Interaction of musleblind, CUG-BP1 and hnRNP H proteins in DM1-associated aberrant IR splicing
2006; Springer Nature; Volume: 25; Issue: 18 Linguagem: Inglês
10.1038/sj.emboj.7601296
ISSN1460-2075
AutoresSharan Paul, Warunee Dansithong, Dongho Kim, John J. Rossi, Nicholas J. G. Webster, Lucio Comai, Sita Reddy,
Tópico(s)RNA Research and Splicing
ResumoArticle31 August 2006free access Interaction of musleblind, CUG-BP1 and hnRNP H proteins in DM1-associated aberrant IR splicing Sharan Paul Sharan Paul Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Warunee Dansithong Warunee Dansithong Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Dongho Kim Dongho Kim Division of Molecular Biology, City of Hope, Duarte, CA, USA Search for more papers by this author John Rossi John Rossi Division of Molecular Biology, City of Hope, Duarte, CA, USA Search for more papers by this author Nicholas JG Webster Nicholas JG Webster Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Lucio Comai Corresponding Author Lucio Comai Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Sita Reddy Corresponding Author Sita Reddy Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Sharan Paul Sharan Paul Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Warunee Dansithong Warunee Dansithong Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Dongho Kim Dongho Kim Division of Molecular Biology, City of Hope, Duarte, CA, USA Search for more papers by this author John Rossi John Rossi Division of Molecular Biology, City of Hope, Duarte, CA, USA Search for more papers by this author Nicholas JG Webster Nicholas JG Webster Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, CA, USA Search for more papers by this author Lucio Comai Corresponding Author Lucio Comai Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Sita Reddy Corresponding Author Sita Reddy Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA Search for more papers by this author Author Information Sharan Paul1, Warunee Dansithong1, Dongho Kim2, John Rossi2, Nicholas JG Webster3, Lucio Comai 1 and Sita Reddy 1 1Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA 2Division of Molecular Biology, City of Hope, Duarte, CA, USA 3Department of Medicine, Division of Endocrinology and Metabolism, University of California, San Diego, La Jolla, CA, USA *Corresponding authors: Institute for Genetic Medicine (IGM), Keck School of Medicine, University of Southern California, Room 240, 2250 Alcazar Street, Los Angeles, CA 90033, USA. Tel.: +1 323 442 2457/3950; Fax: +1 323 442 2764; E-mails: E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2006)25:4271-4283https://doi.org/10.1038/sj.emboj.7601296 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In myotonic dystrophy (DM1), both inactivation of muscleblind proteins and increased levels of CUG-BP1 are reported. These events have been shown to contribute independently to aberrant splicing of a subset RNAs. We demonstrate that steady-state levels of the splice regulator, hnRNP H, are elevated in DM1 myoblasts and that increased hnRNP H levels in normal myoblasts results in the inhibition of insulin receptor (IR) exon 11 splicing in a manner similar to that observed in DM1. In normal myoblasts, overexpression of either hnRNP H or CUG-BP1 results in the formation of an RNA-dependent suppressor complex consisting of both hnRNP H and CUG-BP1, which is required to maximally inhibit IR exon 11 inclusion. Elevated levels of MBNL1 show RNA-independent interaction with hnRNP H and dampen the inhibitory activity of increased hnRNP H levels on IR splicing in normal myoblasts. In DM1 myoblasts, overexpression of MBNL1 in conjunction with si-RNA mediated depletion of hnRNP H contributes to partial rescue of the IR splicing defect. These data demonstrate that coordinated physical and functional interactions between hnRNP H, CUG-BP1 and MBNL1 dictate IR splicing in normal and DM1 myoblasts. Introduction Myotonic dystrophy 1 (DM1) is a multisystem disorder characterized by skeletal muscle disease, cardiac conduction disorders, endocrine dysfunction, which includes insulin resistance and psychiatric disease (Harper, 1989). The genetic defect in DM1 is a CTG repeat expansion, which is located in the 3′untranslated region of a protein kinase, DMPK, and found immediately 5′ of a homeo-domain encoding gene, SIX5 (Brook et al, 1992; Fu et al, 1992; Mahadevan et al, 1992; Boucher et al, 1995). Repeat expansion results both in the aggregation of the mutant DMPK RNA into abnormal nuclear foci and in decreased DMPK and SIX5 levels (Fu et al, 1993; Taneja et al, 1995; Klesert et al, 1997; Thornton et al 1997). Functional inactivation of Dmpk and Six5 in mice demonstrates that decreased levels of either gene results in a unique subset of DM1 features (Reddy and Paul, 2006). These data support the hypothesis that decreased DMPK and SIX5 levels contribute to DM1 pathology. Importantly, several lines of evidence demonstrate that expression of expanded CUG repeats per se plays an important role in the development of DM1 (Mankodi et al, 2000; Liquori et al, 2001; Seznec et al, 2001). Although, the mechanism of CUG RNA toxicity has yet to be completely elucidated, altered splice patterns in a subset of physiologically important RNAs appears to be an important pathological consequence of expanded CUG repeat expression in DM1 cells (Philips et al, 1998; Savkur et al, 2001; Buj-Bello et al, 2002; Charlet-B et al, 2002; Mankodi et al, 2002; Kimura et al, 2005). The toxicity of CUG repeats has been hypothesized to result both from the abnormal sequestration of physiologically important proteins by the expanded CUG repeats and by downstream signaling events, which may alter the levels or function of one or more proteins involved in RNA processing. To date, the muscleblind (MBNL) family of proteins consisting of MBNL1, MBNL2 and MBNL3 have been shown to be sequestered by the expanded CUG repeats and are therefore functionally inactivated in DM1 cells (Miller et al, 2000; Fardaei et al, 2002). MBNL1 and MBNL2 are expressed at relatively high levels in muscle; however, MBNL3 expression is restricted primarily to the placenta (Fardaei et al, 2002). Other data demonstrate that level of the CUG-binding protein (CUG-BP1), the prototype of the CELF family of splicing regulators, is elevated in DM1 myoblasts as a consequence of CUG repeat expression (Timchenko et al, 1996, 2001; Ladd et al, 2001). Importantly, inactivation of MBNL1 or overexpression CUG-BP1 has been shown to result in identical splicing defects in a subset of RNAs (Kanadia et al, 2003; Ho et al, 2004, 2005; Dansithong et al, 2005). However, the numbers of splice regulators that serve to set the aberrant splice patterns in DM1 cells and their possible interactions in normal and DM1 cells are currently unknown. In a previous study, Kim et al (2005) have demonstrated that the splice regulator, hnRNP H, binds in conjunction with a docking protein to both the CUG repeats and a splicing branch point in the DMPK RNA in vitro. In this study, we show that the steady-state level of hnRNP H is elevated in DM1 myoblasts. Increased hnRNP H levels are not causally related to the sequestration and loss of function of the muscleblind proteins in DM1 myoblasts. We demonstrate that elevated hnRNP H levels in normal myoblasts results in altered insulin receptor (IR) exon 11 splicing in a manner that duplicates the defects observed in DM1 myoblasts. These results therefore show that elevated hnRNP H levels contribute to aberrant IR splicing in DM1 myoblasts. Suppression of IR exon 11 splicing by elevated hnRNP H levels requires endogenous CUG-BP1 levels and conversely elevated levels of CUG-BP1 requires endogenous hnRNP H levels for maximum suppression of IR exon 11 inclusion. As both hnRNP H and CUG-BP1 demonstrate RNA-dependent interaction both in vivo and in vitro, these data demonstrate that elevated levels of either hnRNP H or CUG-BP1 results in the formation of an RNA-dependent suppressor complex that is required to maximally inhibit IR exon 11 inclusion in normal myoblasts. We further show that elevated levels of MBNL1 partially rescues the aberrant IR splice pattern resulting from the overexpression of hnRNP H in normal myoblasts. As our results