysomes and producing nascent protein in proximity to phagophores, and finally (iii) ribophagy is undetectable in the time frames we study.\nHow can we perform an experiment to test the impact of ribophagy in a time frame when ribophagy is undetectable? Furthermore, even if ribophagy did meaningfully occur at 2-4 h after mTOR inhibition, if it is non-selective there is no way to specifically interfere with ribophagy without affecting phagophore/autophagosome formation, which is required for the selective recruitment of the mRNAs in our system. This makes it impossible to test the impact of ribophagy.\nRegarding original point 3b)\nThe authors rightly state that a functional impact of localized translation is debatable, also leaving the biological consequences of their own work questionable. Even more so when potential interference from ribophagy is not excluded (as described above). The authors state that they have rewritten key parts of the paper to “temper statements that our findings demonstrate that local translation is required for sustained autophagy”. Yet there is still even speculation on this in the abstract and elsewhere. Please temper further and point out all changes by indicating line number.\nResponse: We have made additional changes in the abstract (line 33 and 35). We have been careful not to firmly conclude that local translation promotes autophagy and revisited the text again to ensure this. Our statements suggest this possibility based on evidence from multiple independent methods that these mRNAs are both selectively enriched and translated there (polysomes, nascent translation proteomics). In this context, it would seem highly unusual to not suggest that local translation could help promote autophagy, while insisting that ribophagy which (i) does not detectably occur in this time frame after mTOR inhibition, (ii) is not selective like our data, and (iii) likely targets disabled ribosomes explains our data. Objectively weighing the literature and our evidence it seems extremely unlikely that ribophagy substantially accounts for our results. We also note that three other expert reviewers are comfortable with our conclusions in the current format.\nRegarding original point 3c)\nThe lack of consistency is still problematic between 6c and 6d because without this consistency, the idea that localized translation of specific mRNA leads to local protein production is simply not supported. The corresponding antibodies for the four transcripts in 6c are widely used, available and suitable for western blotting (as shown extensively in the litterature). This includes DFCP1 that the authors even use themselves (Supp. Fig 1F). The experiments should be done to fullfil this.\nResponse: We have added data showing that RACK1 knockdown also reduces levels of VPS34. More Western blots for more proteins are always possible, but we have already shown the effects on 5 proteins (ATG5-12, LC3, p62 and NBR1, VPS34), including 2 of 4 from Fig.6c (heavy polysome profiling with RACK1 depletion). We believe this adequately demonstrates an impact on translation of proteins involved in autophagy, and the other 3 reviewers concur.\nRegarding original point 4)\nThe previous question is not answered: “the authors mention that they are showing endogenous DFCP1 and LC3B signals in Fig 1c. But in these cells, they are expressing the APEX2-tagged constructs. So how can it be said that the puncta co-localize with endogenous proteins?” Again, please answer: How can the authors claim that the signals come from the endogenous proteins? The Abs surely recognize the APEX-tagged proteins, which the authors even show are present at several fold higher amounts?\nResponse: We encourage the reviewer to consider the full array of data we provide. APEX2 cell lines were generated and validated by our collaborator Dr. Christian Behrends, an expert in APEX2 labeling cell lines. APEX2-DFCP1 biotinylation signal co-localizes tightly with mCherry-DFCP1, with an antibody vs. DFCP1 (Fig.1) and with endogenous p62 (Supplementary Fig.1), which is recruited to the phagophore during its initiation (e.g. PMID:33397898). We have added data confirming the colocalization of APEX2-myc-DFCP1 baits to biotin-positive puncta (labelled with streptavidin) in revised Supplementary Fig.1e. Biotinylated foci are small in agreement with precise localized labeling. In addition, ATG14 proteins were successfully precipitated by streptavidin beads after biotinylation in their generated APEX2-DFCP1 cell lines (revised Supplementary Fig.1f). This construct also biotinylates and selectively enriches a broad collection of proteins involved in autophagosome formation including ATG3, ATG12, ATG5, ATG16L1 and VPS34 (Fig.3h, i), again demonstrating that it localizes appropriately to forming autophagosomes.