EMBO/soda-nel huggingface dataset:
An alternative UPF1 isoform drives conditional remodeling of nonsense-mediated mRNA decay (2022)
Figure 1

Figure 1. Alternative UPF1LL splice isoform contributes to NMD under normal cellular conditions.

A (Top) Schematic representation of alternative 5' splice site usage in exon 7 of mammalian UPF1 that results in two UPF1 protein isoforms that differ in length of the regulatory loop within the helicase core. (Bottom) Ribbon diagram of human UPF1SL helicase core overlaid with that of human UPF1LL. The regulatory loop in domain 1B is indicated for UPF1SL (light blue) and UPF1LL (purple), based on Protein Data Bank accessions 2XZP and 6EJ5 (Chakrabarti et al., 2011; Gowravaram et al., 2018).

B (Top) Semiquantitative RT-PCR of UPF1SL or UPF1LL transcript levels following transfection of HEK-293 cells with a NT siRNA or a siRNA that specifically targets the UPF1LL isoform. (Bottom) Venn diagram (to scale) of overlapping targets identified from RNA-seq following total UPF1 or UPF1LL-specific knockdown. Depicted are genes that increased in abundance at least 1.4-fold (FDR < 0.05) and met read count cutoffs in both datasets. P value indicates enrichment of genes that increased in abundance at least 1.4-fold (FDR < 0.05) with UPF1LL-specific knockdown among those regulated by total UPF1, as determined by Fisher's exact test.

C Heat map of changes in relative mRNA abundance for genes encoding NMD factors, as determined from RNA-seq following transfection of HEK-293 cells with a siRNA that targets both UPF1 isoforms (UPF1total) or a siRNA that specifically targets the UPF1LL isoform.

D Density plot of changes in relative mRNA abundance as determined by RNA-seq in SMG7ko/SMG6kd cells, relative to a parental cell line treated with control siRNAs (Boehm et al., 2021). Genes were categorized as up-regulated by siUPF1total only, siUPF1LL only, or both siUPF1total and siUPF1LL. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons.

E RT-qPCR analysis of indicated transcripts following transfection of HEK-293 cells with the indicated siRNAs. Relative fold changes are in reference to NT siRNA. Asterisk (*) indicates P < 0.05, as determined by two-way ANOVA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed line indicates log2(fold change) of +0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons (Lareau et al., 2007; Ni et al., 2007). See also Dataset EV3 for P values associated with each statistical comparison.

F Gene ontology analysis of 1621 genes that increased in expression at least 1.4-fold upon UPF1LL-specific knockdown in HEK-293 cells under normal cellular conditions. Genes may map to multiple categories.
An alternative UPF1 isoform drives conditional remodeling of nonsense-mediated mRNA decay (2022)
Figure 2

Figure 2. UPF1LL is enriched on NMD-protected transcripts.

A Scheme for the CLIP-UPF1 affinity purification (RIP) assay.

B Scatterplot of CLIP-UPF1LL vs CLIP-UPF1SL RIP-seq efficiency. mRNAs were binned according to 3'UTR length (short, medium, or long).

C Density plot of recovered mRNAs in CLIP-UPF1LL or CLIP-UPF1SL affinity purifications. mRNAs were binned according to 3'UTR length. Statistical significance was determined by K-S test.

D Density plot of recovered mRNAs in CLIP-UPF1LL affinity purifications relative to that of CLIP-UPF1SL. mRNAs were subdivided by PTBP1 and/or hnRNP L motif density within the 3'UTR, as indicated by the gradient triangle. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons.

E RT-qPCR analysis of indicated transcripts from CLIP-UPF1 overexpression RNA-seq experiments. Relative fold changes are in reference to the GFP expressing control line. Significance of CLIP-UPF1SL or CLIP-UPF1LL overexpression was compared to the GFP expressing control line. Asterisk (*) indicates P < 0.05, as determined by two-way ANOVA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. For protected mRNAs, the motif density of PTBP1/hnRNP L within the 3'UTR is indicated. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons (Lareau et al., 2007; Ni et al., 2007). See also Dataset EV3 for P values associated with each statistical comparison.
An alternative UPF1 isoform drives conditional remodeling of nonsense-mediated mRNA decay (2022)
Figure 3

Figure 3. Splicing regulator SRSF1 is required for UPF1LL expression.

A Semiquantitative RT-PCR of UPF1SL or UPF1LL transcript levels following transfection of HEK-293 cells with the indicated siRNAs.