demonstrate that MBNL1 binds to hnRNP H in an RNA-independent manner in normal myoblasts and effectively recruits hnRNP H to intranuclear CUG foci in DM1 myoblasts, these data are consistent with the hypothesis that MBNL1 physically binds to and dampens the inhibitory effect of elevated hnRNP H on IR splicing in normal myoblasts. Consistent with results obtained in normal myoblasts overexpression of MBNL1 in conjunction with siRNA-mediated inactivation of hnRNP H results in a partial rescue of the IR splicing defect in DM1 cells. Taken together, these results demonstrate that finely coordinated physical and functional interactions between hnRNP H, CUG-BP1 and MBNL1 serve to regulate aberrant IR splicing in DM1 myoblasts. Results Steady-state hnRNP H levels are elevated in DM1 myoblasts To test the possible role of hnRNP H in DM1 muscle disease, we measured the levels of hnRNP H in both normal and DM1 myoblasts. We observe that steady-state levels of hnRNP H are ∼2.3- and ∼2.1-fold elevated in two DM1 myoblast lines when compared to the two normal myoblast lines (Figure 1A and E). A similar increase in CUG-BP1 levels (∼2.4- and ∼1.9-fold higher) was observed in the two DM1 myoblast lines studied when compared to normal myoblast lines (Figure 1A and E). The levels of other RNA regulatory proteins including hnRNP A1, SF2/ASF and polyadenylate binding protein nuclear 1 (PABPN1) were measured in both normal and DM1 myoblasts. The steady-state levels of hnRNP A1, SF2/ASF and PABPN1 were however not altered in DM1 myoblasts when compared to normal myoblasts (Supplementary Figure S1). As myoblast lines can be contaminated by fibroblasts, in other experiments, we transduced normal ((CTG)17) and DM1 ((CTG)167 and (CTG)667) fibroblasts with adenoviral vectors expressing both MyoD and green fluorescent protein (GFP) in order to confer a skeletal muscle cell fate on these cells. Fluorescent microscopic observation of fibroblasts 24 h after transduction demonstrated that ∼95% (±5%) of the transduced cells expressed GFP (data not shown). This second approach therefore ensures the homogeneity of the cells that are being examined. Endogenous hnRNP H and CUG-BP1 levels were elevated in DM1 fibroblasts when compared to normal fibroblasts transduced with MyoD expressing viruses. Specifically, both hnRNP H and CUG-BP1 levels increased in a CTG tract length dependent fashion showing ∼1.5- and ∼2.1-fold (hnRNP H) and ∼1.7- and ∼2.5-fold (CUG-BP1) increase in MyoD transduced DM1 fibroblasts encoding CTG167 and CTG667, respectively, when compared to control fibroblasts encoding CTG17 repeats (Figure 1B and E). Figure 1.Steady-state hnRNP H levels are elevated in DM1 myoblasts by mechanisms unlinked to MBNL1 and MBNL2 loss. (A) Endogenous hnRNP H levels were measured in 10 μg of total protein from two normal and two DM1 myoblasts lines by Western blot analyses. The levels of hnRNP H in the DM1 myoblasts lines tested are ∼2.3- and ∼2.1-fold higher than that observed in normal myoblasts. CUG-BP1 levels in the DM1 myoblasts lines are ∼2.4- and ∼1.9-fold higher than that observed in the normal myoblast lines (n=3). (B) Fibroblasts containing 17, 167 and 667 CTG repeats were transduced with adenoviral vectors expressing both MyoD and GFP. Fluorescence microscopy study of fibroblasts 24 h after transduction demonstrated that ∼95% (±5%) of the transduced cells expressed GFP (data not shown). The levels of hnRNP H in the transduced DM1 fibroblasts were ∼1.5 fold (CTG)167 and ∼2.1 fold (CTG)667 elevated when compared with normal transduced fibroblasts (CTG)17 repeats (n=3). CUG-BP1 levels in the transduced DM1 fibroblasts were ∼1.7-fold (CTG)167 and ∼2.5-fold (CTG)667 higher than that observed in the normal transduced fibroblasts (CTG)17 repeats (n=3). (C, D) siRNA-mediated depletion of MBNL1 and MBNL2 in normal myoblasts does not result in increased steady state hnRNP H RNA or protein levels. Northern blot analyses demonstrate that MBNL1 or MBNL2 were ∼95 and ∼93% silenced, respectively. hnRNP H RNA and protein levels in normal myoblasts 5 days after transfection with siRNA directed against MBNL1 and MBNL2 