\nCollectively, this provides a wide array of data from independent methods that show that the DFCP1 proximity labeling system is appropriately and precisely localized.\nRegarding original point 6)\nThe authors explain why APEX-qpcr data is sometimes displayed relative to input or not. 1e should be displayed relative to input, according to this explanation. With statistics, as mentioned above.\nResponse: The reviewer may have misunderstood what we tried to say. Normalizing to input RNA levels can cause decreased cellular levels to be misinterpreted as increased proximal RNA. We believe showing proximity-labeling pulldowns relative to a control pulldown (without H O treatment) is the best way to analyze this data and we have used this strategy throughout the paper including in Fig.1e (note axes labels are “vs omit”). In practice, since 1-minute H O treatment has minimal effects on cellular RNA the results are similar in most cases. The input values are shown in the bars on the left for + H O vs. omit H O in Fig.1b, 1c, 2e, 2f and 2h. This demonstrates that the input values are consistently very close to 1. After showing this many, many times, we did not analyze input RNA in Fig.1e and so this data is not included in Fig.1e.\nRegarding original point 9)\nThe previous question is not answered: “Figure 1d shows enrichment of 18S in the pulldown. Is this also the case for 28S and 5.8S? Please answer. The other findings mentioned here only indirectly address this point.\nResponse: We encourage the reviewer to consider the totality of evidence provided. Collectively, our data uses multiple independent methods to show that complete, active ribosomes are located in proximity to phagophores. In the updated Supplementary Figure.2g, we show enrichment of 28S rRNA in proximity to DFCP1 by RT-qPCR. Our mass spectrometry found that a large number of ribosomal proteins from both large (with the prefix \"RPL\") and small ribosomal subunits (with the prefix \"RPS”) were among the most enriched proteins in proximity to DFCP1 (added in revised supplementary Fig.2d and Source data file). Our polysome profiling demonstrates the presence of heavy polysomes on mRNA in proximity to phagophores again showing that full ribosomes are present. Our functional data demonstrates translation is enriched in proximity to phagophores using fluorescent microscopy and gel analysis of AHA-labeling and proximity pulldown. Collectively, this data demonstrates a large number of protein and RNA components of functional ribosomes are present, these ribosomes are loaded on mRNAs and actively translating near phagophores (pulse-chase MS/MS, fluorescent microscopy)."}, {"round": 3, "reviewer_comments": {"reviewer_1": "Regarding original point 1a)\nT-tests are not normally used for RNA-seq analysis. Seq datasets do not follow normal distribution and have mean-variance dependence, usually rendering t-tests unsuitable. Also, the data must be adjusted for multiple hypothesis testing - eg. using more commonly applied methods for seq analysis such as DESeq2 and EdgeR. The journal may choose to confer with a statistical expert to ensure the correctness of the approach used here.\nThe question then remains whether conclusions are similar if a new analysis is required. Figure 1 should be revised accordingly.\nRegarding original point 1b)\nFig 1b and 1e should be done on the same set of transcripts, for reasons stated previously. The author refers to many repeats and other figures showing similar points. These are all appreciated. But these do not change the fact that this particular key experiment should be properly controlled to ensure that the data quality and robustness lives up to the standard of the journal.\nRegarding original point 1c)\nIn Fig 2h the FIP200 -/- experiment should be performed in comparison to WT cells, for reasons stated previously. Statistics should then be included in the comparison between WT and KO. Again, this concern is unrelated to replicates/other figures with similar points. This is solely to ensure that the quality and conclusions from this particular experiment live up to the standard of the journal.\nRegarding original points 2c&e)\nIt is still suggested to remove the NUFIP data. The concern was based on peer-reviewed literature that was referred to by PMID, not on private discussions.\nRegarding original point 3c)\nThe point is solely about consistency between 6c and 6d. Not about how many proteins are shown in different experiments. The suggestion remains to enhance this consistency to achieve a robust claim for the particular experiment.