B RT-qPCR analysis of indicated transcripts following transfection of a NT siRNA or SRSF1-specific siRNA under conditions of CLIP-UPF1SL or CLIP-UPF1LL overexpression. Relative fold changes are in reference to the GFP expressing control line treated with a NT siRNA. Asterisk (*) indicates P < 0.05, as determined by unpaired Student's t-test. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). See also Dataset EV3 for P values associated with each statistical comparison.

C Density plot of changes in relative mRNA abundance as determined by RNA-seq following SRSF1 overexpression (Data ref: Caputi et al, 2019). Genes were categorized as up-regulated by siUPF1total only, siUPF1LL only, or both siUPF1total and siUPF1LL. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons.

D Density plot as in (C), with genes binned according to enrichment in the CLIP-UPF1LL or CLIP-UPF1SL affinity purifications.
An alternative UPF1 isoform drives conditional remodeling of nonsense-mediated mRNA decay (2022)
Figure 4

Figure 4. UPF1LL overcomes translocation inhibition by PTBP1.

A Scheme of the fluorescence-based unwinding assay to monitor UPF1 translocation in real time (Fritz et al., 2020). An RNA substrate harboring a high-affinity PTBP1 binding site is incubated with highly purified UPF1ΔCH in the absence and presence of equal molar amounts of highly purified PTBP1. Upon the addition of ATP, UPF1 translocation results in a decrease in fluorescence due to displacement of a labeled, duplexed oligonucleotide and subsequent quenching by a trap strand.

B UPF1SLΔCH translocation along an RNA substrate harboring a high-affinity PTBP1 binding site in the absence and presence of PTBP1. Time to 50% unwound and relative total % unwound at end-of-assay (1200 sec) are indicated. Results of four technical replicates are shown for each dataset and represent at least three independent experiments. Shaded region indicates SD.

C Results as in (B) but with UPF1LLΔCH.
An alternative UPF1 isoform drives conditional remodeling of nonsense-mediated mRNA decay (2022)
Figure 7

Figure 7. Translational repression promotes the decay of mRNAs by UPF1LL in the NMD pathway and down-regulates normally protected transcripts.

A mRNA decay measurement using Roadblock-qPCR (Watson et al., 2020). RNA was isolated from HEK-293 cells at indicated timepoints following transfection with indicated siRNAs and treatment with 400 µM 4sU and 50 µg/mL puromycin. mRNA half-lives were estimated by fitting the data to a single-phase exponential decay model. Puromycin treatment was compared to the vehicle control and siUPF1LL was compared to the siNT in the absence and presence of puromycin treatment using the extra-sum-of-squares F test. Error bars indicate mean±SD (n = 4 biological replicates). See also Dataset EV3 for P values associated with each statistical comparison.

B RT-qPCR analysis of indicated transcripts following transfection of HEK-293 cells with indicated siRNAs and treatment with 50 µg/mL puromycin for 4hr. Relative fold changes are in reference to vehicle-treated, NT siRNA. Black dots represent individual data points and error bars indicate mean±SD (n = 3 biological replicates). Dashed lines indicate log2(fold change) of ±0.5. PTC+ indicates the use of primers specific to transcript isoforms with validated poison exons (Lareau et al., 2007; Ni et al., 2007). See also Dataset EV3 for P values associated with each statistical comparison.

C Density plot of changes in relative mRNA abundance as determined from RNA-seq following treatment of HEK-293 cells with 50 µg/mL of puromycin for 4hr. mRNAs were subdivided by PTBP1 and/or hnRNP L motif density within the first 400 nt of 3'UTR. Statistical significance was determined by K-W test, with Dunn's correction for multiple comparisons.

D Density plot as in (C), following UPF1LL-specific knockdown.
Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNAREs (2019)
Figure 1

Figure 1. Many SNAREs bind mammalian Atg8 proteins

Peptide array dot blot analysis to identify LIR motifs in the indicated SNARE proteins. Amino acids for the identified LIRs from positive signals are marked on each SNARE. Amino acids are denoted as amino acid single letter codes in the blot.

Schematics of the functional domains of Stx16/Vti1a/Stx6 cognate SNAREs with the positions of LIR motifs. The LIR motif marked in green in syntaxin 16 indicates established LIR based on following analyses.

Co-immunoprecipitation (Co-IP) analysis of interactions between FLAG-tagged Stx16/Vti1a/Stx6 and EGFP-tagged LC3B or GABARAP overexpressed in HEK293T cells. * indicates mouse IgG heavy chain of precipitated mouse-anti-FLAG antibody.

