EMBO/soda-nel huggingface dataset:
Small Noncoding Vault RNA2-1 Disrupts Gut Epithelial Barrier Function via Interaction with HuR (2022)
Figure 1

Figure 1. Mucosal vtRNA expression associated with intestinal pathologies.

(A) Levels of mucosal vtRNA and uc.417 in the colons of mice treated with 3% DSS for 5 days, as measured by RT-Q-PCR analysis. Values are the means ± SEM (n = 5 mice per group). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with control mice.

(B) Levels of mucosal vtRNA and uc.417 in the small intestines of mice exposed to CLP for 24 h. Values are the means ± SEM (n = 5 mice per group). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with sham mice.

(C) Levels of mucosal vtRNA2-1, vtRNA1-1, vtRNA1-2, and vtRNA1-3 in the colon of patients with active ulcerative colitis (UC). Values are the means ± SEM (n = 6 per group). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with controls.

(D) Levels of tissue vtRNAs in the ileal mucosa of patients with Crohn's disease (CD). Values are the means ± SEM (n = 6 per group). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with controls.
Small Noncoding Vault RNA2-1 Disrupts Gut Epithelial Barrier Function via Interaction with HuR (2022)
Figure 2

Figure 2. Ectopically overexpressed vtRNA2-1 disrupts intestinal epithelial barrier function in vitro.

(A) Levels of vtRNAs in Caco-2 cells 48 h after transfection with vtRNA2-1 expression vector (vt2-1). Values are the means ± SEM (n = 6 or 3 biological replicates). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with control vector.

(B) Expression levels of claudin 1, occludin, E-cadherin, ZO-1, JAM-A, and MVP in cells described in A. Hsc70 immunoblotting was performed as an internal control for equal loading.

(C) Distribution of claudin 1, occludin, E-cadherin, and ZO-1 in cells treated as described in A. Forty-eight hours after transfection, cells were fixed, permeabilized, and incubated first with antibodies against different intercellular junction proteins and then with FITC-conjugated anti-IgG. Original magnification, ×500. Three separate experiments were performed and showed similar results.

(D) Changes in TEER (left) and FITC-dextran paracellular permeability (right) in cells treated as described in A. TEER assays were performed on 12-mm Transwell filters; paracellular permeability was assayed by adding the membrane-impermeable trace molecule FITC-dextran to the insert medium. Values are the means ± SEM (n = 3 biological replicates). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with control vector.
Small Noncoding Vault RNA2-1 Disrupts Gut Epithelial Barrier Function via Interaction with HuR (2022)
Figure 3

Figure 3. vtRNA2-1 silencing improves intestinal epithelial barrier function in vitro.

(A) Levels of vtRNAs in Caco-2 cells 48 h after transfection with the siRNA targeting vtRNA2-1 (si-vt2-1) or control siRNA (C-siRNA). Values are the means ± SEM (n = 3 biological replicates). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with C-siRNA.

(B) Immunoblots of claudin 1, occludin, E-cadherin, ZO-1, and JAM-A in cells treated as described in A. Hsc70 immunoblotting served as an internal control for equal loading. Three separate experiments were performed and showed similar results.

(C) Changes in TEER (left) and FITC-dextran paracellular permeability (right) in cells treated as described in A. Values are the means ± SEM (n = 3 biological replicates). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with C-siRNA.
Small Noncoding Vault RNA2-1 Disrupts Gut Epithelial Barrier Function via Interaction with HuR (2022)
Figure 4

Figure 4. Ectopically overexpressed vtRNA2-1 decreases intercellular junction levels and lowers epithelial renewal and Paneth cell function ex vivo.

(A) Levels of vtRNA2-1 in primarily cultured intestinal organoids 48 h after transfection with vtRNA2-1 expression vector (vt2-1). Organoids were derived from proximal small intestine and grown in the indicated conditions. Values are the means ± SEM (n = 6 biological replicates). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with control vector.

(B) Images of immunofluorescence staining of claudin 1, occludin, E-cadherin, and ZO-1 in intestinal organoids treated as described in A. Scale bars, 100 µm. Three separate experiments showed similar results.