\nRegarding original point 4)\nThe authors again do not answer the question. The robustness of the DFCP1 proximity labeling system was not questioned. The question was about statements in the manuscript claiming “co-localization with endogenous DFCP1 or LC3B” (both in the main text and legend), which did not make sense. It is apparent that these statements have now been removed by the authors in the newest version, although this was not mentioned by the authors in their reply.\nRegarding original point 9)\nThe referral to other supportive data is fine, but it is unfortunate that the precise question for fig 1d still remains unanswered.", "reviewer_2": "The authors have addressed all of my main scientific criticisms. Overall, I believe the story is quite complete and novel, supported by a variety of complementary results.\nMy main criticism is more about the Figure aesthetics. The font usage seems inconsistent and in some cases very small. For a final version of the paper, perhaps the authors could move some of the sub-panels to the supplemental figures to allow sufficient space to increase font sizes, etc.", "reviewer_5": "Autophagy requires the coordinated activity of numerous autophagy-related proteins. In this study, Xue and co-workers employ APEX2-mediated proximity labeling to profile RNAs and proteins localized near forming autophagosomes. The authors demonstrate that mRNAs encoding key autophagy regulators (e.g., ATG3, ATG12) are enriched at autophagosome formation sites, as evidenced by APEX2-DFCP1/LC3B proximity labeling and single-molecule FISH. Polysome profiling and nascent proteomics further confirm active translation of these mRNAs at these loci, mediated by the ribosome-associated protein RACK1, which facilitates mRNA recruitment to phagophores. Overall, the manuscript presents a compelling mechanism whereby RACK1-dependent local translation ensures an energy-efficient supply of autophagy machinery components. While the study is well-structured and addresses a critical gap in understanding spatial regulation of autophagy, the following issues should be addressed to strengthen the rigor and interpretability of the findings.\n1. The authors analyzed RNAs proximal to autophagosomes using APEX-seq but focused the heatmap (Fig. 1e) on preselected \"candidate Atg genes.\" To enhance transparency and provide a global perspective, a volcano plot comparing enrichment levels (fold change) and statistical significance (p-values) across all transcripts would better illustrate transcriptome-wide specificity. For example, DEseq analyses should be performed to compare the APEX-seq data from APEX2-DFCP1 versus each of the three controls (APEX2-cytoV5, APEX2-perilipin, APEX2-NPM1). This approach would clarify whether autophagy-related mRNAs are uniquely enriched or part of a broader RNA localization pattern.\n2. In Figures 1b/c, the authors claim that EGFR mRNA is not enriched near autophagosomes. However, EGFR exhibits higher fold changes in APEX2-DFCP1/LC3B pulldowns compared to negative controls (e.g., H1.1). The use of a two-way ANOVA is briefly mentioned, but justification for this choice over simpler tests (e.g., Student’s t-test) is lacking. Clarifying the rationale for the statistical method and confirming whether conclusions hold under alternative analyses would strengthen confidence in the specificity claims.\n3. The authors applied both polysome profiling and nascent proteomics to investigate localized translation. A direct comparison of these datasets would reinforce the conclusion that translationally active mRNAs are functionally prioritized. Specifically, correlating RNA enrichment (APEX-seq/polysome profiling) with nascent protein synthesis (SILAC H/L ratios) would yield useful information. Highlighting overlaps or discrepancies between these methods would provide deeper mechanistic insight.\n4. The dependency of mRNA localization on RACK1 and active translation raises key questions about its molecular role. One possibility is that RACK1 may act as an RNA-binding protein to directly recruit transcripts. Alternatively, RACK1 may recognize nascent peptide motifs (similar to signal recognition particle-mediated targeting) to indirectly recruit ribosome-bound RNAs? For the benefit of the readers, the authors should elaborate how their findings agree or disagree with these models."}, "author_response": "Response: Thank you for your suggestion. We have re-analyzed the RNA-seq data from the APEX2- proximity labeling experiment using DESeq2 and generated updated dot plots (Figure.1d) to replace the original Figure 1f. The revised figure reflects this new analysis and supports conclusions consistent with our original findings.