were measured by Northern blot and Western blot analyses respectively. In both cases, the membranes were stripped and re-probed for GAPDH RNA and protein in parallel as an internal control. (E) Relative steady-state levels of hnRNP H and CUG-BP1 in DM1 cells when compared to normal controls is shown. Download figure Download PowerPoint Elevated hnRNP H levels in DM1 myoblasts is unlinked to the functional inactivation of MBNL1 and MBNL2 The muscleblind proteins, MBNL1 and MBNL2, are functionally inactivated in DM1 cells due to their aberrant sequestration by the expanded CUG repeats located in the mutant DMPK RNA. To test if elevated hnRNP H levels result as a consequence of the functional inactivation of MBNL1 and MBNL2 in DM1 cells, we measured the steady-state levels of hnRNP H in normal myoblasts in which MBNL1 and MBNL2 mRNA levels were depleted by the cognate siRNAs. In these experiments, the steady-state RNA and protein levels of hnRNP H were not elevated as a consequence of reduced MBNL1 or MBNL2 levels (Figure 1C and D). Thus, the increased steady-state levels of hnRNP H in DM1 myoblasts do not result from the inactivation of MBNL1 and MBNL2. These data suggest that increased hnRNP H levels may be a consequence of signaling events occurring downstream of CTG repeat expansion in DM1 myoblasts. Increased hnRNP H levels result in aberrant IR splicing in normal myoblasts The 36 nt exon 11 of the alpha subunit of the IR RNA is alternatively spliced in normal myoblasts, with isoform A (IR-A), in which exon 11 is excluded or isoform B (IR-B), in which exon 11 is included being produced in approximately equal amounts (Figure 2A; Seino and Bell, 1989; Savkur et al, 2001; Dansithong et al, 2005). In contrast, DM1 cells demonstrate preferential exclusion of IR exon 11 (Savkur et al, 2001; Ho et al, 2004; Dansithong et al, 2005). To test the role of hnRNP H in the aberrant splice patterns observed in DM1, we studied the effect of altered hnRNP H dosage on IR exon 11 splicing in normal myoblasts and compared and contrasted these results with those observed for CUG-BP1 and the MBNL proteins. Figure 2.Overexpression of hnRNP H induces abnormal IR splicing in normal human myoblasts. (A) Schematic of the IR genomic sequence encoding exons 10, 11 and 12 is shown. Primers used to amplify the IR-B (167 nt; exon 11 is included) and the IR-A (131 nt; exon 11 is excluded) isoforms are indicated. (B) siRNA-mediated downregulation of hnRNP H does not alter IR splicing in normal myoblasts. Normal myoblasts were transfected with siRNAs directed against hnRNP H, CUG-BP1, MBNL1 and MBNL2, and total RNA was isolated 5 days after transfection and subjected to RT–PCR analysis using IR primers indicated. IR-B and IR-A levels were measured by densitometry analyses and % IR-B was calculated as described in Materials and methods. Levels of IR-B obtained in the experiment shown in (i) are indicated. (ii) siRNA-mediated silencing was studied by analyzing of 10 μg of protein by Western blot analyses for hnRNP H and CUG-BP1. 1.0 μg of mRNA was subjected to Northern blot analyses to measure the silencing achieved for MBNL1 and MBNL2. (C) Overexpression of Flag-hnRNP H results in decreased IR exon 11 splicing. Normal myoblasts were transfected with Flag tagged-MBNL1, MBNL2, hnRNP H and CUG-BP1, and total RNA was isolated 48 h later and subjected to RT–PCR analyses using IR primers as indicated. Levels of IR-B obtained in the experiment shown in (i) are indicated. (ii) Total protein (10 μg) was analyzed by Western blots to measure the relative expression of MBNL1, MBNL2, hnRNP H and CUG-BP1 using the anti-Flag, anti-hnRNP H and anti-CUG-BP1 antibodies, respectively. The blots were stripped and re-probed for GAPDH protein (Western blot) or GAPDH mRNA (Northern blot) expression as an internal control. For the RT–PCR analyses, GAPDH RNA was amplified as an internal control. (D) The results of three independent experiments of altered hnRNP H, CUG-BP1, MBNL1 and MBNL2 dosage on IR exon 11 splicing in normal myoblasts are tabulated. The asterisk (*) represents significant differences from the control (Student's