GST pull-down analysis of interactions between radiolabeled Myc-Stx16 and GST-tagged mAtg8 proteins.

GST pull-down analysis of interactions between wild type (WT) or LIR-mutant (L219A/V222A) Stx16 and GST-tagged LC3C or GABARAP. Lower panel shows percentages of WT or LIR-mutant Stx16 bound to GST-LC3C or GST-GABARAP. Data shown as means ± SEM of precipitated Stx16, n = 3.

GST pull-down analysis of interactions between WT or different types of LIR-mutant Stx16 and GST-tagged LC3C or GABARAP. Lower panel shows percentages of WT or LIR-mutant Stx16 bound to GST-LC3C or GST-GABARAP.

GST pull-down analysis of interactions between radiolabeled Myc-Stx16 and WT or LDS-mutant GABARAP (GABARAP-Y49A and -Y49A/F104A). Lower panel shows percentages of Stx16 bound to WT or LDS-mutant GABARAP. Data shown as means ± SEM of precipitated Stx16, n = 3.
Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNAREs (2019)
Figure 2

Figure 2. The mAtg8-interacting SNAREs Stx16 and Stx17 are required for efficient bulk autophagic flux

WT or STX17-knockout (STX17KO) HeLa cells were starved with or without the presence of bafilomycin A1 (Baf A1, 100 nM) for 2 h, and cell lysates were subjected to Western blot analysis of LC3B and p62.

Quantifications of LC3B-II levels normalized to β-actin from cells treated as in (A); Data shown as means ± SEM of LC3B-II and β-actin ratios, n = 3; *, p < 0.05 (one-way ANOVA).

WT, STX16KO, STX17KO or STX16/STX17 double KO (STX16/STX17DKO) HeLa cells were starved with or without the presence of Baf A1 (100 nM) for 2 h, and cell lysates were subjected to Western blot analysis of LC3B.

Quantifications of LC3B-II levels normalized to β-actin from (C); Data shown as means ± SEM of LC3B-II and β-actin ratios, n = 3; †, not significant; **, p < 0.01 (one-way ANOVA).

WT or STX16/STX17DKO HeLa cells were starved in EBSS for 2 h, and subjected to ultrastructural analysis of the autophagic vesicles (AV) with electron microscopy. AVi: initial autophagic vacuoles; AVd: degradative autophagic vacuoles; G: Golgi apparatus. Image acquisition and counting as in Methods. Scale bars, 1 μm and 0.5 µm (top sections).

Quantifications of autophagic vesicles in WT and STX16/STX17DKO HeLa cells treated as (E); Data shown as means ± SEM of AV profiles relative to cytoplasmic area; †, not significant; *, p < 0.05 (two-way ANOVA). AV profiles from 47 images of each samples were counted.
Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNAREs (2019)
Figure 3

Figure 3. Stx16 and Stx17 are required for selective autophagy of mitochondria, peroxisomes, and M. tuberculosis

WT or STX16/STX17DKO HeLa-YFP-Parkin cells were treated with CCCP (10 µM) or oligomycin A (5 µM) and antimycin A (10 µM) (OA) for 16 hours, and subjected to high-content microscopy (HCM) analysis of mitochondria clearance. Masks: blue, nuclei; red, mitochondria stained with mitochondrial DNA (mtDNA) antibody. Scale bar: 20 µm.

Quantifications of mtDNA by object count or total area per cell in WT or STX16/STX17DKO HeLa-YFP-Parkin cells treated as in (A); Data shown as means ± SEM of mtDNA object count (upper panel) or object total area (lower panel) per cell; minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).

WT or STX16/STX17DKO Huh7 cells were treated with H2O2 (0.4 mM) for indicated time points, and autophagic clearance of peroxisomes was measured by the protein levels of PMP70, PEX14 and p62.

Quantifications of peroxisomal proteins PMP70 and PEX14 for WT or STX16/STX17DKO Huh7 cells treated as in (C); Data shown as means ± SEM of PEX14 or PMP70 and β-actin ratios, n = 3; †, not significant; *, p < 0.05; **, p < 0.01 (two-way ANOVA).

WT, STX16KO, STX17KO or STX16/STX17DKO THP-1 cells were infected with Mycobacterium tuberculosis (Mtb), followed by starvation to induce autophagy. Autophagic clearance of infected Mtb was measured by Mtb colonies grown on Middlebrook 7H11 agar plates; Data shown as means ± SEM of Mtb colonies, n = 4; *, p < 0.05; **, p < 0.01 (two-way ANOVA).