\nRegarding original point 1b)\nFig 1b and 1e should be done on the same set of transcripts, for reasons stated previously. The author refers to many repeats and other figures showing similar points. These are all appreciated. But these do not change the fact that this particular key experiment should be properly controlled to ensure that the data quality and robustness lives up to the standard of the journal.\nResponse: We have repeated the experiment and performed the analysis in the same manner as in Figure 1b and 1c. All mRNAs that showed enrichment in proximity to DFCP1 or LC3B were tested with the CytoV5-APEX2 and Perilipin-APEX2 systems and did not show enrichment in proximity to these negative control constructs. These updated results are now included in Supplementary Figure 2b.\nRegarding original point 1c)\nIn Fig 2h the FIP200 -/- experiment should be performed in comparison to WT cells, for reasons stated previously. Statistics should then be included in the comparison between WT and KO. Again, this concern is unrelated to replicates/other figures with similar points. This is solely to ensure that the quality and conclusions from this particular experiment live up to the standard of the journal.\nResponse: We repeated this experiment with WT and FIP200-/- cells as requested. The conclusions remain the same. This shows that many of the mRNAs encoding proteins involved in autophagy are enriched in proximity to WT cells but not in FIP200-/- cells (Fig.2e, below). In addition, the manuscript already provides multiple datasets that demonstrate that the mRNAs involved in autophagy are enriched at phagophores/autophagosomes without relying on this data with FIP200-/- cells. These include:\n(i) Enrichment with proximity labeling with DFCP1 by both RT-qPCR and RNAseq\n(ii) Enrichment with proximity labeling with LC3B\n(iii) Lack of enrichment with two control non-phagophore proximity-labeling proteins\n(iv) Induction of enrichment by Torin1 treatment that induces phagophores\n(v) Enrichment in proximity to phagophore markers WIPI1 and DFCP1 by FISH\n(vi) Enrichment with proximity labeling with DFCP1 by polysome profiling\n(vii) Enrichment with proximity labeling with DFCP1 by nascent peptide labeling proteomics\nCumulatively, this data makes a very strong case for localization of these mRNAs to phagophores/autophagosomes already with three independent markers of phagophores or autophagosomes (DFCP1, WIPI2, LC3B) and with multiple independent negative controls. We have used multiple independent methods (RT-qPCR, RNAseq, FISH, polysome profiling, nascent peptide proteomics) to show this mRNA localization including unbiased proteomic and RNAseq methods.\nRegarding original points 2c&e)\nIt is still suggested to remove the NUFIP data. The concern was based on peer-reviewed literature that was referred to by PMID, not on private discussions.\nResponse: We removed the NUFIP1 data.\nRegarding original point 3c)\nThe point is solely about consistency between 6c and 6d. Not about how many proteins are shown in different experiments. The suggestion remains to enhance this consistency to achieve a robust claim for the particular experiment.\nResponse: We appreciate the reviewer’s suggestion regarding the need for consistency between Figures 6c (now 6a) and 6d (now 6b). In response, we performed additional experiments to quantify the protein level changes of ATG3 and DFCP1 in cells transfected with RACK1 siRNAs (Fig.6e, f). These experiments showed that ATG3 protein levels are significantly reduced, while DFCP1 levels are also reduced, though not to a statistically significant degree (Fig.6f). We believe these additional data improve the consistency between the panels and strengthen the robustness of our conclusions for this specific experiment.\nRegarding original point 4)\nThe authors again do not answer the question. The robustness of the DFCP1 proximity labeling system was not questioned. The question was about statements in the manuscript claiming “co-localization with endogenous DFCP1 or LC3B” (both in the main text and legend), which did not make sense. It is apparent that these statements have now been removed by the authors in the newest version, although this was not mentioned by the authors in their reply.\nResponse: We revised the text to: “These formed discrete foci only in cells treated with H O , and these colocalized with DFCP1 or LC3B, respectively” in line 126-128 and legend of Fig.1a. We added data that APEX2-DFCP1 biotinylated foci also co-localize with endogenous p62 (Supp. Fig.1d) which is recruited early to phagophore formation sites.\nRegarding original point 9)\nThe referral to other supportive data is fine, but it is unfortunate that the precise question for fig 1d still remains unanswered.\nResponse: The Fig.1d, is now Fig.1b and 1c.