two-tailed t-test; P<0.05). Download figure Download PowerPoint To assess the role of endogenous hnRNP H on IR splicing, we decreased the levels hnRNP H, CUG-BP1, MBNL1 and MBNL2 using the cognate siRNAs in normal myoblasts. A reduction in the levels of hnRNP H and CUG-BP1 did not alter the equilibrium of IR exon 11 inclusion in normal myoblasts. In contrast, as previously reported, siRNA-mediated depletion of MBNL1 or MBNL2 resulted in the inhibition of IR exon 11 splicing, with MBNL1 depletion achieving almost complete repression of IR exon 11 inclusion and MBNL2 being slightly less efficient at inhibiting IR exon 11 splicing (Figure 2B and D). The relative levels of IR-B and IR-A were measured by RT–PCR analyses using the primers indicated in Figure 2A. These results demonstrate first, an absolute requirement of MBNL1 for IR exon 11 splicing and second, that endogenous hnRNP H and CUG-BP1 levels do not influence the equilibrium of IR exon 11 inclusion in normal myoblasts. In a mirror image experiment hnRNP H, CUG-BP1, MBNL1 and MBNL2 were overexpressed in normal myoblasts. In these experiments, Flag-MBNL1, Flag-MBNL2, Flag-hnRNP H and Flag-CUG-BP1 were transfected into normal myoblasts and 48 h later the relative levels of IR-A and IR-B were measured by RT–PCR. Importantly, we observe that the percent of IR-B produced is significantly lower in normal myoblasts that express either ∼2.3-fold higher levels of hnRNP H (∼14.1%; P=0.0001) or ∼2.4-fold higher levels of CUG-BP1 (∼10.3%; P=0.0001) when compared to untransfected controls (∼46.6%) (Figure 2C and D). Inhibition of IR exon 11 inclusion achieved by hnRNP H was in the same range achieved by CUG-BP1, although overexpression of Flag-CUG-BP1 was slightly more efficient at inhibiting IR exon 11 splicing. Significantly, overexpression of the MBNL proteins was not sufficient to increase the efficiency of IR exon 11 splicing. Thus, these data demonstrate that the aberrant IR splice pattern in DM1 myoblasts can be recapitulated in normal myoblasts when either hnRNP H or CUG-BP1 levels are increased to approximate those observed in DM1 myoblasts. Both hnRNP H and CUG-BP1 are required to maximally inhibit IR exon 11 splicing As elevated levels of either hnRNP H or CUG-BP1 result in the reduction of IR-B levels in normal myoblasts, we tested if both proteins act in a coordinate fashion to suppress IR splicing in normal myoblasts. Thus, in a first set of experiments, we simultaneously down regulated CUG-BP1 levels by ∼97% using siRNAs and overexpressed hnRNP H in normal myoblasts. Significantly, the reduction in IR-B levels observed resulting from elevated hnRNP H levels (∼14.5%) was abolished in normal myoblasts that overexpress hnRNP H when endogenous CUG-BP1 levels are depleted (∼42.3%) (Figure 3A and C). In these experiments, a small increment (∼10–20%) in endogenous CUG-BP1 levels was observed when hnRNP H was overexpressed in normal myoblasts (Figure 3A(ii)). Figure 3.Abnormal IR splicing achieved by the overexpression of hnRNP H and CUG-BP1 in normal myoblasts requires endogenous levels of CUG-BP1 and hnRNP H. (A) Normal myoblasts were first transfected with siRNAs directed against CUG-BP1 and 3 days later the cells were re-transfected with vector or vector expressing Flag-hnRNP H. Following a 48-h incubation the cells were harvested for analyses. In parallel, normal myoblast cultures were transfected with Flag-hnRNP H and harvested 2 days post-transfection. In all cases, the harvested cells were split into two aliquots. From one aliquot, total RNA was isolated and subjected to RT–PCR analysis using IR primers. GAPDH RNA was amplified in parallel as an internal control. Levels of IR-B obtained in the experiment shown in (i) are indicated. (ii) Total protein (10 μg) from the second aliquot was analyzed by Western blots to measure the silencing achieved for CUG-BP1 using anti-CUG-BP1 mab staining. Relative levels of hnRNP H were measured using anti-hnRNP H or anti-Flag antibodies. (B) Normal myoblasts were first transfected with siRNAs directed against hnRNP H and 3 days later the cells were re-transfected with vector or