EMBO/soda-nel huggingface dataset:
A stress-induced tyrosine-tRNA depletion response mediates codon-based translational repression and growth suppression (2020)
Figure 7

Figure 7. Functional effects of interactions between hnRNPA1 and tRFTyrGUA.

(A) Schematic depicting the expected visualization of a cross-linked immunoprecipitation by autoradiogram. Bands corresponding to smRNA-RBP interactions (no RNase digestion) or a smear representing mRNA-RBP interactions (low RNase digestion) by autoradiogram were processed for HITS-CLIP.

(B) IGV plots from an hnRNPA1 HITS-CLIP (Huelga et al., 2012) reveals interactions with tRFTyrGUA.

(C) IGV plots representing SSB interacting with the tRFTyrGUA in samples that were treated with low levels of RNase digestion. SSB bound tRFTyrGUA reads mapped to multiple loci encoding tRNATyrGUA.

(D) Similar to the IGV plots shown in (C), but depicting SSB interactions with tRFTyrGUA loci in samples without RNase digestion.

(E) A cumulative distribution in control and hnRNPA1 depleted cells of the stability levels for mRNA transcripts with 3' UTR hnRNPA1 CLIP binding (Huelga et al., 2012). Transfection of tRFTyrGUA led to a significant right-shift in the expression levels of 3' UTR bound hnRNPA1 transcripts. Statistical significance was measured using the Kolmogorov-Smirnov test.

(F) Cumulative distribution as in (E). Transfection of locked nucleic acid against tRFTyrGUA and treatment with 200µM H2O2 led to a significant left-shift in mRNA stability of 3'UTR bound hnRNPA1 transcripts. Statistical significance was assessed using the Kolmogorov-Smirnov test.

(G) Model of tRNATyrGUA-dependent gene regulatory response to oxidative stress.
An E3 ligase-independent function for RNF123 in RIG-I like receptors-mediated antiviral signaling (2016)
Figure 1

Figure 1. RNF123 Overexpression inhibits RNA virus-induced responses.

A RNF123 suppresses the SeV- and EMCV-induced but not the cGAS and STING-mediated activation of the IFN-β promoter. HEK293T cells (3×105) were transfected with the IFN-β reporter plasmid (100 ng), the pRL-TK Renilla luciferase plasmid (100 ng), the expression plasmid for cGAS (50 ng) and STING (50 ng) or empty vector (100 ng), and 0, 20, 50, or 100 ng (wedges) of RNF123 for 12 h, and then were infected with SeV or EMCV or left uninfected. Luciferase assays were performed after 18 h and results are presented relative to the luciferase activity in control cells.

B RNF123 inhibits the SeV-, but not the cGAS and STING-induced activation of the NF-κB promoter. HEK293T cells (3×105) were transfected with the NF-κB reporter plasmid (100 ng), the pRL-TK Renilla luciferase plasmid (100 ng), the expression plasmids for cGAS (50 ng) and STING (50 ng), or empty vector (100 ng), and 0, 20, 50 or 100 ng (wedges) of RNF123 for 12 h, and were then infected for 24 h with SeV or left uninfected, followed by analysis as described in A.

C, D RNF123 inhibits the SeV-induced, but not the cGAS and STING-mediated RANTES and IFN-β transcription. The transfection and treatment were as in A. Total RNA was isolated and RT-PCR (C) or Q-PCR (D) was performed using the indicated primers.

E RNF123 inhibits the SeV-induced, but not the cGAS and STING-mediated IFN-β secretion. The transfection and treatment were as in A. Bioassays for type I interferon in the supernatants of HEK293T cells were performed and the results are presented relative to untreated control cells.

F Overexpression of RNF123 enhances NDV-eGFP replication. HEK293T cells (1×105) were transfected with the expression plasmids for Flag-RNF123 or empty vector (2 µg). At 24 h after transfection, cells were infected with NDV-eGFP. At 30 h after infection, viral replication was determined by GFP expression visualized by fluorescence microscopy and analyzed by western blot.

G RNF123 enhances EMCV replication. HeLa cells (1×105) were transfected with the expression plasmids for Flag-RNF123 or empty vector (2 µg). At 24 h after transfection, cells were infected with EMCV or given DMEM with no virus. At 18 h after infection, viral replication was determined by VP1 expression, which was analyzed by western blot.

Each histogram shows the mean ± SD of three independent experiments done in triplicate. ***P <0.001; **P <0.01; * P <0.1; ns, P >0.05.
An E3 ligase-independent function for RNF123 in RIG-I like receptors-mediated antiviral signaling (2016)
Figure 2

Figure 2. Knockdown of RNF123 potentiates anti-RNA virus signaling.