\nWe encourage the reviewer to consider the totality of evidence provided. Collectively, our data uses multiple independent methods to show that complete, active ribosomes are located in proximity to phagophores. These include polysome profiling data showing heavy polysomes in proximity to DFCP1 including the mRNAs of interest. In the updated Supplementary Figure.3h, we show enrichment of 18S rRNA and 28S rRNA in proximity to DFCP1 by RT-qPCR. Our mass spectrometry found that a large number of ribosomal proteins from both large (with the prefix \"RPL\") and small ribosomal subunits (with the prefix \"RPS”) were among the most enriched proteins in proximity to DFCP1 (added in revised supplementary Fig.3a and Source data file). Together these provide multiple independent pieces of robust evidence for the presence of both RNA and protein components of ribosomes and their active translation at the site.\nReviewer #2 (Remarks to the Author):\nThe authors have addressed all of my main scientific criticisms. Overall, I believe the story is quite complete and novel, supported by a variety of complementary results.\nMy main criticism is more about the Figure aesthetics. The font usage seems inconsistent and in some cases very small. For a final version of the paper, perhaps the authors could move some of the sub-panels to the supplemental figures to allow sufficient space to increase font sizes, etc.\nResponse: We appreciate your observation about the inconsistent font usage and small font sizes in some panels. We revised the figures to ensure consistency and appropriately sized fonts throughout. Additionally, we moved some sub-panels to the supplementary figures to create more space and improve clarity in the main figures.\nReviewer #5 (Remarks to the Author):\nAutophagy requires the coordinated activity of numerous autophagy-related proteins. In this study, Xue and co-workers employ APEX2-mediated proximity labeling to profile RNAs and proteins localized near forming autophagosomes. The authors demonstrate that mRNAs encoding key autophagy regulators (e.g., ATG3, ATG12) are enriched at autophagosome formation sites, as evidenced by APEX2-DFCP1/LC3B proximity labeling and single-molecule FISH. Polysome profiling and nascent proteomics further confirm active translation of these mRNAs at these loci, mediated by the ribosome-associated protein RACK1, which facilitates mRNA recruitment to phagophores. Overall, the manuscript presents a compelling mechanism whereby RACK1-dependent local translation ensures an energy-efficient supply of autophagy machinery components. While the study is well-structured and addresses a critical gap in understanding spatial regulation of autophagy, the following issues should be addressed to strengthen the rigor and interpretability of the findings.\n1. The authors analyzed RNAs proximal to autophagosomes using APEX-seq but focused the heatmap (Fig. 1e) on preselected \"candidate Atg genes.\" To enhance transparency and provide a global perspective, a volcano plot comparing enrichment levels (fold change) and statistical significance (p-values) across all transcripts would better illustrate transcriptome-wide specificity. For example, DEseq analyses should be performed to compare the APEX-seq data from APEX2-DFCP1 versus each of the three controls (APEX2-cytoV5, APEX2-perilipin, APEX2-NPM1). This approach would clarify whether autophagy-related mRNAs are uniquely enriched or part of a broader RNA localization pattern.\nResponse: Thank you for this helpful suggestion. In response, we re-analyzed the APEX2-seq data using DESeq2 to compare APEX2-DFCP1 with each of the controls (APEX2-CytoV5 and APEX2-Perilipin). Based on this analysis, we generated updated dot plots, now included in the revised Figure 1d, which display the fold changes and p-values for selected autophagy-related mRNAs as colored dots.\nTo provide a more global analysis we have added Volcano plots for DFCP1, CytoV5 and Perilipin proximity labeling (see below and Supplementary Fig.2c). This demonstrates that virtually no RNAs are significantly enriched in the CytoV5 and Perilipin proximity labellings above background. This re-iterates that these are good negative controls for non-specific labeling. After careful analysis we removed data with NPM1 because it has RNA-binding activity with poorly understood specificity and therefore may pulldown mRNAs encoding proteins involved in autophagy, making it a poor negative control.