vector expressing Flag-CUG-BP1. Following a 48-h incubation the cells were harvested for analyses. In parallel, normal myoblast cultures were transfected with Flag-CUG-BP1 and harvested 2 days post-transfection. Total RNA was isolated from harvested cells and subjected to RT–PCR analysis using IR and GAPDH primers (internal control). Levels of IR-B obtained in the experiment are shown in (i). (ii) Silencing achieved for hnRNP H and the relative levels of CUG-BP1 measured by Western blots using anti-hnRNP H, anti-CUG-BP1 and anti-Flag antibodies are shown. The blots were re-probed for GAPDH protein using anti-GAPDH polyclonal antibodies as a loading control. (C) Results from three independent experiments are tabulated. The asterisk (*) represents significant differences from the control (Student's two-tailed t-test; P<0.05). Download figure Download PowerPoint In a second set of experiments, IR splicing was studied in normal myoblasts in which CUG-BP1 was overexpressed in conjunction with siRNA-mediated silencing of hnRNP H (silencing achieved for hnRNP H was ∼83%). In these experiments, we observed that the suppression of exon 11 inclusion was not as effective when CUG-BP1 was overexpressed in conjunction with the downregulation of endogenous hnRNP H levels (∼22.6%) when compared to the overexpression of CUG-BP1 alone (∼11.6%) (Figure 3B and C). Overexpression of CUG-BP1 did not result in altered steady-state hnRNP H levels in normal myoblasts (Figure 3B(ii)). These data demonstrate that both hnRNP H and CUG-BP1 are required to maximally suppress IR exon 11 splicing in normal myoblasts. hnRNP H and CUG-BP1 form an RNA-dependent suppressor complex in normal myoblasts To test if hnRNP H and CUG-BP1 interact in vivo, normal myoblasts were transduced with recombinant adenoviral vectors designed to express either Flag-CUG-BP1 or GFP. To detect both RNA-dependent and -independent interactions, extracts from normal myoblasts cultures overexpressing Flag-CUG-BP1 and GFP were first incubated with the anti-Flag antibody beads. Subsequently, the immunoprecipitates were divided into two aliquots, which were treated either with or without RNAse A as described in Materials and methods. After extensive washing, the bound proteins were eluted by competition with the Flag peptide, and the eluted proteins were immunoblotted and stained with Flag and hnRNP H antibodies, respectively. Immunoprecipitation experiments were carried out in triplicate and representative panels are shown in Figure 4. Expression of Flag-CUG-BP1 was confirmed by immunostaining with the anti-Flag monoclonal antibody (mab) (Figure 4A). In parallel, the eluted proteins were immunostained with anti-hnRNP H antibodies (Figure 4B). Endogenous hnRNP H co-immunoprecipitated with Flag-CUG-BP1 in RNase-free immunoprecipitates. Interaction between CUG-BP1 and hnRNP H was however lost upon RNAse A treatment. To verify the specificity of the RNA-dependent interaction observed between CUG-BP1 and hnRNP H, we studied the CUG-BP1 immunoprecipitates by Western blot analyses using antibodies directed against hnRNP A1, SF2/ASF and PABPN1. hnRNP A1, SF2/ASF and PABPN1 did not co-immunoprecipitate with Flag-CUG-BP1 either under non-RNAse or RNAse treatment conditions (Supplementary Figure S4). Thus, these data are consistent with the formation of an RNA-dependent suppressor complex consisting of both hnRNP H and CUG-BP1 in normal myoblasts that overexpress CUG-BP1. Figure 4.hnRNP H interacts with CUG-BP1 in RNA-dependent manner in vivo. Normal myoblasts were transduced with recombinant adenoviruses expressing Flag-CUG-BP1 or GFP. At 48 h post infection, total cell extracts were prepared and incubated with anti-Flag beads to immunoprecipitate proteins under non-RNAse and RNAse treatment conditions as described in Materials and methods. The eluted proteins from each immunoprecipitation were analyzed by Western blot staining with anti-Flag mab (A), anti-hnRNP H polyclonal antibodies (B). Staining with anti-Flag mab demonstrates the precipitation of Flag-CUG-BP1 (A). (B) Staining with anti-hnRNP H polyclonal antibodies demonstrates that hnRNP