A HEK293T cells with stable knockdown of RNF123 were generated using shRNA. Western blot was performed with anti-RNF123, anti-VISA (as a control), and anti-GAPDH antibodies after infection with five lentiviruses encoding a shRNA control (GFPsh) and four shRNAs targeting RNF123 (RNF123 sh #1, #2, #3, and #4) in HEK293T cells.

B, C Knockdown of RNF123 facilitated the activation of IFN-β and NF-κB promoters induced by SeV and EMCV but not by cGAS and STING. Stable HEK293T cells with GFPsh or RNF123 shRNAs (2×105) were transfected with the indicated reporter plasmid (100 ng), pRL-TK Renilla luciferase plasmid (100 ng), cGAS (50 ng), and STING (50 ng), or empty vector (100 ng). At 12 h after transfection, they were infected for 12 h with SeV or EMCV or left uninfected, followed by analysis as described in the legend of Fig 1A.

D, E Knockdown of RNF123 enhances the IFN-β and RANTES transcription induced by SeV and EMCV, but not that by cGAS and the STING. HEK293T cells were transfected with cGAS (1 μg) and STING (1 μg) or empty vector for 12 h, and then infected with SeV or EMCV for 12 h or left uninfected. Total RNA was isolated and RT-PCR (D) and Q-PCR (E) were performed using the indicated primers.

F, G Time-course of potentiation of SeV-induced IFN-β transcription (F) and secretion (G) by knockdown of RNF123. HEK293T cells were infected with SeV for 6, 12, 18, or 24 h, total RNA was isolated, and RT-PCR was performed using the indicated primers (F). Bioassays of type-I interferon in the supernatants of HEK293T cells were performed, and the results are presented relative to untreated control cells (G).

H HeLa cells with stable knockdown of RNF123 were generated by shRNA. Western blot was performed as described in Fig 2A.

I, J Knockdown of RNF123 enhances SeV- and EMCV- induced IFN-β expression in HeLa cells. HeLa cells were infected with SeV or EMCV for 12 h, total RNA was isolated, and RT-PCR (I) or Q-PCR (J) was performed using the indicated primers.

K Knockdown of RNF123 suppresses NDV-eGFP replication. HEK293T cells (2×105) were infected with NDV-eGFP. At 30 h after infection, viral replication was determined by GFP expression visualized by fluorescence microscopy and analyzed by western blot with GFP antibody.

L Knockdown of RNF123 inhibits EMCV replication. HeLa cells (2×105) were infected with EMCV for 0, 12, or 18 h.

Viral replication was determined by VP1 expression which was analyzed by western blot. Each histogram shows the mean ± SD of three independent experiments done in triplicate. ***P <0.001; **P <0.01; *P <0.1; ns, P >0.05.
An E3 ligase-independent function for RNF123 in RIG-I like receptors-mediated antiviral signaling (2016)
Figure 3

Figure 3. RNF123 targets RIG-I and MDA5 in the RLR signaling pathway.

A, B RNF123 inhibits the activation of the IFN-β (A) and NF-κB (B) promoters induced by the overexpression of RIG-I, RIG-I CARD, MDA5 CARD, and VISA, but has no effect on the TBK1- and IRF3-5D-induced activation of the IFN-β promoter (A) and the TAB2- and IKKβ-induced activation of the NF-κB promoter (B). HEK293T cells (2×105) were transfected with the indicated plasmids (100 ng), IFN-β or NF-κB promoter luciferase plasmid (100 ng), and pRL-TK Renilla luciferase plasmid (100 ng), together with an empty vector and RNF123 construct (100 ng). Luciferase assays were performed 24 h after transfection.

C Knockdown of RNF123 potentiates the activation of the IFN-β promoter induced by RIG-I, RIG-I CARD, MDA5, and MDA5 CARD, but not that by VISA, TBK1, and cGAS/STING. 293T-GFPsh, shRNF123 #1, #3, and #4 stable cell pools (2×105) were transfected with the indicated plasmids. Luciferase assays were performed 24 h after transfection.

D RNF123 interacted with RIG-I and MDA5, but not VISA, TBK1, and cGAS. HEK293T cells (1×106) were transfected with Flag-RNF123 (F-RNF123) together with HA-RIG-I, HA-MDA5, HA-VISA, HA-TBK1, or HA-cGAS plasmids (8 μg each). Cell lysates were immunoprecipitated (IP) with anti-HA (αHA). The immunoprecipitates were analyzed by western blotting (immunoblotting [IB]) with anti-Flag (αF) or anti-HA antibody (αHA). Whole-cell lysates (WCL) were analyzed by western blotting with anti-HA and anti-Flag antibodies to determine the expression levels of transfected plasmids.

