EMBO/soda-nel huggingface dataset:
DDX60 selectively reduces translation off viral type II internal ribosome entry sites (2022)
Figure 6

Figure 6. Polysome profile of wild type (type I IRES) poliovirus versus chimeric (type II IRES) poliovirus in the presence of DDX60.

(A) Schematic of polysome profiling strategy. HeLa cells stably expressing wild type DDX60 were infected with parental poliovirus (type I IRES) or chimeric poliovirus (type II IRES). 6 hpi cells were treated with 100 μg/mL of cycloheximide for 15 minutes to arrest polysomes and subjected to polysome profiling by ultracentrifugation through 15%-50% sucrose gradients. Amount of poliovirus VP4 mRNA from polysome fractions was determined by RT-qPCR.

(B) Cells depicted in (A) were subjected to polysome profiling by lysing in the presence of cycloheximide and protease and phosphatase inhibitors, applying cell lysates to 15%-50% sucrose gradients, and subjecting to ultracentrifugation. Centrifuged gradients were run through a fractionator and total RNA in each fraction was measured by UV absorbance (254 nm).

(C) Representative results of RNA electrophoresis analysis of individual fractions. Shown are results from one replicate of DDX60-transfected, EMCV-IRES-PV-infected cells. RNA was analyzed by Bioanalyzer to visualize 28S, and 18S ribosome subunit distribution. RIN, RNA integrity number.

(D) Effect of DDX60 expression on parental poliovirus (type I IRES, purple circles) versus chimeric poliovirus (type II IRES, red squares) VP4 mRNA polysomes (left) and cellular GAPDH mRNA polysomes in the same samples.

Data information: Mean percent ± SEM of poliovirus VP4 or GAPDH mRNA in each fraction relative to VP4 or GAPDH mRNA in all fractions from n=3 replicate plates per condition.

Expanded Figure Legends
Widespread use of unconventional targeting signals in mitochondrial ribosome proteins (2021)
Figure 2

Fig. 2. The non-canonical targeting and translocation signal of Mrp17 is located between amino acids 30 and 60.

(A) In vivo characterization of mitochondrial targeting capacity of different Mrp17 truncations fused to GFP visualized by fluorescent microscopy with MitoTracker Orange straining. Scale bar for all micrographs is 10 µm.

(B) Characterization of Mrp17 translocation signal using an in vitro import assay: shown are autoradiographs of full-length Mrp17 or its truncations fused to DHFRmut, translated in vitro with radiolabeled amino-acids, incubated with isolated yeast mitochondria for 2, 5, or 10 min, treated with proteinase K (PK) to remove nonimported proteins and visualized by 16% SDS-PAGE/autoradiography. As a negative control, mitochondria were treated with valinomycin, antimycin and oligomycin (VAO) that eliminate membrane potential. For comparison, 20% of the protein used per import reaction was loaded on the first lane.
Widespread use of unconventional targeting signals in mitochondrial ribosome proteins (2021)
Figure 3

Fig. 3. Positive charge and Tom20-binding motifs are important features of the Mrp17 targeting signal.

(A) Primary sequence of Mrp17 highlighting charged amino-acids, Tom20-binding motifs (TBM1, TBM2) and secondary structure (from PDB:5MRC);

(B) In vitro mitochondria translocation capacity of WT Mrp17, Mrp17K-R with all lysines (K) substituted with arginines (R), and Mrp17K-A with all lysines (K) substituted with alanines (A) fused to DHFRmut. Import was performed as described in Fig. 2 legend.

(C) In vivo mitochondrial targeting capability of WT Mrp17, Mrp17K-R, and Mrp17K-A fused to GFP. Scale bar is 10 µm.

(D) Substitution of lysines with alanines in the regions 1-60 or 30-60 of Mrp17 abolishes mitochondrial import capacity: (top) schematics of substitution positions, TBMs are in green, targeting signal outlined in grey, substitutions are denoted by arrows; (bottom) in vitro translocation of the mutants.