H co-immunoprecipitates with Flag-CUG-BP1 when the immunoprecipitates are not treated with RNAse. Co-immunoprecipitation of hnRNP H with Flag-CUG-BP1 does not occur when immunoprecipitates are treated with RNAse. Download figure Download PowerPoint Overexpression of MBNL1 partially rescues the IR splicing defect resulting from elevated levels of hnRNP H in normal myoblasts We next tested if elevated levels of MBNL1 can rescue the IR splicing defect resulting from the overexpression of hnRNP H in normal myoblasts. Thus MBNL1 was overexpressed in conjunction with hnRNP H in normal myoblasts and the relative levels of IR-B and IR-A was measured by RT–PCR analyses. The percent IR-B produced was significantly higher in normal myoblasts which overexpress both Flag-MBNL1 and Flag-hnRNP H (∼21.3%), when compared with myoblasts that overexpress Flag-hnRNP H alone (∼11.9%) (Figure 5A and B). Thus, overexpression of MBNL1 can partially rescue the inhibitory effect resulting from elevated levels of hnRNP H on IR exon 11 inclusion in normal myoblasts. As overexpression of MBNL1 does not increase the amount of IR-B produced in normal myoblasts (Figure 5A and B), these data suggest that MBNL1 may partially repress the inhibitory activity of elevated hnRNP H levels on IR exon 11 inclusion by physical interaction. Figure 5.Overexpression of MBNL1 partially rescues the IR splicing defect resulting from elevated levels of hnRNP H in normal myoblasts and MBNL1 interacts with hnRNP H in an RNA-independent manner in vivo. (A) Normal myoblasts were transfected with Flag-MBNL1, Flag-hnRNP H or Flag-MBNL1 and Flag-hnRNP H in combination. Total RNA was isolated 48 h post-transfection and subjected to RT–PCR analyses using IR primers. GAPDH RNA was amplified in parallel as an internal control. Levels of IR-B obtained in the experiment shown in (i) are indicated. (ii) Total protein (10 μg) was analyzed by Western blots to measure the levels of expressed MBNL1 and hnRNP H using anti-Flag and anti-hnRNP H antibodies, respectively. The blots were re-probed for GAPDH as a loading control. (B) The results of three independent experiments are tabulated. The asterisk (*) represents significant differences from the control (Student's two-tailed t-test; P<0.05). (C) Normal myoblasts were transduced with recombinant adenoviruses expressing Flag-MBNL1 or GFP. At 48 h postinfection, total cell extracts were prepared and incubated with anti-Flag beads to immunoprecipitate proteins under non-RNAse and RNAse treatment conditions. The eluted proteins from each immunoprecipitation were analyzed by Western blot staining with anti-Flag mab (i), anti-hnRNP H polyclonal antibodies (ii), and anti-CUG-BP1 mab (iii). Staining with anti-Flag mab demonstrates the precipitation of Flag-MBNL1 (i). (ii) Staining with anti-hnRNP H polyclonal antibodies demonstrates that endogenous hnRNP H co-immunoprecipitates with Flag-MBNL1 both under non-RNAse and RNAse treatment conditions. (iii) CUG-BP1 mab staining demonstrates that CUG-BP1 co-immunoprecipitates with Flag-MBNL1 only under non-RNAse treatment conditions. Download figure Download PowerPoint MBNL1 and hnRNP H demonstrate RNA-independent interaction in vivo To test if MBNL1 interacts with hnRNP H in vivo, normal myoblasts were transduced with adenoviral vectors expressing Flag-MBNL1 or GFP. Flag antibodies were used to immunoprecipitate MBNL1 and the interaction of endogenous hnRNP H with flag-MBNL1 was tested under both RNase A and RNAse A free treatment conditions as described in Materials and methods. Immunoprecipitation experiments were carried out in triplicate and the representative panels are shown in Figure 5C. Expression of Flag-MBNL1 was confirmed by immunostaining with the anti-Flag mab (Figure 5C(i)). RNA-independent interaction between Flag-MBNL1 and hnRNP H and RNA-dependent interaction between Flag-MBNL1 and CUG-BP1 was observed in these experiments (Figure 5C(ii) and (iii)). As MBNL1 interacts with hnRNP H in an RNA-independent manner these data support the hypothesis that MBNL1 physically interacts with hn
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