(C) Sizes of intestinal organoids at different times after vtRNA2-1 overexpression. Left, bright field microscopy analysis of growth of organoids; right, confocal analysis of BrdU (yellow) and DAPI (blue) on day 3 after culture. Scale bars, 100 µm.

(D) Quantitative data of surface area of organoids (left) and BrdU (right) in organoids treated as described in C. Values are means ± SEM (n = 4 biological replicates). Unpaired, two-tailed Student's t test was used. * P< 0.05 compared with control vector.

(E) Immunostaining (left) of Paneth (lysozyme-positive) cells and quantitative data (right) in intestinal organoids treated as described in A. Lysozyme: green. Scale bars, 100 µm. Values are means ± SEM (n = 4 biological replicates). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with control vector.
Small Noncoding Vault RNA2-1 Disrupts Gut Epithelial Barrier Function via Interaction with HuR (2022)
Figure 5

Figure 5. Elevation of mucosal vtRNA2-1 enhances gut barrier dysfunction in mice exposed to septic stress.

(A) Levels of vtRNA2-1 in the small intestinal mucosa of mice on day 5 after intraperitoneal injection with a recombinant vtRNA2-1 lentiviral expression vector (Lenti-vt2-1) or empty control lentiviral vector (vector). Values are means ± SEM (n = 6 biological replicates). Unpaired, two-tailed Student's t test was used. * P< 0.05 compared with vector.

(B) Immunoblots of claudin 1, occludin, JAM-A, ZO-1, and E-cadherin in the mucosa from mice treated as described in A. GAPDH immunoblotting served as an internal control for equal loading.

(C) Changes in gut permeability in sham- and CLP (septic stress)-mice. CLP was induced on day 5 after injection with lenti-vt2-1 or control vector. FITC-dextran was given orally 20 h after CLP, and blood samples were collected 4 h later. Values are means ± SEM (n = 5 biological replicates). Unpaired, two-tailed Student's t test was used. * P< 0.05 compared with vector.

(D) Proliferating cells as measured by BrdU labeling assays in the mucosa from mice treated as described in A. BrdU (1 h post-injection, S phase): green. Scale bars, 25 µm. Experiments were repeated in four mice injected with either lenti-vt2-1 or control vectors and showed similar results.

(E) Paneth cells (lysozyme-positive cells) in the small intestinal mucosa from mice treated as described in A. Lysozyme: red. Scale bars, 25 µm.
Small Noncoding Vault RNA2-1 Disrupts Gut Epithelial Barrier Function via Interaction with HuR (2022)
Figure 6

Figure 6. vtRNA2-1 inhibits translation of claudin 1 and occludin and enhances decay of E-cadherin mRNA.

(A) Levels of claudin-1, occludin, and E-cadherin mRNAs in Caco-2 cells 48 h after transfection with vtRNA2-1 expression vector. Values are means ± SEM (n = 3 biological replicates). Unpaired, two-tailed Student's t test was used. *P < 0.05 compared with control vector.

(B) Stability of the mRNAs in cells treated as described in A. The mRNA levels were examined at different times after administration with actinomycin D. Values are means ± SEM (n = 3 biological replicates). Unpaired, two-tailed Student's t test was used. *P < 0.05 compared with control vector.

(C) Newly synthesized claudin 1 and occludin proteins in cells treated as described in A. After cells were exposed to L-azidohomoalaine (AHA), cell lysates were incubated with the reaction buffer containing biotin/alkyne reagent; the biotin-alkyne-azide-modified protein complex was pulled down by paramagnetic streptavidin-conjugated dynabeads.

(D) Quantitative data of immunoblots shown in C. Values are means ± SEM (n = 3 biological replicates). Unpaired, two-tailed Student's t test was used. * P < 0.05 compared with control vector.

(E) Distribution of claudin 1 (left), occludin (center), and GAPDH (right) mRNAs in each gradient fraction of polysomal profiles prepared from cells treated as described in A.
Small Noncoding Vault RNA2-1 Disrupts Gut Epithelial Barrier Function via Interaction with HuR (2022)
Figure 7

Figure 7. vtRNA2-1 inhibits translation of claudin 1 and occludin via interaction with HuR.

