P68 RNA helicase (DDX5) alters activity of cis- and trans-acting factors of the alternative splicing of H-Ras

Abstract

Background: H-Ras pre-mRNA undergoes an alternative splicing process to render two proteins, namely p21 H-Ras and p19 H-Ras, due to either the exclusion or inclusion of the alternative intron D exon (IDX), respectively. p68 RNA helicase (p68) is known to reduce IDX inclusion.

Principal findings: Here we show that p68 unwinds the stem-loop IDX-rasISS1 structure and prevents binding of hnRNP H to IDX-rasISS1. We also found that p68 alters the dynamic localization of SC35, a splicing factor that promotes IDX inclusion. The knockdown of hnRNP A1, FUS/TLS and hnRNP H resulted in upregulation of the expression of the gene encoding the SC35-binding protein, SFRS2IP. Finally, FUS/TLS was observed to upregulate p19 expression and to stimulate IDX inclusion, and in vivo RNAi-mediated depletion of hnRNP H decreased p19 H-Ras abundance.

Significance: Taken together, p68 is shown to be an essential player in the regulation of H-Ras expression as well as in a vital transduction signal pathway tied to cell proliferation and many cancer processes.

Figure 1. Recently proposed model for the regulation of alternative splicing for H-Ras pre-mRNA by Guil et al. .

A) RasISS1 (blue brackets) is the silencer downstream of the alternative intron D exon (IDX), showed as an yellow exon. Arrow-headed lines indicate protein-RNA interactions. Red factors activate IDX inclusion (SC35 and SRp40); blue factors inhibit IDX inclusion. Concerning to the green factors, only direct/indirect binding to both IDX and rasISS1 sequences has been reported. P68 RNA helicase also showed binding to both IDX and rasISS1 sequences. For clarity, FUS/TLS- and hnRNP H-RNA bindings are separately showed below on the right. B) Detail of the IDX and rasISS1 sequences. “Δ” indicates the region deleted in ΔrasISS1. C) In vitro splicing reaction of 1N pre-mRNA (containing exon3-D1-IDX-rasISS1) activated with 400 ng of recombinant SRp40 (lanes 1–4). For lanes 2, 3 and 4, 100, 200 and 400 ng of recombinant p68 was added, respectively. The “*” indicates that ³²P-labeled RNA was used in the assay. D) Proposed secondary structure of the IDX-rasIS1 sequence as previously published . S. Guil et al. also demonstrated that this structure is conserved in hamster, mouse, rat and humans, and that some silent mutations are compensated by a silent one on the opposite strand across the four species . The “Δ” indicates the region deleted in the ΔrasISS1.

Figure 2. P68 RNA helicase unwinds IDX-rasISS1 in vitro.

A) Winding activity assay between IDX and rasISS1 RNAs. The “*” indicates that ³²P-labeled RNA was used. The other RNAs were added unlabeled (cold). All lanes contain 18 ng IDX. Lane 1: no additional RNA; lanes 2, 3, 4 and 5: 1, 10, 50 and 100 ng cold rasISS1 RNA, respectively; lanes 6, 7, 8 and 9: 1, 10, 50 and 100 ng cold ΔrasISS1, respectively. dsRNA: double-stranded RNA; ssRNA: single-stranded RNA. Samples were run on a 8% (w/v) native polyacrylamide gel. B) Winding activity assay of the IDX-rasISS1 RNA (1 pmol) in the absence (lane 1) or presence of increasing amounts of p68 (100, 200 and 400 ng in lanes 2, 3 and 4, respectively). P68 RNA helicase was added after winding assays followed by 1 h at 37°C. Samples were run on a 8% (w/v) native polyacrylamide gel. C) On the left, binding-shift assay of labeled rasISS1, ΔrasISS1 and IDX RNAs (1 pmol each) with increasing amounts of recombinant GST-fused hnRNP H (GST-H). Lanes 1, 5 and 9 did not contain GST-H; lanes 2–4, 6–8 and 10–12 contained 100, 200 and 400 ng, respectively, of recombinant GST-H. HMW (high molecular weight). On the right, similar experiment with GST-FUS is showed. D) Binding-shift assay of labeled rasISS1 RNA in nuclear extract (lanes 2–8) or without extract (in Roeder D buffer, lanes 9–14). Lane 1 did not contain nuclear extract. To obtain supershift complexes, 0.5, 1 and 2 µl anti-hnRNP H serum (lanes 2, 3 and 4, respectively) and 0.5, 1 and 2 µl anti-FUS (lanes 6, 7 and 8, respectively) were added to the reaction. Lane 5 contained nuclear extract only. Lanes 9–10, 11–12 and 13–14 contain 100, 200 and 400 ng of recombinant GST-H and GST-FUS, respectively. Samples were run on a 6% (w/v) native polyacrylamide native gel. HMW (high molecular weight). E) Pre-winded ³²P-IDX-rasISS1 RNA was incubated with 0.6 and 1.2 µg recombinant p68 (lanes 2 and 3, respectively), with 160, 320 and 640 ng recombinant hnRNP H (lanes 4, 5 and 6, respectively). Lanes 7, 8 and 9 contained 160, 320 and 640 ng recombinant hnRNP H, respectively plus 1.2 µg of recombinant p68. Samples were run on a 6% (w/v) native polyacrylamide gel. F) Crosslinking assay between hnRNP H (640 ng) and ³²P-IDX-rasISS1 RNA (1 pmol) (lanes 2–7). Lane 1 did not contain hnRNP H. Lanes 3–7 contained 100, 200, 400, 800 and 1200 ng p68, respectively. Samples were run on a 10% SDS-polyacrylamide protein gel and autoradiographed.