(E) Mrp17 variants rescue ∆mrp17 strain growth defect on respiratory media: the indicated variants or empty vector (EV) were introduced in WT yeast and then the genomic MRP17 was disrupted by knock out, the resulting mutants were serially diluted 10× and spotted on media containing glucose or glycerol as a sole carbon source.
Widespread use of unconventional targeting signals in mitochondrial ribosome proteins (2021)
Figure 4

Fig. 4. Mrp17 uses a similar translocation route into mitochondria as MTS containing proteins.

(A) The in vitro import of Mrp17-DHFRmut is sensitive to the elimination of outer mitochondrial membrane proteins by trypsinization (left), more so than the model import substrate Atp1 (right). Mitochondria were incubated with the indicated concentrations of trypsin and the import assay was performed as described in the legend for Fig. 2. Proteinase K (PK) was not added to the sample without mitochondria.

(B) In vitro translocation of Mrp17-DHFRmut into mitochondria isolated from WT and ∆tom20 yeast showing reduced translocation in ∆tom20 background.

(C) Quantification of the experiment in panel (B) repeated 3 times, whiskers correspond to minimum and maximum values.

(D) Expression of Mrp17-GFP in ∆tom20 yeast shows mislocalization to the nucleus compared to WT cells (Full micrographs and WT control shown in Appendix Figure S6A). Scale bar is 10 µm.

(E) Import of Mrp17-DHFRmut into purified ∆tom22 mitochondria, control import into WT mitochondria is shown on Appendix Figure S6B

(F) Substitution of both Tom20-binding motifs (TBM) with alanines (TBM1,2mut) abolishes Mrp17 mitochondrial import capacity while substituting of a single motif (TBM1mut, TBM2mut) reduces import.

(G),(H) Import of Mrp17-DHFRmut and control proteins Atp1 and Aac1 into WT, tim17-ts (G) and tim22-ts (H) mitochondria. Import was performed at indicated temperatures, proteinase K was added to all samples except the loading control in the first lane of each autoradiograph (20% of protein amount used for each import reaction). The full autoradiograph for Aac1 with molecular weight markers is shown in Appendix Figure S6D.
Reversing neural circuit and behavior deficit in mice exposed to maternal inflammation by Zika virus (2021)
Figure 1

Figure 1. ZIKV infection during pregnancy induces MIA and inflammatory responses in fetal brains.

A ELISA measurement of serum concentrations of maternal IL-17a at E14.5. Mock n=8; ZIKV n=6 mice.

B RT-PCR analysis of IL-17Ra mRNA levels in the embryonic brains at E14.5. Mock n=7; ZIKV n=8 embryos.

C Schematic of the E14.5 brain. LV ChP, lateral ventricular choroid plexus.

D Schematic of morphological states of quiescent and activated Iba-1+ macrophages.

E The representative images of IF-IHC staining of E14.5 embryonic brains with antibodies against Iba-1 (green) and DAPI (blue). Scale bar, 200 μm.

F Quantification of numbers of Iba-1+ cells on the meningeal regions. Mock n=5; ZIKV n=7 embryos.

G The representative images of IF-IHC staining of choroid plexus of E14.5 embryonic brains with antibodies against Iba-1 (green) and DAPI (blue). Scale bar, 50 μm.

H Quantification of total number of Iba-1+ cells per ChP area.

I Percentage of Iba-1+ cells on the surface of ChP.

J Percentage of round-shaped Iba-1+ cells.

K The representative images of IF-IHC staining of choroid plexus of E14.5 embryonic brains with antibodies against Iba-1 (green), CD68 (red) and DAPI (blue). Scale bar, 50 μm.

L Percentage of CD68+; Iba-1+ cells out of total Iba-1+ cells.

Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM. Mock n=5; ZIKV n=6 biological replicates in (H-J).
Reversing neural circuit and behavior deficit in mice exposed to maternal inflammation by Zika virus (2021)
Figure 4

Figure 4. Dysregulation of inhibitory neurons and synapses in ZIKV-affected offspring mice.

A Representative images of PV+, SST+ and CAL+ cells in the prelimbic (PL, upper panel) and infralimbic (IL, lower panel) cortex. Scale bars: 200 μm.

B Quantification of PV+ cells in the PL and IL cortex.

C PV+ interneuron numbers plotted against social preference.