(A) Association of vtRNA2-1 with HuR in Caco-2 cells as measured by biotin-labeled RNA pull-down assays. After cytoplasmic lysates were incubated with biotinylated vtRNA2-1, levels of HuR and other proteins in the pull-down material were assessed. Three separate experiments showed similar results.

(B) Association of endogenous HuR with endogenous vtRNA2-1 (top) and vtRNA1-1 (bottom). After immunoprecipitation (IP) of RNA-protein complexes from cell lysates using anti-HuR antibody (AB) or control IgG, RNAs were isolated and measured by RT-Q-PCR analysis. Values are means ± SEM (n = 3 biological replicates). Unpaired, two-tailed Student's t test was used. *P < 0.05 compared with IgG.

(C) Effect of increasing the levels of vtRNA2-1 on HuR binding to claudin 1 (CDN 1; top) and occludin (bottom) mRNAs. Twenty-four hours after cells were transfected with vtRNA2-1 expression vector, the levels of CLDN1 and occludin mRNAs in the materials pulled down by anti-HuR AB were examined. Values are means ± SEM (n = 3 biological replicates). One-way ANOVA was utilized with Tukey's post hoc test. *,+ P < 0.05 compared with IgG or cells transfected with control vector, respectively.

(D) Changes in the expression levels of claudin-1, occludin, and E-cadherin proteins 48 h after cells were transfected with HuR expression vector alone or co-transfected with HuR and vtRNA2-1 expression vectors.

(E) Changes in TEER (left) and FITC-dextran paracellular permeability (right) in cells treated as described in D. Values are the means ± SEM (n = 4 biological replicates). One-way ANOVA was utilized with Tukey's post hoc test. *,+ P < 0.05 compared with cells transfected with control vector or cells transfected with HuR expression vector alone, respectively.
A Golgi rhomboid protease Rbd2 recruits Cdc48 to cleave yeast SREBP (2016)
Figure 1

Figure 1. Rbd2 is a Golgi-resident rhomboid protease

A. Plot of correlation coefficients generated from comparison of the genetic profiles from sre2Δ and SPCC790.03Δ (rbd2Δ) to all other profiles from the genetic interaction array. Note the high correlations among dsc1-4, sre2, and rbd2.

B. Protein structural predictions for S. pombe Rbd2 (blue) and E. coli GlpG (red). Protein structures were predicted using the Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2). Rbd2 model structure was aligned to GlpG using PyMOL.

C. Protein sequence alignment of E. coli GlpG, human RHBDL4, S. pombe Rbd2, and S. cerevisiae Rbd2. Alignments were generated using Clustal Omega server (http://www.ebi.ac.uk/Tools/msa/clustalo). Predicted transmembrane domains (TM) and shared sequence motifs were used for alignment. Positions of functionally important, conserved rhomboid amino acid residues (including predicted catalytic dyad serine and histidine) are shaded in red. Residues boxed in green were identified as required for Sre1 activation from a genetic selection and were analyzed in Figures 2E and F. The starting residue numbers of the indicated sequences within their full-length proteins are given. Asterisks mark residues identical in all sequences and colons denote conservative substitutions.

D. Topology model for S. pombe Rbd2 with the conserved six-pass TM. Both the N- and C-termini face the cytoplasm. The protease active site serine and histidine form a catalytic dyad between TM4 and TM6. A SHP-box, predicted to bind Cdc48, is positioned at the Rbd2 C-terminus.

E. Cells carrying a plasmid expressing rbd2-6xmCherry and either of two Golgi proteins dsc2-6xGFP (dsc2-6xGFP rbd2-6xmCherry) or Sec72-6xGFP (Sec72-6xGFP rbd2-6xmCherry) were imaged by confocal fluorescence microscopy.
A Golgi rhomboid protease Rbd2 recruits Cdc48 to cleave yeast SREBP (2016)
Figure 2

Figure 2. SREBP activation requires rbd2

A. Wild-type (WT), rbd2Δ or sre1Δ yeast (200 cells) containing either empty vector (EV) or a plasmid expressing sre1N (Sre1 amino acids 1-440) were grown on rich medium in the presence or absence of cobalt chloride (CoCl2).