E Endogenous interaction of RNF123 with RIG-I upon SeV infection. HEK293T cells (3×106) were treated with SeV for 12 h or left untreated. Cell lysates were immunoprecipitated with rabbit anti-RNF123 antibody or control IgG. The immunoprecipitates were analyzed by western blotting with rabbit anti-RIG-I antibody. The expression levels of the endogenous proteins were analyzed by western blotting with anti-RIG-I and anti-RNF123 antibodies.

F Endogenous interaction of RNF123 with MDA5 upon EMCV infection. HeLa cells (3×106) were treated with EMCV for 12 h or left untreated. Cell lysates were immunoprecipitated with rabbit anti-RNF123 antibody or control IgG. The immunoprecipitates were analyzed by western blotting with rabbit anti-MDA5 antibody. The expression levels of the endogenous proteins were analyzed by western blotting with anti-MDA5 and anti-RNF123 antibodies. Each histogram shows the mean ± SD of three independent experiments done in triplicate. ***P <0.001; **P <0.01; *P <0.1; ns, P >0.05.
An E3 ligase-independent function for RNF123 in RIG-I like receptors-mediated antiviral signaling (2016)
Figure 4

Figure 4. The E3 ligase activity of RNF123 is not required for its inhibitory action on the RLR signaling pathway.

A Schematic of the structures of RNF123 and the mutants used in this work.

B The SPRY and coiled-coil domains are both indispensable for inhibiting IFN-β promoter activation. HEK293T cells (3×105) were transfected with the IFN-β reporter plasmid (100 ng), the pRL-TK Renilla luciferase plasmid (100 ng), the expression plasmid for RNF123 and its mutants (100 ng), and the empty vector or RIG-I CARD/MDA5 CARD (100 ng). Reporter assays were performed 24 h after transfection.

C The SPRY domain and two coiled-coil domains of RNF123 are both required for interaction with RIG-I CARD. HEK293T cells (1×106) were transfected with HA-RIG-I CARD together with F-RNF123 or its mutants (8 μg each). Cell lysates were immunoprecipitated (IP) with anti-Flag (α-F). The immunoprecipitates were analyzed by western blotting (immunoblotting [IB]) with anti-HA (α-HA) or anti-Flag (α-F). Whole-cell lysates (WCL) were analyzed by western blotting with anti-HA and anti-Flag antibodies.

D KPC2 has no effect on the activation of the IFN-β reporter induced by RIG-I CARD and MDA5 CARD. HEK293T cells (3×105) were transfected with the IFN-β reporter plasmid (100 ng), the pRL-TK Renilla luciferase plasmid (100 ng), and the indicated expression plasmids. Reporter assays were performed 24 h after transfection.

E Effect of shKPC2 plasmids on endogenous KPC2. RT-PCR was performed with the indicated primers after HEK293T cells were infected with six lentiviruses encoding an shRNA control (GFPsh) and five shRNAs targeting kpc2 (Kpc2 #1-#5).

F KPC2 is not required for the inhibitory action of RNF123. HEK293T cells (2×105) with GFPsh or KPC2 sh (#1, #2, or #4) were transfected with the IFN-β reporter plasmid (100 ng) and the pRL-TK Renilla luciferase plasmid (100 ng) with the empty vector or RNF123 (100 ng). Twenty-four hours after transfection, cells were infected with SeV or left uninfected. Reporter assays were performed 18 h after infection.

G Knockdown of KPC2 had no effect on SeV-induced IFN-β expression. HEK293T cells with GFPsh or KPC2 sh #4 were infected with SeV for the indicated times, total RNA was isolated, and RT-PCR experiments were performed.

Each histogram shows the mean ± SD of three independent experiments done in triplicate. ***P <0.001; **P <0.01; *P <0.1; ns, P >0.05.
An E3 ligase-independent function for RNF123 in RIG-I like receptors-mediated antiviral signaling (2016)
Figure 5

Figure 5. RNF123 inhibits RIG-I/MDA5 binding with VISA.

A, B RNF123 interacts with RIG-I/MDA5 CARD. HEK293T cells (1×106) were transfected with expression plasmids for Flag-RNF123 together with HA-RIG-I or its truncations (8 μg each). Co-immunoprecipitation and whole-cell lysates were analyzed as described in the legend of Fig. 3D.

C, D RNF123 competes with VISA for RIG-I/MDA5 CARD binding. HEK293T cells (1×106) were transfected with expression plasmids for HA-RIG-I CARD/MDA5 CARD alone or together with Flag-VISA or with Flag-VISA and Flag-RNF123 (8 μg each). Co-immunoprecipitation and whole-cell lysates were analyzed as described in Fig. 3D.