Schematics for the requirement of Stx16 and Stx17 in autophagic clearance of damaged mitochondria, surplus peroxisomes and invaded bacterial pathogens.
Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNAREs (2019)
Figure 4

Figure 4. Stx16 and Stx17 cooperate in ribophagy

Schematic of ribophagy detection with Keima-based reporter system; FP: fluorescent protein.

Validation of STX16/STX17 DKO by Western blot analysis in HEK293 cells stably expressing RPS3-Keima (left panel) and HCT116 cells stably expressing RPL28-Keima (right panel).

WT or STX16/STX17DKO HEK293 RPS3-Keima cells were cultured in full medium or starved for 8 hours, and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm. Masks: white, cells identified based on nuclei; red, Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm, respectively; lower panel shows Keima puncta without masks. Scale bar: 20 µm.

Quantifications of autolysosomal Keima puncta in WT or STX16/STX17DKO HEK293 RPS3-Keima cells treated as in (C). Data shown as means ± SEM of Keima puncta per cell, minimum 1000 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).

WT or STX16/STX17DKO HCT116 RPL28-Keima cells were cultured in full medium or starved for 8 hours, and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm. Masks: white, cells identified based on nuclei; red, Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm, respectively; lower panel shows Keima puncta without masks. Scale bar: 20 µm.

Quantifications of autolysosomal Keima puncta in WT or STX16/STX17DKO HCT116 RPL28-Keima cells treated as in (E). Data shown as means ± SEM of Keima puncta per cell, minimum 1000 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant; **, p < 0.01 (two-way ANOVA).
Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNAREs (2019)
Figure 5

Figure 5. Stx16 is required for the maintenance of lysosomal homeostasis and mTOR activity

(A) WT or STX16KO HeLa cells were starved in EBSS for 2 h and subjected to HCM analysis of LAMP2 and LC3 puncta. Masks: white, cells identified based on nuclei; red, LC3 puncta; green, LAMP2 puncta; yellow, overlap of LC3 and LAMP2. Scale bar: 20 µm.

(B-D) Quantifications of LC3 puncta per cell (B), LC3 and LAMP2 overlap area per cell (C), and LAMP2 puncta per cell (D) in WT or STX16KO cells treated as in (A). Data shown as means ± SEM of puncta or overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (two-way ANOVA).

(E) Western blot analysis of LAMP1 and LAMP2 protein levels in WT and separate clones of STX16KO HeLa, Huh7, and U2OS cells.

(F) Western blot analysis of LAMP2 protein levels in WT and separate clones of STX6KO or VTI1AKO HeLa cells.

(G) Time course of mTOR inactivation during EBSS starvation in WT vs. STX16KO HeLa cells analyzed by Western blotting for mTOR substrates ULK1 and 4E-BP1.

(H) WT or STX16KO HeLa cells were starved in EBSS for 1 h and subjected to HCM analysis of mTOR puncta. Masks: white, cells identified based on nuclei; red, mTOR puncta. Scale bar: 20 µm.

(I) Quantifications of mTOR puncta in WT or STX16KO cells treated as in (H). Data shown as means ± SEM of mTOR puncta per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).
Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNAREs (2019)
Figure 6

Figure 6. Stx16 regulates proper distribution of acidified compartments in the cell

WT or STX16KO HeLa cells were starved in EBSS for 1 h, followed by starvation with the presence of LysoTracker Red DND-99 (LTR) (100 nM) for additional 30 min, and subjected to HCM analysis of LTR. Scale bar: 20 µm.

Quantifications of LTR count per cell or total area per cell in WT and STX16KO HeLa cells treated as in (A). Data shown as means ± SEM of LTR puncta per cell or object total area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant (two-way ANOVA).

WT or STX16KO HeLa cells were starved in EBSS for 1 h, followed by starvation with the presence of LTR (100 nM) for additional 30 min, and subjected to HCM analysis of the colocalization between LTR and TGN46. Masks: white, cells identified based on nuclei; red, LTR puncta; green, TGN46 puncta; yellow, overlap between LTR and TGN46. Scale bar: 20 µm.

Quantifications of overlaps between LysoTracker Red and TGN46 in WT and STX16KO cells treated as in (C). Data shown as means ± SEM of LTR and TGN46 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).