B. Western blot was probed with anti-Sre1 IgG of phosphatase-treated, whole cell lysates from WT and the indicated mutants grown for the indicated time in the absence of oxygen. Dsc5 serves as a loading control and was detected by chemiluminescence. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.

C. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre1 cleavage. Western blot was performed using anti-Sre1 or anti-Flag IgG of phosphatase-treated, whole cell lysates from cells grown for 3 hr in the presence or absence of oxygen.

D. Indicated yeast strains expressing rbd2 or chromosomal Flag-tagged rbd2, rbd2-S130A or rbd2-H182A were analyzed for Sre2 cleavage by western blot probed with anti-Sre2 serum or anti-Flag IgG of phosphatase-treated, whole cell lysates from cells grown in the presence of oxygen.

E. Sre1 cleavage assay was performed as in (C). G128V, A127D and A186T mutant strains were obtained from our previous mutagenesis screening (Stewart et al, 2012). Dsc5 serves as a loading control.

F. Sre2 cleavage assay for indicated yeast strains was performed as in (D).

G. A model substrate of Sre2 containing amino acids 423-793 (Sre2MS) with an N-terminal 3xFlag epitope tag was expressed on a plasmid under the control of a CaMV promoter in sre2Δ, dsc1Δsre2Δ or rbd2Δsre2Δ yeast strains. Whole cell lysates from indicated yeast strains were analyzed by western blot with anti-Flag antibody. Three independent isolates (A-C) are shown for each strain. Empty vector (EV) transformation serves as a negative control.
A Golgi rhomboid protease Rbd2 recruits Cdc48 to cleave yeast SREBP (2016)
Figure 3

Figure 3. Uncleaved SREBP is degraded in a proteasome-dependent and Dsc E3 ligase-dependent manner in the absence of rbd2

A. Wild-type (WT) and the indicated mutant strains were grown for 3 hr at the non-permissive temperature (36°C) to inactivate Mts3. Phosphatase-treated cell lysates were analyzed by western blot with anti-Sre2 serum and imaged using chemiluminescence. P and N denote Sre2 precursor and cleaved nuclear forms, respectively.

B. WT, rbd2Δ and sre2Δ cells were treated with bortezomib (Bz) for 3 hr, and phosphatase-treated, whole cell lysates were analyzed by western blot with anti-Sre2 serum.

C. WT, dsc1Δ and rbd2Δ cells carrying either empty vector (EV) or a plasmid expressing 3xFlag-Sre2MS (MS) were treated with bortezomib (Bz) for 3 hr. Whole cell lysates were analyzed by western blot with anti-Flag antibody.

D. Whole cell lysates from the indicated strains were analyzed by western blot with anti-Sre2 serum.

E. Catalytically dead Dsc1 E3 ligase (dsc1-C634A) mutant and other indicated yeast strains were analyzed by western blot probed with anti-Sre2 serum and imaged using chemiluminescence.
A Golgi rhomboid protease Rbd2 recruits Cdc48 to cleave yeast SREBP (2016)
Figure 4

Figure 4. Rbd2 is an active rhomboid protease

A. Human HEK293T cells were transfected with plasmids expressing different 3xHA-tagged rhomboid proteases: S. pombe rbd2, S. pombe rbd2 recoded for human expression, human RHBDL2, D. melanogaster Rho1, and P. falciparum ROM4. Whole cell lysates were analyzed by western blot to detect the HA-tagged rhomboid [asterisks indicate loading control bands detected with antibody ab179726 (abcam)].

B. Human HEK293T cells were transfected with plasmids expressing the indicated rhomboid substrates and either recoded S. pombe rbd2, D. melanogaster Rho1, or P. falciparum ROM4. Conditioned media and cell lysates were analyzed by western blot. P and C denote precursor and cleaved products, respectively.