E RNF123 does not compete with REUL for RIG-I CARD binding. The methods of transfection and co-immunoprecipitation were as described in Fig. 5C,D.

F Knockdown of RNF123 facilitates the endogenous interaction between RIG-I and VISA in 293T cells. 293T cells stably infected with SeV were treated for the indicated times. Cell lysates were immunoprecipitated with anti-VISA antibody. The immunoprecipitates and lysates were analyzed by western blotting with the indicated antibody.

G Knockdown of RNF123 facilitates the endogenous interaction between MDA5 and VISA in HeLa cells. HeLa cells were stably infected with EMCV for the indicated times. Co-immunoprecipitation and whole-cell lysates were analyzed as described in (F).
A deep proteome and transcriptome abundance atlas of 29 healthy human tissues (2019)
Figure 2

Figure 2 | Analysis of protein and transcript expression levels within and across tissues. (A) Distribution of global transcript and protein abundance in all tissues. It is apparent that the dynamic range of protein expression (iBAQ scale) exceeds that of mRNA expression (FPKM scale; see Appendix Fig S10 for the corresponding plot for RNA and protein copy numbers). (B) Protein to mRNA abundance plot for brain tissue. The slope of the regression line indicates that high abundance mRNAs give rise to more protein copies per mRNA than low abundance mRNAs. (C) Ranked abundance plot of proteins and transcripts in human heart. While the 10 most abundant transcripts cover almost 70% of all transcripts in this tissue, the corresponding proteins only represent about 20% of the total protein. (D) Analysis of the number of genes that are shared among the 100 most abundant transcripts and proteins. Regardless of the tissue, the fraction of shared genes rarely exceeds 20%. (E) Correlation analysis of protein to RNA abundance (in log10 scale) across tissues, resulting in almost 90% positive correlations. The protein highlighted in the next panel are marked. (F) Examples for proteins that show high (SYK, left panel) or no (EIF4A3, right panel) correlation of protein/RNA ratios across tissues. While the former indicates that different tissues express different quantities of SYK, EIF4A3 expression appears to be similar in all tissues.
STIM1-mediated calcium influx controls antifungal immunity and the metabolic function of Th17 cells (2020)
Figure 1

Figure 1. Mutation of L374 in the C terminus of STIM1 abolishes SOCE and causes CRAC channelopathy.

(A) Pedigree of the patients P1 (II-1) and P2 (II-2) presenting with CRAC channelopathy. Filled circles (females) and squares (males) indicate homozygous patients; dotted symbols indicate heterozygous asymptomatic carriers; symbols with "?" indicate asymptomatic family members not available for DNA sequencing.

(B) Sanger sequencing of genomic DNA isolated from PBMC of the mother (I-2), P1 and P2.

(C) STIM1 and STIM2 mRNA expression in PBMC of P1, P2, mother and a healthy donor (HD) that were left unstimulated or stimulated with anti-CD3/CD28 for 24 hours. mRNA levels of STIM1/2 normalized to 18S. Graph shows the mean ± SEM of duplicates from one experiment.

(D) STIM1 protein expression in CD4+ and CD8+ T cells from P1, P2, the mother and a HD analyzed by flow cytometry. Shaded histograms: polyclonal rabbit IgG control antibody; open histograms: polyclonal anti-STIM1 antibody. Bar graphs show the delta MFI calculated as MFISTIM1 - MFIIgG control that was normalized to HD T cells. Bar graphs are mean ± SEM of 2 independent repeat experiments.

(E) STIM1 protein expression in expanded human T cells analyzed by immunoblotting. One representative Western blot of 3 is shown. Bar graphs are the mean ± SEM of STIM1 expression normalized to actin from 3 independent experiments.

(F) Ca2+ influx in Fura-2 loaded PBMC of P1, P2, the mother and a HD. T cells were stimulated with 1 μM thapsigargin (TG) in the absence of extracellular Ca2+ followed by addition of 1 mM extracellular Ca2+. Bar graphs show the integrated Ca2+ influx response (AUC, area under the curve) from 400-800 sec and the peak Ca2+ influx normalized to baseline Ca2+ levels (F340/380) at 400 sec. Data represent the mean ± SEM from 2 experiments. Statistical analysis by unpaired Student's t-test. **p<0.01, ***p<0.001.
STIM1-mediated calcium influx controls antifungal immunity and the metabolic function of Th17 cells (2020)
Figure 2

Figure 2: L374P mutation abolishes STIM1 puncta formation and colocalization with ORAI1.