D Quantification of SST+ cells in the PL and IL cortex.

E SST+ interneuron numbers plotted against social preference.

F Quantification of CAL+ cells in the PL and IL cortex. Mock, n=4 mice; ZIKV, n=4 mice in B, D, and F. P = 0.0490 (PL in B), 0.0253 (IL in B), 0.0113 (PL in F).

G CAL+ interneuron numbers plotted against social preference. Black solid lines represent regression line; grey lines indicate 95% confidence intervals in C, E, and G. Mock (black circles), n=4 mice; ZIKV (red circles), n=4 mice. P = 0.0054 (PL in C), 0.0226 (IL in C), 0.1765 (PL in E), 0.5840 (IL in E), 0.2983 (PL in G), 0.2109 (IL in G); linear regression.

H IHC staining of mPFC stained with antibodies against vGLUT1. Scale bars, 5 μm.

I Quantification of vGLUT1+ puncta numbers out of 100 μm2 region. Mock, n=4 mice; ZIKV, n=4 mice.

J IHC staining of mPFC stained with antibodies against vGAT. Scale bars, 5 μm.

K Quantification of vGAT+ puncta numbers out of 100 μm2 region. Mock, n=4 mice; ZIKV, n=4 mice.

Data information: All data are presented as mean ± SEM. *P<0.05, **P<0.01. Two-tailed unpaired t-test unless described otherwise.
Reversing neural circuit and behavior deficit in mice exposed to maternal inflammation by Zika virus (2021)
Figure 5

Figure 5. Hyperconnectivity of vHIP-mPFC circuit in mice exposed to maternal ZIKV infection.

A Schematic of mPFC-projecting ventral hippocampus (vHIP) neurons. AAVretro-Cre represents a retrograde-transporting AAV virus expressing Cre; DIO-mCherry is a Cre-dependent mCherry reporter.

B Representative confocal images of the vHIP stained with mCherry (red) and c-Fos (green).

C Quantification of mCherry and c-Fos double positive cells out of total mCherry-positive cells in the vHIP CA1 regions. Scale bar, 50 μm. Mock (n= 5 mice) vs. ZIKV (n= 5 mice), p=0.0013.

D Slice recording paradigm. AAV-CaMKII-hChR2-mCherry was injected into vHIP CA1 region and LED (365 nm) was applied to the mPFC.

E Top, mCherry expression at the injection site. Bottom, mCherry-labeled axons in the mPFC. Scale bars, 200 μm and 10 μm in the zoomed areas.

F Representative EPSC traces of the pyramidal neurons in the mPFC.

G Quantification of EPSC amplitude of the pyramidal neurons in the mPFC (PL and IL). Mock (n=17 neurons from 5 mice) vs. ZIKV (n=19 neurons from 5 mice), p=0.0122.

Data information: Data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.
Reversing neural circuit and behavior deficit in mice exposed to maternal inflammation by Zika virus (2021)
Figure 6

Figure 6. Synaptic connection and pattern of vHIP innervation of mPFC neurons.

A Schematic tracing of presynaptic boutons from vHIP neurons using AAV-DIO-GFP virus.

B Representative examples of GFP-labeled presynaptic boutons. Scale bars: 2.5 μm.

C Quantification of presynaptic bouton density and weighted size in the mPFC (PL and IL). Mock: n= 57 axons from 6 mice; ZIKV: n= 63 axons from 7 mice.

D Quantification of presynaptic bouton weighted size in the mPFC (PL and IL). Mock: n= 2974 boutons from 6 mice; ZIKV: n= 3486 boutons from 7 mice.

E Trans-synaptic tracing of vHIP-derived neurons in the mPFC. AAV1-hSyn-Cre injection in the ventral hippocampus (vHIP) of Ai14 mouse followed by examination of neuronal types in the mPFC.

F Representative confocal images of co-labeled tdTomato and neuronal markers including CaMKII, PV, CAL, and SST in the mPFC. Scale bar, 50 μm.

G Quantification of the percentages of mPFC neuron subtypes innervated by vHIP afferent axons.