C. Human HEK293T cells were transfected with plasmids expressing the GFP-TatA-Flag substrate and the indicated rhomboid proteases. Whole cell lysates were analyzed by western blot. P, CN, CC denote precursor, N-terminal cleaved and C-terminal cleaved products, respectively.

D. Human HEK293T cells were transfected with plasmids expressing the indicated GFP-TatA-Flag substrates and either wild-type or mutant recoded S. pombe rbd2. Whole cell lysates were analyzed by western blot.

E. Human HEK293T cells were transfected with plasmids expressing the indicated GFP-TatA-Flag substrates and different rhomboid proteases. Whole cell lysates were analyzed by western blot.

F. Human HEK293T cells were transfected with plasmids expressing the GFP-TatA (A8L)-Flag and recoded S. pombe rbd2. C-terminal cleavage products were purified using anti-Flag antibodies and purified protein was analyzed by mass spectrometry.
A Golgi rhomboid protease Rbd2 recruits Cdc48 to cleave yeast SREBP (2016)
Figure 5

Figure 5. Rbd2 cleavage requires Lys 743

A. Diagram of Sre2MS substrate (aa 423-793). The enlarged model shows predicted positions of TM1 and TM2 denoting the 26 amino acids tested for mutagenesis by colored squares. Numbers indicate the number of amino acid residues. Mutants showing a block in cleavage due to defects in Dsc1 function are indicated in orange, Rbd2 function in pink, a combination of defects in Dsc1 and Rbd2 in blue (intermediate), and residues showing normal cleavage are indicated in gray.

B. Whole cell lysates from WT, rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag-Sre2MS WT or indicated mutants were analyzed by western blot with anti-Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively.

C. Model outlining the predicted fate of Sre2MS in different yeast strains. U denotes either mono-ubiquitin or poly-ubiquitin.

D-E. Whole cell lysates from WT, rbd2Δ and dsc1Δ cells carrying a plasmid expressing 3xFlag-Sre2MS WT or indicated mutants were analyzed by western blot with anti-Flag antibody. P and N denote Sre2MS precursor and cleaved forms, respectively. Asterisks indicate non-specific, loading control bands, which were used to normalize the intensities of P and N in Figure 5F. Two independent isolates, A and B, are shown for each strain.

F. Intensities of individual P and N bands from western blots were quantified using LI-COR software. Relative intensity of P and N to corresponding non-specific band (asterisk) in each lane was normalized to that of P level in isolate A of WT strain for each mutagenesis. The ratio of N to P or relative P level was presented in bar graphs.

G. The average ratio of N to P level for different Sre2MS mutants in wild-type cells (Appendix Fig S2) was plotted. Cleavage was scored as normal if the ratio was >2.

H. For Sre2MS mutants showing defects in cleavage, the average fold-increase in P level in dsc1Δ cells compared to WT cells from two isolates (Appendix Fig S2) was plotted. Mutants were grouped into three phenotypic classes (intermediate, dsc1Δ, and rbd2∆) according to observed precursor accumulation. Precursor accumulation was defined as dsc1Δ if <1.5 (orange), rbd2Δ if >3 (pink), and intermediate if in between (blue).
A Golgi rhomboid protease Rbd2 recruits Cdc48 to cleave yeast SREBP (2016)
Figure 6

Figure 6. Rbd2 binds Cdc48 through a C-terminal SHP-box

A. Alignment of SHP-box sequences from human UFD1, human p47, S. cerevisiae Dfm1, and S. pombe Rbd2 C-terminal SHP-box (aa 242-251). Asterisks denote residues identical in all sequences, colons mark conservative substitutions, and dots mark semiconservative substitutions.

B. Recombinant proteins GST-fused Rbd2 C-terminus (Rbd2200-251), Dsc5 UBX (Dsc5323-425), Dsc2 UBA (Dsc2298-372), and GST-HA-V5 control were bound to GST magnetic beads and incubated with S. pombe cytosol fraction from wild-type cells. Equivalent amounts of unbound and bound fractions were probed for anti-Cdc48, anti-ubiquitin and anti-GST IgG.