(A) Domain architecture of STIM1. Abbreviations: CAD: CRAC activation domain; CC1-3: coiled-coil domains 1-3; CCb9, coiled-coil fragment b9; cEF, canonical EF-hand; nEF, non-canonical EF-hand; PB, polybasic domain; P/S, proline/serine-rich region; SAM: sterile alpha motif; non-canonical EF-hand; S, signal peptide; SOAR: STIM1-ORAI1 activation region; TM: transmembrane domain.

(B,C) Homology models of the STIM1 p.L374P mutation based on published crystal (Yang et al., 2012) and NMR (Stathopulos et al., 2013) structures of STIM1. (B) Location of the M374P mutation (red sticks) in the context of the triple mutant (L374M/V419A/C437A) human CAD/SOAR/CCb9 dimer structure relative to CC2 (light blue ribbon) and CC3 (light green ribbon). The template structure without the M374P mutation (grey ribbon) and the movement of the apex positions of both subunits are indicated. (C) Location of the L374P mutation in the context of the NMR structure of a bimolecular complex of two STIM1 CC1TM-distal-CC2 peptides (CC1TM-distal region shown as light pink ribbon, and CC2 shown as light blue ribbon) and two ORAI1 C-terminal (aa 272-292) peptides (yellow ribbons) (Stathopulos et al., 2013). The loss of the backbone amide hydrogen (NH) due to the L374P mutation results in an altered hydrogen bond network near the mutation (small red dashed box). The zoomed view of this area (large red dashed box) shows the backbone hydrogen bond network for the L374P protein (red dashed lines) compared to the WT protein (black dashed lines). The maintained backbone atom hydrogen bonds in this region for the L374P mutant compared to the WT protein are listed in red text while the lost hydrogen bonds are listed in black text.

(D-H) GFP-ORAI1 expressing HEK293 cells were transfected with mCherry-STIM1 (WT or L374P) and left unstimulated or stimulated with 1 μM TG in Ca2+-free Ringer's solution. (D,E) Representative TIRF microscopy images from one of two repeat experiments. Scale bars, 10 μm. (F) Percentages of WT or mutant mCherry-STIM1 present at the plasma membrane (i.e. visible in the TIRFM evanescent field) before TG stimulation. Data are the mean ± SEM calculated from 550 (WT) and 830 (L374P) cells from two independent experiments. (G) Change of mCherry-STIM1 (WT or L374P mutant) fluorescence [F(t)/F(0)] in the TIRFM evanescence field. Fluorescence normalized to values at the time of TG addition in Ca2+-free Ringer's buffer at 0 sec. Traces show mean ± SEM of 40-50 cells from two independent experiments. (H) Colocalization of mCherry-STIM1 (WT or L374P mutant) and GFP-ORAI1 from the time of TG addition at 0 sec. Colocalization measured using the Pearson's coefficient for GFP and mCherry. Traces show mean ± SEM of 27 cells from two independent experiments. Data information: Statistical analysis by unpaired Student's t-test (F, G) and Mann Whitney U test (H). *p<0.05; ***p<0.001.
STIM1-mediated calcium influx controls antifungal immunity and the metabolic function of Th17 cells (2020)
Figure 3

Figure 3. STIM1 p.L374P mutation causes defect in T cell proliferation and cytokine production.

(A,B) Cell size (A) and proliferation (B) of CD4+ T cells from P1 (red), P2 (blue), the mother (grey) and HD (black) stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (10 μg/ml) in the presence or absence of 1 μM FK506 for 24 hours. (A) Representative histograms of FSC (left panel) and percentages of T cell blasts (defined as cells to the right of the dotted vertical line) analyzed by flow cytometry (right panel). (B) Representative histograms of CFSE dilution (left panel) and percentages of proliferating cells (defined as cells to the left of the dotted vertical line) (right panel). Bar graphs in A and B are the mean ± SEM from two independent experiments.

(C,D) Cytokine production by PBMC from P1, P2, the mother and an unrelated HD after stimulation with PMA (40 ng/ml) and ionomycin (500 ng/ml) for 4 hours. Cytokines were analyzed by flow cytometry following surface staining with antibodies against CD3, CD4 and CD45RO, permeabilization and intracellular cytokine staining for GM-CSF, IL-22 and IL-17A. Representative flow cytometry plots (C) and quantification of Th17 (GM-CSF, IL-22, IL-17A), Th1 (TNF-α, IFN-γ) and Th2 (IL-4) cytokines (D). Data represent the mean ± SEM from two independent experiments.

Data information: Statistical analysis by unpaired Student's t-test. *p<0.05, **p<0.01, ***p<0.001.