H Comparisons of percentages of vHIP-innervated mPFC (PL and IL) neuron subtypes. Mock, n=4 mice; ZIKV, n=7 mice. P= 0.5343 in CaMKIIa, 0.0117 in PV, 0.7491 in CAL, 0.4484 in SST, 0.1519 in other uncharacterized neuron subtypes.

I Schematic tracing of a retrograde dye CTB488-labeled vHIP neurons projecting towards mPFC. CTB488 was injected into the mPFC of vGLUT1-Cre::Ai14 mice followed by IHC staining in vHIP.

J Representative confocal images of CTB488-labeled tdTomato-positive neurons in the vHIP. White arrowheads indicate CTB488 single positive neurons. Scale bars: 200 μm.

K Quantification of the percentage of tdTomato;CTB488 double-positive cells out of total CTB488-positive cells. Mock, n= 7 mice; ZIKV, n=7 mice.

Data information: All data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.
Reversing neural circuit and behavior deficit in mice exposed to maternal inflammation by Zika virus (2021)
Figure 7

Figure 7. Reducing vHIP-mPFC circuit activity rescued social memory deficit in ZIKV-affected offspring mice.

A Schematic of AAVretro-Cre and AAV-DIO-hM4Di-mCherry or AAV-DIO-mCherry injections.

B mCherry-labeled axons in the vHIP. Scale bars, 200 μm (left panel), and 50 μm (right panel).

C Representative animal tracks in three-chamber test.

D Discrimination indices of the social memory session of three-chamber test. mCherry mock, n=8 mice; mCherry ZIKV, n=10 mice; hM4Di ZIKV n=10 mice, mCherry mock vs. mCherry ZIKV p=0.0041; mCherry ZIKV vs. hM4Di ZIKV p=0.0115; and mCherry mock vs. hM4Di ZIKV p=0.7076.

E Representative confocal images of mCherry (red) and c-Fos (green) double-labeled cells in the vHIP CA1 regions. Scale bars, 50 μm.

F. Quantification of the percentage of c-fos;mCherry double positive cells out of total mCherry cells. mCherry mock, n=6 mice; mCherry ZIKV, n=4 mice; hM4Di ZIKV n=6 mice.

Data information: All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey post hoc test.

EXANDED VIEW FIGURE LEGENDS
TRAF6 directs FOXP3 localization and facilitates regulatory T cell function through K63-linked ubiquitination (2019)
Figure 1

Figure 1. TRAF6 is highly expressed by Treg subsets and plays an important role in maintaining immune homeostasis.

A TRAF6 expression in differentiating CD4+ T cells. Naïve CD4+ T cells were obtained from wild type C57BL/6 mice by FACS and activated with anti-CD3/CD28 (1µg, and 2µg/ml) for the indicated times in the presence of distinct Thelper lineage-directing cytokines or under neutral activation conditions (Th0). After total RNA extraction and cDNA conversion, RT-PCR determined of Traf6 mRNA in differentiating Th0, Th1, Th17 and iTregs was determined.

B TRAF6 mRNA expression by human Tregs and non-Treg CD4+ T cell. Human Tregs (CD3+/CD4+/CD8-/CD25HIGH/CD127low/CD39+) and non-Treg CD4+ T cells (CD3+/CD4+/CD8-/CD25-) were obtained from the peripheral blood of healthy donors by FACS after Ficoll-Paque PLUS gradient centrifugation and magnetic bead enrichment of CD4+ T cells. TRAF6 mRNA was measured by qRT-PCR.

C, D Evidence of lymphoproliferative disease in Traf6fl/flFoxp3Cre+ mice. (C) Spleens and lymph nodes were recovered from Traf6fl/flFoxp3Cre+ mice and Traf6fl/fl littermates at 8 weeks of age (Scale bars: 5 mm). (D) The cellularity of the lymphoid tissues of Traf6fl/flFoxp3Cre+ mice and their Traf6fl/fl Foxp3Cre- littermates was determined (8 mice/group).

E, H Effect of Treg-specific TRAF6-deficiency on baseline T cell activation. The frequencies of effector cells (CD44high/CD62Llow), memory cells (CD44high/CD62Lhigh) and naïve cells (CD44low/CD62Lhigh) in the CD4+ T cell compartments of Traf6fl/fl and Traf6fl/flFoxp3Cre+ mice were determined by flow cytometry (5 mice/group).