\nAdditional mRNAs beyond those involved in autophagy are enriched in DFCP1 proximity labeling. Supplementary Table 1 contains the full RNAseq dataset so readers to perform other queries. First, we note that in this complete list there are several other mRNAs enriched in proximity to DFCP1 that are also involved in autophagy like SMCR8, CCGP1 and OSBPL8. Text has been added to the Results section concerning this. In addition, we performed a GO-term analysis of all mRNAs enriched in proximity to DFCP1. GO-terms related to chromosome replication and cell divison were enriched (Supplementary. Figure 3b. Bar graph of enriched terms across gene lists, colored by p-values.). Nascent peptides from these mRNAs involved in cell division were not detected by proteomics suggesting these mRNAs are not translated. mTOR inhibition slows cell growth and division, suggesting translation of these mRNAs are likely disfavored. These mRNAs may localize somewhere in proximity to the ER or phagophores as part of a post-transcriptional repression program, or potentially for degradation by autophagy. These possible interpretations have been included in the discussion. Investigating these other mRNAs further is the subject of another research project, as these mRNAs are not locally translated and beyond the scope of the current article.\n2. In Figures 1b/c, the authors claim that EGFR mRNA is not enriched near autophagosomes. However, EGFR exhibits higher fold changes in APEX2-DFCP1/LC3B pulldowns compared to negative controls (e.g., H1.1). The use of a two-way ANOVA is briefly mentioned, but justification for this choice over simpler tests (e.g., Student’s t-test) is lacking. Clarifying the rationale for the statistical method and confirming whether conclusions hold under alternative analyses would strengthen confidence in the specificity claims.\nResponse: EGFR mRNA, which encodes a transmembrane receptor, is translated on the endoplasmic reticulum (ER), near where DFCP1-positive phagophores originate. This spatial overlap likely explains why some small amounts of EGFR mRNA could be detected in proximity to DFCP1, despite not being specifically involved in autophagy. This difference would be significant if a Student’s t-test was used, but this statistical test does not account for the testing of multiple mRNAs. We selected an ANOVA for Figures 1b/c to account for the repeated testing of multiple mRNAs. Nearly all of the mRNAs involved in autophagy are not transmembrane or secreted proteins, so this could not account for their enrichment there.\n3. The authors applied both polysome profiling and nascent proteomics to investigate localized translation. A direct comparison of these datasets would reinforce the conclusion that translationally active mRNAs are functionally prioritized. Specifically, correlating RNA enrichment (APEX-seq/polysome profiling) with nascent protein synthesis (SILAC H/L ratios) would yield useful information. Highlighting overlaps or discrepancies between these methods would provide deeper mechanistic insight.\nResponse: As requested, we have now added a correlation analysis between RNA enrichment (APEX2-DFCP1-seq) and nascent protein synthesis (SILAC H/L ratios), in Figure.3d (below). The correlation for key autophagy-related mRNAs/nascent proteins that appeared in both datasets is apparent. Notably, ATG3, ATG5, ATG12, and PIK3C3 were significantly enriched in the APEX2-DFCP1-seq data and also exhibited strong labeling in the SILAC data, suggesting active translation at the site of RNA enrichment. Some mRNAs exhibited high levels of nascent peptides like p62 (SQSTM1) whereas others showed lower levels of nascent peptides like ZFYVE1 (DFCP1). These mRNAs with weaker correlation can likely be attributed to differences in the rate of translation of individual mRNAs. Translation rates can vary to a large degree due to specific RNA sequences, structures and RNA-binding protein regulators.\n4. The dependency of mRNA localization on RACK1 and active translation raises key questions about its molecular role. One possibility is that RACK1 may act as an RNA-binding protein to directly recruit transcripts. Alternatively, RACK1 may recognize nascent peptide motifs (similar to signal recognition particle-mediated targeting) to indirectly recruit ribosome-bound RNAs? For the benefit of the readers, the authors should elaborate how their findings agree or disagree with these models.\nResponse: Thank you for raising this important point. Our findings support a model in which RACK1 contributes to mRNA localization and translation at forming autophagosomes in a puromycin- and translation-dependent manner. This supports a model in which RACK1 facilitates mRNA localization by associating with translating ribosomes. We don’t believe that our data can distinguish whether RACK1 does this by binding to the mRNAs via its positioning near the mRNA exit tunnel, or by binding to nascent peptides. We have discussed these possible mechanistic models in the article."