Figure 3. FUS stimulates IDX inclusion.

Lane 1: In vitro splicing reaction of ³²P-1 N pre-mRNA (containing exon3-D1-IDX-rasISS1); lanes 2, 3 and 4: 65, 130 and 260 ng recombinant GST-H,; lanes 5 and 6: 90 and 180 ng recombinant GST-FUS. Samples were run on a 10% urea denaturing polyacrylamide gel.

Figure 4. RNAi-mediated depletion of FUS and hnRNP H downregulates p19 H-Ras expression.

A) Western blot on HeLa extracts from cells specifically depleted by means of RNAi-mediated depletion of p68 (lane 2), hnRNP A1 (lane 3), FUS/TLS (lane 4) and hnRNP H (lane 5). Lanes 1: HeLa cells transfected with empty pSuper vector (negative control). Antibodies against actine or GAPDH were used as loading controls. B) Western blot on extracts similar as in (A), lane 4, with RNAi-mediated depletion of FUS/TLS (lanes RNAi FUS) as compared to cells transfected with empty vector, lanes (-). Cells were transfected with 6 µg or 4 µg of the pSuper plasmids. Samples were run on 12.5% SDS denaturing polyacrylamide gels. Blots were incubated with anti-p19 and anti-FUS antibodies. Anti-GAPDH was used in all lanes as internal loading control and also as a fixed value of 1 to which all calculations for each lane were standardized. Blots subjected to chemiluminescence (peroxidase-luminol) and then quantified with the imaging software Multi Gauge V3.0 (Fujifilm) using LAS 3000 apparatus (Fujifilm). The RNAi-mediated depletion of FUS/TLS induced a drop on the protein abundance of 52–64% on FUS and 27–35% on p19. C) Western blot on extracts with siRNA-mediated depletion of hnRNP H with a siRNA to hnRNP H (Santa Cruz Biotechnology, sc-35579). Lane (-) contains the siRNA-B control (Santa Cruz Biotechnology, sc-44230). The siRNA depletion of hnRNP H induced a drop on the protein abundance of 50% on both hnRNP H and p19 proteins.

Figure 5. Downregulation of p68 RNA helicase alters the dynamic localization of the SC35 splicing factor.

A) Indirect immunofluorescence detection of HeLa cells containing the pSuper-RNAi-p68 RNA helicase vector (-p68); pSuper-RNAi-A1 vector (-A1); pSuper-RNAi-H vector (-H); pSuper-RNAi-FUS vector (-FUS). As negative controls, cells were transfected with empty pSuper (-). Anti-SC35 primary antibody diluted 1/600 (mouse) and secondary anti-mouse Alexa Fluor® 633 F(ab')₂-labeled antibody . B) Indirect immunofluorescence detection of HeLa cells containing the pSuper-RNAi-p68 RNA helicase vector (-p68) or pSuper empty vector (-) incubated with anti-B” (1/20, mouse), anti-Coilin (1/100, rabbit), anti-SMN (1/1000, mouse) and FITC-labelled anti-PARP (1/100, mouse). Primary mouse and rabbit antibodies were incubated with secondary anti-mouse Alexa Fluor® 633 or 488, respectively, F(ab')₂-labeled antibody. C) Flow cytometric experiments estimating apoptosis levels in HeLa cells transfected either with empty pSuper or pSuper-RNAi-p68 RNA helicase vectors, and stained with annexin V-FITC (Annexin LOG) and propidium iodide (IP LOG). R3 represents % of viable cells and R1+R2 % of dead plus apoptotic cells.