F, I Effect of Treg-specific TRAF6-deficiency on baseline T cell activation. The frequencies of effector cells (CD44high/CD62Llow), memory cells (CD44high/CD62Lhigh) and naïve cells (CD44low/CD62Lhigh) in the CD8+ T cell compartments of Traf6fl/fl Foxp3Cre- (wild type) and Traf6fl/flFoxp3Cre+ mice were determined by flow cytometry (5 mice/group).

G, J Impact of TRAF6-expression on in vitro Treg differentiation. As in A, naïve CD4+ T cells were isolated from Traf6fl/flFoxp3Cre+ and Traf6fl/fl mice and differentiated into iTregs. Conditions of sub-optimal TGFβ concentrations (0.5ng/ml, 0.05ng/ml) were tested as well and intracellular FOXP3 was measured after 4 days.

Data information: Panels (A, B, D, H, I and J) represent mean results +/-SEM. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, no significance, by an unpaired t test. A, C, E-G are representative findings from at least 3 experiments. Panels A and J represent the means of 3 replicates.
TRAF6 directs FOXP3 localization and facilitates regulatory T cell function through K63-linked ubiquitination (2019)
Figure 2

Figure 2. Expression of TRAF6 by Tregs is required for in vivo function and suppression of anti-tumor immunity.

A In vivo suppressive function of WT and Traf6fl/flFoxp3Cre+ Tregs. Tregs from the indicated mice (CD45.2+) were isolated by FACS, as were naïve (CD62Lhigh/CD25-) CD4+ responder T cells from congenically distinct (CD45.1+) donor mice. Tregs and Tresponders were mixed at a 1:5 (2x105:10x105) ratio before injection into Rag2-/- mice. 7 days later, spleens were harvested, and the relative frequencies and absolute numbers of each transferred cell populations were determined by flow cytometry.

B Implanted B16 melanoma growth in Traf6fl/flFoxp3Cre+ and wild type (Traf6fl/flFoxp3Cre-) mice. 1x105 B16F10 cells were injected subcutaneously (s.c.) into the shaved flanks of the indicated mice (n= 5/group). Tumor volumes were monitored every 3 days.

C, D Proinflammatory cytokine production in tumor-bearing mice with and without Treg-specific TRAF6 expression. Cell suspensions of the tumor-draining lymph node and tumor-infiltrating leukocytes (TILs) were recovered after 21 days of tumor growth. Ex vivo stimulation with PMA and ionomycin in the presence of Golgistop for 5 hour preceded intracellular staining for IFNγ and IL-17 and flow cytometry analysis.

E, F FOXP3 expression by CD4+ T cells in tumor bearing Traf6fl/flFoxp3Cre+ and wild type (Traf6fl/flFoxp3Cre-) mice. The frequency of FOXP3+ cells in the CD4+ cells of tumor-draining lymph nodes (dLN), peripheral, non-tumor draining lymph nodes (pLN), spleens, and TILs were determined by flow cytometry.

Data information: A (right panels), B, D, E and F denote the average results from at least 3 independent experiments +/- SEM (3-5 replicates).*P<0.05; **P<0.01; ***P<0.001 by an unpaired t test. Panels A, left; C; and E show representative findings from these experiments.
TRAF6 directs FOXP3 localization and facilitates regulatory T cell function through K63-linked ubiquitination (2019)
Figure 3

Figure 3. TRAF6 physically interacts with FOXP3 and induces K63-linked polyubiquitination of the transcription factor.

A Assessment of K63-type ubiquitination of FOXP3 upon expression of wild type and catalytically deficient TRAF6 mutants. 293T cells were transfected with the given combinations of vectors encoding wild type TRAF6, the enzymatically deficient L74H or C70A TRAF6 mutants, HA-FOXP3, and FLAG-labeled ubiquitin molecules possessing a single lysine residue (K63) that restrain the possible ubiquitin monomer linkages on polyubiquitinated proteins to the K63-type only. Cells were lysed and proteins modified by K63-ubiquitin chains were recovered from lysates by pulling down using a bead-immobilized K63-specific TUBE reagent. The presence of FOXP3 among these modified proteins was determined by immunoblotting as were levels of FOXP3 and TRAF6 in the pre-IP whole cell lysate (WCL).