}, {"round": 4, "reviewer_comments": {"reviewer_5": "The authors have addressed the questions raised in the previous round of review. In the APEX-CytoV5 data, it is quite surprising that so few RNAs were enriched. It is usually expected that cytoplasmic APEX2 could label many RNAs. The data presented in this study contradicts with previous APEX-seq studies. Do the authors have any control experiments demonstrating that the CytoV5 labeling and the subsequent RNA enrichment were successful? To help readers better understand the method, these control data should be included and the low level of enrichment of APEX-CytoV5 should be explicated discussed."}, "author_response": "Response: While Proximity-labeling of RNA with APEX2 is in principle a powerful way to assess RNA localization, there have been very few publications using it since the original reports in 2019. The ones that have used the method reported weak enrichment of about 2-fold or less for most mRNAs (PMID: 34875090 see Fig.4c and 3d, PMID: 33306092 see RIP or PFA-RIP in Fig.2d). Others have begun to use alternative approaches including fixation of cells, alternative enzymes etc. This suggests it is likely a very challenging system requiring substantial optimization, as we also experienced. Corroborating this idea, at the International RNA Society meeting and elsewhere when I presented this research, I had many people come ask me about the APEX2 method for RNA pulldown because no one could get it to work even if they had expert-level experience with proximity-labeling of proteins. In this context, I think we should expect weak enrichment, and our results are not substantially different from the few papers reporting similar experiments in literature.\nWe provide the following evidence that CytoV5 labeling and RNA enrichment worked.\n(i) Fluorescent microscopy validation that CytoV5-APEX2 labeling is active (Supplementary Fig.2a).\n(ii) We re-analyzed the APEX2-CytoV5 RNAseq data using the limma package, that is frequently used for analysis of RNAseq datasets. In this analysis we normalized each replicate to the control performed with the same cells at the same time to reduce variability. Using this method, we observe a standard volcano plot pattern showing significant enrichment and under-representation of RNAs in expected patterns. The plot on the left is with adjusted p-value (Supplementary Fig.2d) and the plot on the right is unadjusted p-value.\n(iii) Analysis of RNAs enriched and under-represented in CytoV5 labeling show expected patterns:\na. Under-representation of nuclear lncRNAs including NEAT, XIST. Many of the most under-enriched mRNAs are lncRNAs or anti-sense RNAs which are usually nuclear (blue dots in plot above from Supplementary Fig.2d, Supplementary Table) \nb. GO term analysis shows CytoV5 depleted RNAs involved in chromatin-binding, DNA binding, transcription and other nuclear functions. Fazal et al. also noted this in their APEX2-seq data for nuclear-enriched mRNAs. (Supplementary Fig.2e, below). We further note that a set of mRNAs enriched in the nucleus in the analysis of Fazal et al. is under-enriched in our cytosolic APEX2-seq data, as expected (Supplementary Fig.2f, below).\nLog2-fold enrichment in CytoV5-APEX2seq of nuclear mRNAs from Fazal et. al. PMID: 31230715\n0.1\n0\n-0.1\n-0.2\n-0.3\n-0.4\n-0.5\nc. GO term analysis shows CytoV5 labeling enriches mRNAs encoding proteins from the cell periphery\nd. Assessment of the enrichment of Autophagy-related mRNAs in cytoplasmic APEX2seq datasets from other papers (PMID: 31230715, APEX2-OMM (outer mitochondrial membrane) and APEX2-NES (FLAG-APEX2-NES uses a nuclear export signal (NES)) shows no substantial enrichment of this set of mRNAs compared to that with DFCP1.\ne. RT-qPCR data of Beta-Actin in the CytoV5-APEX2 pulldown showing weak enrichment (Supplementary Fig.2b).\nActin\n2.5\n2.0\ne\ng n a )tim 1.5\nho\nc\nd .s 1.0\nlov\n(\nF\n0.5\n0.0\nInput\nPulldo\nwn\nThis analysis demonstrates that while the CytoV5-APEX2 system was functional and enriched or depleted the RNAs it would be expected to, the enrichment of these RNAs was weak and somewhat variable, impairing the statistical analysis of these results. While mRNA enrichment with CytoV5-APEX2 was weak, the enrichment of Autophagy-related mRNAs in proximity to DFCP1 was much larger and statistically significant with adjusted p values. This underscores that the enrichment of these mRNAs in proximity to DFCP1 is substantial."}, {"round": 5, "reviewer_comments": {"reviewer_5": "The authors have addressed the APEX-seq issue raised in the previous round of review. I support publication at Nature Communications."}, "author_response": ""}]

Decision letter
[Editorial Note: See attachment]