C. GST pulldown assay was performed as in (B) using truncated forms of Rbd2 C-terminus; Rbd2200-251, Rbd2200-225, Rbd2225-251 and Rbd2200-240. Unbound (1x) and bound (5x) fractions were analyzed by western blotting

D. Single residue mutants of conserved glycine residues in Rbd2200-251 (G244R and G246R) were tested for Cdc48 binding as in (C). Western blots (B-D) were imaged using chemiluminescence.
A Golgi rhomboid protease Rbd2 recruits Cdc48 to cleave yeast SREBP (2016)
Figure 7

Figure 7. APEX2 system detects Rbd2-Cdc48 binding in cells

A. Detergent-solubilized yeast extracts from cells containing chromosomal Flag-tagged rbd2 or rbd2-G246R in rbd2Δ background were prepared, and proteins associated with Rbd2-Flag were immunopurified using anti-Flag magnetic beads. Equal amount of total (lanes 1-3) along with 25x bound fractions (lanes 4-6) were analyzed by western blot with anti-Cdc48 serum and anti-Flag IgG.

B. Illustration outlining APEX2-catalyzed biotin-phenol labeling. An Rbd2-Flag-APEX fusion protein oxidizes biotin-phenol to biotin-phenoxyl radical that covalently labels binding partners.

C. Biotin-phenol labeling was performed for 1 min with H2O2 treatment as described in Materials and Methods. WT cells or rbd2Δ cells carrying rbd2-Flag-APEX2 (rbd2-F-AX) or rbd2-G246R-Flag-APEX2 (G246R-F-AX) plasmid were lysed after biotin-labeling reaction, and proteins were denatured by heating the cells in lysis buffer containing 1%SDS. Biotinylated proteins were then enriched using streptavidin magnetic beads. Lysates and 50x-enriched eluates were analyzed by western blot with IRDye 800CW Streptavidin, anti-Cdc48 serum, or anti-Flag IgG.
A Golgi rhomboid protease Rbd2 recruits Cdc48 to cleave yeast SREBP (2016)
Figure 8

Figure 8. SREBP activation requires Cdc48 binding to Rbd2

A. Yeast strains (200 cells) containing chromosomal Flag-tagged rbd2, rbd2-G244R, rbd2-G246R or rbd2-S130A in rbd2Δ background and sre1Δ yeast were grown on rich medium in the absence or presence of cobalt chloride (CoCl2).

B. Western blot of phosphatase-treated, whole cell lysates from strains in (A) grown for 3 hr in the presence or absence of oxygen was probed with anti-Sre1 or anti-Flag IgG. P and N denote Sre1 precursor and cleaved nuclear forms, respectively.

C. Sre2 cleavage was assayed by western blot using anti-Sre2 serum or anti-Flag IgG of phosphatase-treated, whole cell lysates from the indicated strains. N denotes Sre2 cleaved nuclear form.

D. Whole cell lysates from indicated yeast strains carrying a plasmid expressing 3xFlag-Sre2MS were analyzed by western blot with anti-Flag antibody. Two independent isolates, A and B, are shown for each strain. Asterisk denotes non-specific band.

E. Diagram of four Rbd2 chimeras fused to the SHP-box from S. pombe Der1-like protein (SPBC365.08c). The SHP-boxes of Rbd2 and Der1-like protein are shaded red and green, respectively. Asterisks denote point mutation in the SHP-box.

F. Yeast strains containing Flag-tagged rbd2-SHP, rbd2-SHP*, rbd2-G246R-SHP, and rbd2-G246R-SHP* were generated in rbd2Δ background by chromosomal integration of the rbd2 fusion proteins depicted in (E). Indicated yeast strains were then assayed for Sre1 cleavage as in (B).

G. Indicated strains were assayed for Sre2 cleavage as in (C).

H. Wild-type, sre2Δ or strains containing chromosomal Flag-tagged rbd2, rbd2-G246R or rbd2-S130S in rbd2Δ background (lane 7-12) were treated with bortezomib (Bz) for 2 hr, and phosphatase-treated, whole cell lysates were analyzed by western blot with anti-Sre2 serum or anti-Flag IgG.