B Co-immunoprecipitation (co-IP) of FOXP3 with TRAF6 and its facilitation of K63-linked ubiquitination. 293T cells expressing the indicated constructs encoding HA-FOXP3 and FLAG-TRAF6 were lysed and incubated with bead immobilized anti-FLAG antibodies. FOXP3 molecules co-IPed in this manner were resolved by SDS PAGE and detected by immunoblotting with anti-HA antibodies.

C　293T cells were transfected with the indicated combinations of expression constructs encoding HA-FOXP3, Myc-TRAF6, and FLAG-tagged ubiquitin molecules - either wild type (FLAG-Ub), or a variant with a K-to-R mutation at either lysine residue 48 (K48R) or 63 (K63R) responsible for preventing K48- and K63-type polyubiquitination, respectively. .

D Endogenous, reciprocal co-IP of FOXP3 and TRAF6 in murine iTreg lysate. Naïve CD62Lhigh/CD25-/CD4+ T cells were obtained by FACS and activated with anti-CD3/CD28 antibodies (1ug and 2ug/ml, respectively) in the presence of IL-2 and TGFβ (100U/ml and 5ng/ml, respectively) for four days before lysis. Antibodies against TRAF6 (left) or FOXP3 (right) were used to pull down target proteins and their interaction partners, which were resolved under denaturing conditions and visualized by immunoblotting.

E Degree of K63 ubiquitination in the cellular FOXP3 pools of Traf6fl/flFoxp3Cre+ and Traf6wt/wtFoxp3Cre+mice. CD4+/YFP+ cells from Traf6fl/flFoxp3Cre+ ("Traf6-/-") or wild type Traf6wt/wtFoxp3Cre+mice ("Traf6+/+") were isolated by FACS, lysed and either total FOXP3 (right) or K63-ubiquitin-modified proteins (left) were immunoprecipitated. Levels of polyubiquitinated FOXP3 were observed in each by probing with antibodies specific for anti-K63-ubiquitin and FOXP3, respectively, and levels of total FOXP3 and actin were also measured by immunoblot analysis of whole cell lysate.

Data information: Shown are the representative findings of 3 independent experiments. Where indicted, levels of FOXP3, TRAF6, or actin (loading control) were measured in the pre-IP WCL input as well.
TRAF6 directs FOXP3 localization and facilitates regulatory T cell function through K63-linked ubiquitination (2019)
Figure 4

Figure 4. TRAF6 targets lysine residue 262 on FOXP3 for ubiquitination.

A Immunoblot analysis of TRAF6-mediated ubiquitination of wild type, and mutant FOXP3 molecules. 293T cell lines were transfected with a normal HA-tagged Foxp3 construct, another encoding a ubiquitination resistant mutant in which all lysine residues were replaced by arginines (20R), or one of 20 single lysine-containing constructs with only the indicated lysine residue available for modification. These cell lines also received expression vectors encoding TRAF6 and ubiquitin molecules labeled with Myc and FLAG tags, respectively. Negative controls did not receive TRAF6 or expressed FOXP3 alone. Labeled FOXP3 proteins were pulled down from cell lysates (anti-HA) and ubiquitinated species were visualized by immunoblotting for FLAG.

B Assessing the impact of K262 mutation on FOXP3 ubiquitination by TRAF6. As in A, 293T cells were transfected with combinations of expression constructs encoding Myc-TRAF6, FLAG-ubiquitin, and an HA-tagged FOXP3 molecule that was either normal (HA-FOXP3) or possessed a K-to-R mutation at residue 262 (K262R). FOXP3 proteins were pulled down with anti-HA beads and either ubiquitinated proteins or total FOXP3 proteins were detected by probing for FLAG and HA, respectively.

Data information: Shown are representative blots from 3 experiments. Expression of FOXP3 and TRAF6 by transfectants pre-IP WCL was confirmed (input).