Identification of novel non-canonical RNA-binding sites in Gemin5 involved in internal initiation of translation

Abstract

Ribonucleic acid (RNA)-binding proteins are key players of gene expression control. We have shown that Gemin5 interacts with internal ribosome entry site (IRES) elements and modulates initiation of translation. However, little is known about the RNA-binding sites of this protein. Here we show that the C-terminal region of Gemin5 bears two non-canonical bipartite RNA-binding sites, encompassing amino acids 1297-1412 (RBS1) and 1383-1508 (RBS2). While RBS1 exhibits greater affinity for RNA than RBS2, it does not affect IRES-dependent translation in G5-depleted cells. In solution, the RBS1 three-dimensional structure behaves as an ensemble of flexible conformations rather than having a defined tertiary structure. However, expression of the polypeptide G51383-1508, bearing the low RNA-binding affinity RBS2, repressed IRES-dependent translation. A comparison of the RNA-binding capacity and translation control properties of constructs expressed in mammalian cells to that of the Gemin5 proteolysis products observed in infected cells reveals that non-repressive products accumulated during infection while the repressor polypeptide is not stable. Taken together, our results define the low affinity RNA-binding site as the minimal element of the protein being able to repress internal initiation of translation.

Figure 1.

Identification of the IRES-binding site in Gemin5. (A) Schematic of HIS-tagged polypeptides used in RNA-binding assays. Numbers indicate amino acid residues referred to the full-length protein. Grey ovals depict WD motifs located within the N-terminal region of the protein. A triangle within the C-terminal region of the protein depicts the position of the epitope recognized by Gemin5 antibody. (B) UV-crosslinking (UV-XL) assay conducted with increasing amounts of purified HIS-tagged Gemin5 polypeptides depicted at the top and radiolabelled domain 5, fractionated in SDS-PAGE and visualized by autoradiography. In each case, the mobility of the protein detected by WB using anti-Gemin5 or Coomassie Blue staining of the purified protein is shown on the right. Mobility of Mw markers is indicated at the left. (C) Gemin5-RNA binding assay. Autoradiograph of denaturing 6% acrylamide gels, 7 M Urea loaded with RNAs isolated from Ni-agarose beads coupled to the indicated proteins (0.07 pmol). D5 and RNAc are used for radiolabelled domain 5 and total cytoplasmic RNA, respectively.

Figure 2.

Determination of the RNA-binding capacity of Gemin5 truncated polypeptides. (A) Gel-shift assay conducted with increasing amounts of purified His-tagged Gemin5 polypeptides depicted at the top and radiolabelled domain 5 RNA, fractionated in native gels and visualized by autoradiography. (B) Gemin5 C-terminal region structure prediction. Jpred3 program (Jnet version: 2.2, www.compbio.dundee.ac.uk) was used for the secondary structure prediction analysis. Helical prediction scores below 7 are shown in light grey and scores between 8 and 9 in dark grey. High scores represent higher probability of adopting a helical conformation. Coil-coiled regions are displayed as a grey thick line and labelled in white Cs. Blue and brown boxes represent the boundaries of the two regions analyzed; dark brown box depicts the overlapping sequence.

Figure 3.

(A) Schematic of Gemin5 polypeptides expressed in transfected cells. Numbers indicate amino acid residues referred to in the text. Ovals depict WD motifs located within the N-terminal region of the protein. A triangle within the C-terminal region of the protein depicts the position of the epitope recognized by Gemin5 antibody. (B) Analysis of the optimal time of expression of Gemin5 polypeptides in HEK293 cells. Plasmids expressing the indicated proteins were transfected in HEK293 cells; transfected (+) and mock-transfected (−) cells were harvested at the indicated time (h) in lysis buffer. Equal amount of total protein was loaded in SDS-PAGE (8% G5_845–1436, and G5_845–1508, 15% G5_1287–1508, G5_1383–1508) and immunoblotted with anti-Gemin5 antibody. Arrows depict the position of the full-length Gemin5 (G5) as well as the expressed proteins. Asterisks depict unspecific products. (C) Expression of G5_1297–1412 determined by anti-Xpress WB; loading control was assessed by anti-tubulin. (D) Ratio of the level of expression of Gemin5 polypeptides relative to the amounts of intracellular Gemin5 protein, detected by WB on the same membrane by anti-Gemin5 antibody. For G5_1297–1412 (which does not contain the anti-G5 epitope) the intensity of bands detected with anti-Xpress was normalized to the intensity observed with the same antibody in a parallel experiment with G5_1287–1508, blotted on the same membrane.