WT or STX16/STX17DKO HeLa cells were starved in EBSS for 2 h, and subjected to ultrastructural analysis of the Golgi apparatus with electron microscopy. VF of Golgi: volume fraction of Golgi relative to total cytoplasmic area. Scale bar, 2 μm. Red arrows indicate Golgi apparatus.
Mammalian Atg8 proteins regulate lysosome and autolysosome biogenesis through SNAREs (2019)
Figure 7

Figure 7. Mammalian Atg8s regulate Stx16 localization and acidification of endolysosomal organelles

WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 90 min and subjected to HCM analysis of overlaps between LAMP2 and Stx16. Masks: white, cells identified based on nuclei; green, LAMP2 puncta; red, Stx16 puncta; yellow, overlap of LAMP2 and Stx16. Scale bar: 20 µm.

Quantifications of overlaps between LAMP2 and Stx16 in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells treated as in (C). Data shown as means ± SEM of LAMP2 and Stx16 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (one-way ANOVA).

Quantifications of overlaps between LBPA and Stx16 in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells grown in full medium or treated as in (A). Data shown as means ± SEM of LBPA and Stx16 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (one-way ANOVA). Representative images are shown in Appendix Fig. S5A.

WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 1 hour, followed by starvation with the presence of LTR (100 nM) for additional 30 min, and subjected to HCM analysis of LTR. Scale bar: 20 µm.

Quantifications of LTR puncta in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells treated as in (D). Data shown as means ± SEM of LTR puncta per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant; *, p < 0.05; **, p < 0.01 (one-way ANOVA).

Schematic model of this study. Mammalian Atg8 proteins directly bind Stx16 through its LIR motif, which controls the proper localization of the Stx16 SNARE complex at the endolysosomal compartments. The Stx16 SNARE complex functions in the maintenance of lysosomal homeostasis upon autophagy induction, thus controlling mTOR activity.
Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and -independent mechanisms (2021)
Figure 1

Figure 1: dTor and dLap1 differentially affect the fat body lipidome

A) GL and GPL and enzymes. Full abbreviations in Appendix Fig. S1A.

B) dTor and dLap1 topology.

C - E) Subcellular localization of dLap1mGFP and dTormGFP-E177Q in the wild-type

(w-) or dLap1+/∆11 fat body of 3rd instar fly larvae, when expressed as UAS transgenes.

F - H) Control, dTorKO and dLap1-/- larvae with pseudo-colored red fat body.

I) The relative abundance of individual GL and GPL classes in the wild-type fat body, as detected by mass spectrometry (MS). n=10 individual analyses.

J) TAG content of 3DO larvae measured by enzymatic assay. Bars show mean ± SEM measured in three sets of thirty larvae. One-Way ANOVA with Tukey's multiple comparison test. **** p < 0.0001.

K) The abundance of individual lipid classes (mean ± SEM) within the set of six membrane GL and GPL extracted from 4DO fat body samples. w- n = 7, dTorKO n = 7, dLap1-/- n = 4. One-Way ANOVA with Bonferroni's post hoc test. * p < 0.05 and ** p < 0.01.

L - O) Neutral lipid staining of the 5DO larval fat body. White arrows (O) indicate nuclear-localized neutral lipid staining, blue signal shows DAPI.

P) % of adult females that hatch after crossing female dTor+/- ;; dLap1-/TM6C with male y/FM7i ;; dLap1-/TM6C flies. Two-tailed T-Test. **** p < 0.0001, compared with expected Mendelian ratio (n≥3).
Torsin and NEP1R1-CTDNEP1 phosphatase affect interphase nuclear pore complex insertion by lipid-dependent and -independent mechanisms (2021)
Figure 2

Figure 2: The dTor membrane domain and homo-oligomerization motif are important for dTor function in the fat body

A) dTor mutants and chimeras. Sig Seq, ER targeting signal sequence.

B) Wild-type and mutant dTormGFP proteins expressed in dTorKO fat body cells.

C, D) Size of individual fat body cells (C) and LD density of individual fat body cells (D). Bars show the group mean, light grey highlights 2x SD of the control mean (animals expressing the Sec61b control ER protein), and dotted red lines show dTorKO mean. These are results of n≥3 larvae (of 3 independent crosses). One-way ANOVA, with Bonferroni's post hoc test. ** p < 0.01, *** p < 0.001 and **** p <0.0001.

E - M) GFP, Phalloidin and LipidTOX detect the expression of dTormGFP chimeras (green), cell boundaries (red), and LD (red), respectively, in dTorKO fat body cells.