Rab1b and ARF5 are novel RNA-binding proteins involved in FMDV IRES–driven RNA localization

Figures

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  Figure 1.. Identification of proteins associated with transcripts encompassing the subdomains of domain 3. (A) Schematic representation of the modular domains of the FMDV IRES element. Subdomains of domain 3 are highlighted by color lines surrounding the corresponding secondary structure. The following color code is used: purple for SL3a, blue for SL3abc, green for SL123, and orange for D3. Numbers indicate the nucleotide position included on each transcript. (B) Overview of the RNA-binding protein purification protocol. A representative image of silver-stained gel loaded with proteins associated with control RNA, SL3abc, and SL3a transcripts after streptavidin-aptamer purification is shown. (C) Venn diagram showing the number of factors associated with each subdomain.

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  Figure S1.. Purification of RNAs. Images of denaturing acrylamide gel loaded with RNAs SL3a and SL3abc, in parallel to the control RNA (8% acrylamide, 7 M urea), and SL123 and D3 (6% acrylamide, 7 M urea). Red arrows point to the RNA obtained by streptavidin purification (+); ribosomal RNAs (rRNA and 5S RNA) are detected only in the input sample.

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  Figure S2.. Proteins associated with domain 3 subdomains. (A) Representation of the proteins identified in two biological replicate samples. (B) Functional classification (PANTHER) of proteins associated with the different subdomains. The graph represents the % of factors identified with the transcripts following filtering by score (>10%) and control RNA subtraction. (C) Number of proteins associated with the indicated subdomains according to their annotated function.

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  Figure S3.. Representation of the log10 score of proteins bound to the different subdomains against each other. Representation of the functional groups (log10score) associated with SL3a versus SL3abc (A), SL123 versus D3 (B), SL3a versus D3 (C), and SL3abc versus D3 (D). Proteins belonging to functional cellular processes are colored as indicated in the legend.

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  Figure 2.. Functional networks of proteins associated with SL3a, SL3abc, SL123, and D3 transcripts. Circles depict functionally related nodes obtained with the application BiNGO (Cytoscape platform). The area of a node is proportional to the number of proteins in the test set annotated to the corresponding GO category, and the color intensity indicates the statistical significance of the node according to the colored scale bar. White nodes are not significantly overrepresented; they are included to show the coloured nodes in the context of the GO hierarchy. Arrows indicate branched nodes. Networks are shadowed blue, pink, or grey, according to the functional process. The mean statistical significance (P value) of the networks obtained for each domain relative to a complete human proteome is indicated on the bottom panel. A dash depicts networks with P values > 10−2.

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  Figure 3.. Rab1b and ARF5 are RNA-binding proteins. Gel-shift assays performed with increasing concentration of purified His-Rab1b alone (A) or in the presence of competitor RNA SL123, SL3abc, or control RNA (B), His-ARF5 alone (C) or in the presence of competitor RNA SL123, SL3abc, or control RNA (D). Probes are colored as shown in the legend. Band shift conducted for His-Rab1b and His-ARF5 with a control RNA (E), His-PCBP2 (F), and His-Ebp1 (G) using the indicated probes. The graphs represent the adjusted curves obtained from the quantifications of the retarded complex relative to the free probe (mean ± SD) from two independent assays for each probe. Gel images are representative examples of one assay. For competition experiments (B) and (D), the % of retarded probe relative to the lane without competitor RNA was measured in triplicated assays using a probe: competitor RNA ratio 1:200 for Rab1b and 1:500 for ARF5.

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  Figure S4.. Representative examples of gel-shift images performed with the indicated combination of increasing amounts of the purified proteins and the labeled transcripts (A), or adding a competitor unlabeled RNA (B). Representative images of band shift assays performed with different labeled transcripts and proteins Rab1b, ARF5, PCBP2, or Ebp1 (A). Competition assays conducted with SL3abc RNA and a control RNA of retarded complex formation with Rab1b or ARF5 with SL123 probe (B).

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  Figure 4.. Effect of Rab1b or ARF5 depletion on IRES activity. (A) The levels of Rab1b and ARF5 were determined by Western blot using anti-Rab1b or anti-ARF5 in comparison with siRNAcontrol-transfected cells. Tubulin is used as loading control. Rab1b- and ARF5-depleted cells were used to monitor relative IRES-dependent translation using bicistronic constructs. The effect on protein synthesis was calculated as luciferase activity/chloramphenicol acetyl transferase activity relative to the control siRNA. Each experiment was repeated three times. Values represent the mean ± SD. Asterisks (P = 0.0024) denotes statistically significant differences between cells treated with the siRNAcontrol and siARF5 RNA. (B) GFP-Rab1b and GFP-ARF5 colocalize with the Golgi compartment, but expression of the dominant-negative GFP-Rab1b DN disorganizes the Golgi. Representative images of HeLa cells transfected side by side with GFP-Rab1b, GFP-Rab1b DN, or GFP-ARF5; fixed 30 h post-transfection; and permeabilized. Immunostaining of the Golgi was carried out using anti-GM130 antibody (bar = 10 μm). White arrows denote colocalization of GM130 and GFP-tagged proteins Rab1b or ARF5 in transfected cells, whereas white…