Figure 4.

Effect of Gemin5 depletion on protein synthesis. (A) WB analysis of HEK293 cell extracts transfected with siRNA G5.3 targeted to Gemin5 or a control siRNA with no target sequence in mammalian mRNAs. Tubulin was used as loading control. (B) Gemin5-depleted cells were used to monitor IRES- and cap-dependent translation upon transfection with equal amounts of the bicistronic plasmid (diagram at the top). Each experiment was repeated at least three times in duplicate wells. Effect on protein synthesis was calculated as the% of CAT (striped bars) and LUC (black bars) values observed in the control siRNA. Values represent the mean ± SD. (***P < 0.005).

Figure 5.

Differential effect of Gemin5 C-terminal truncated polypeptides on IRES activity. (A) Diagram of the silencing and co-transfection assay with indication of harvesting time (h), depending on the expressed Gemin5 construct. Effect of expression of G5_1287–1508 (B), G5_1297–1412 (C) and G5_1383–1508 (D) on IRES-dependent translation (striped bars) monitor by LUC activity, relative to the values observed in control siRNA treated cells (empty bars). Black bars depict the effect of Gemin5 silencing on IRES-dependent translation. Values represent the mean ± SD (***P < 0.005; **P < 0.01). A Gemin5 WB (top panel) shows the silencing (Gemin5, p170 band) and the expression of the indicated G5 construct separated on 15% SDS-PAGE. Tubulin is used as loading control. Mobility of MW markers is indicated at the left of each WB.

Figure 6.

Effect of G5_845–1508 and G5_845–1436 polypeptides on IRES activity. (A) Diagram of the silencing and co-transfection assay with indication of harvesting time (h). Effect of expression of G5_845–1508 (B) and G5_845–1436 (C) on IRES-dependent translation (striped bars) monitor by LUC activity, relative to the values observed in control siRNA treated cells (empty bars). Black bars depict the effect of Gemin5 silencing on IRES-dependent translation. Values represent the mean ± SD (***P < 0.005; **P < 0.01). A Gemin5 WB (top panel) shows the silencing (Gemin5, p170 band) and the expression of the indicated G5 construct separated on 8% SDS-PAGE. Tubulin is used as loading control. Mobility of MW markers is indicated at the left of each WB.

Figure 7.

Mutational analysis of L protease cleavage site on Gemin5. (A) Amino acid sequence alignment of candidate L protease recognition motif near the C-terminal sequence of Gemin5 (G5-II) with known L^pro substrates (G5-p85, L-VP4 FMDV polyprotein and Daxx). (B) Diagram of the double amino acid substitutions introduced in the wild-type Gemin5. A dot in the sequence alignment is used to indicate no change with respect to the wild-type sequence. Plasmids expressing G5_{1289–1508FLAG} (WT) (p40) or the substitution mutants (carrying substitutions indicated at the top) in IRES-dependent manner were co-transfected with increasing amounts of a plasmid expressing the FMDV L protease in BHK-21 cells. Cell extracts were analysed by 12% SDS-PAGE and WB using Gemin5 antibody. A slight decrease in the mobility of the mutated proteins was observed. Arrows indicate the position of L-induced cleavage product (p25), which was detected in the WT sequence loaded in parallel.

Figure 8.

Hypothesized model for the differential effect of Gemin5 truncated polypeptides on translation control. The C-terminal region of Gemin5 starting at residue 845 is shown in pale blue. Within this region, dark blue barrels depict the RNA-binding sites RBS1 (diamonds) and RBS2 (wavy line), respectively. The striped rectangle located between these sites depicts the overlapping region of the polypeptides analysed in this study, which was partially deleted in construct G5_{Δ1365–1394}. Arrows depict the RKAR (840) and TKRL (1435) recognition sites of the FMDV L protease. Domain 5 of the FMDV IRES is shown as a red or orange hairpin, indicating the higher or lower efficiency of binding to the Gemin5 polypeptides based on gel-shift or UV-crosslinking assays. In this model, we hypothesize that the IRES RNA binds to RBS2 on the most C-terminal fragment G5_1383–1508 and the full-length Gemin5 protein reducing translation efficiency, while binding of the IRES RNA to RBS1 in proteins G5_845–1508, G5_1287–1508 and G5_1297–1412 does not adversely affect internal initiation of translation. Details are provided in the text.