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  Figure S5.. Rab1b or ARF5 depletion affects IRES activity. (A) The levels of Rab1b and ARF5 were determined by WB using anti-Rab1b or anti-ARF5 relative to siRNAcontrol-transfected cells. Tubulin was used as loading control. Rab1b- and ARF5-depleted cells were used to monitor relative IRES-dependent translation using monocistronic constructs. Each experiment was repeated three times. The effect on protein synthesis was calculated as % of luciferase activity/μg of protein relative to the control siRNA. Values represent the mean ± SD. An asterisk denotes statistically significant differences (P = 0.034) between cells treated with the siRNAcontrol or siARF5 RNA.

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  Figure 5.. The mRNA bearing the IRES element is arranged in cytoplasmic clusters. (A) Schematic representation of IRES-luc and cap-luc mRNAs (top), and luciferase activity (RLU/μg protein) in transfected HeLa cells (bottom). Values represent the mean ± SD obtained in triplicate assays. (B) Representative images of RNA-FISH assays conducted with cells transfected side by side with plasmids expressing IRES-luc mRNA, cap-luc mRNA, or pluc (a control plasmid lacking the CMV promoter but containing the luciferase cDNA sequence). Cells were fixed 30 h post-transfection, permeabilized, and incubated with the probe targeting the luciferase-coding region. Cell nucleus was stained with DAPI. White rectangles denote images enlarged on the right panels. (C) Quantification of RNA clusters in cells expressing IRES-luc or cap-luc RNA. The number of RNA spots in single cells (positive luciferase RNA expression) was determined and represented as a percentage of total transfected cells according to their degree of association (≥3 spots in 3 μm). Three independent experiments were conducted. In total, 257 and 162 RNA groups/cell were counted in cells expressing IRES-luc or cap-luc RNA, respectively (P = 3.7 ×…

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  Figure 6.. Juxtaposition of GFP-Rab1b with IRES-containing RNA. (A) Overview of the RNA–protein localization protocol. Representative images of RNA-FISH assays conducted with HeLa cells cotransfected with plasmid expressing GFP-Rab1b (B), or GFP-Rab1b DN (C) and IRES-luc mRNA, or cap-luc mRNA. Triplicate experiments were conducted side by side. The cells were fixed 30 h post-transfection, permeabilized, and incubated with the probe targeting the luciferase-coding region (white signals on the left panels). Cell nucleus was stained with DAPI. White squares denote images enlarged on the right panels (bar = 10 μm; crop image, 5 μm). White arrows denote examples of IRES-luc colocalizing with GFP-Rab1b in transfected cells. Same symbols are used for GFP-Rab1b DN. For completeness, cap-luc RNA is also marked with white arrows. Quantification of the RNA–protein juxtapositioning according to Manders’ coefficient M1 is shown on the right panel (n = 60). P values for Rab1b and Rab1b DN with RNA IRES-luc: P = 0.001 and RNA cap-luc: P = 0.027. P values for IRES-luc and cap-luc in cells cotransfected with Rab1b or Rab1b DN: P = 0.0003, P = 0.470, respectively.

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  Figure S6.. Juxtapositioning of ARF5 with IRES-luc mRNA. (A) Representative images of RNA-FISH conducted with HeLa cells cotransfected side by side with plasmids expressing GFP-ARF5 and IRES-luc mRNA, or GFP-ARF5 and cap-luc mRNA, in triplicate experiments. The cells were fixed 30 h post-transfection, permeabilized, and incubated with the probe targeting the luciferase-coding region (white spots). White rectangles denote images enlarged on the right panels, with and without DAPI (bar = 10 μm overlap image; crop image, 5 μm). White arrows denote examples of IRES-luc or cap-luc RNA signals colocalizing, or not, with GFP-ARF5 in transfected cells. RNA–protein juxtapositioning according to Manders’ coefficient M1 (red signals [RNA] on GFP protein signals) in double-transfected cells is shown on the right panel (n = 60) (P = 0.004). (B) RNA–protein juxtapositioning according to Manders’ coefficient M2 (GFP protein signals overlapping with red signals [RNA]) in double-transfected cells. P values: GFP-Rab1b (P = 0.005), Rab1b DN (P = 0.160), and ARF5 (P = 0.050). (C) Quantification of the GFP-Rab1b overlap with RNA signals in cells transfected with pIRES-luc (n = 277) or pCAP-luc (n = 70).…