Interaction of muscleblind, CUG-BP1 and hnRNP H proteins in DM1-associated aberrant IR splicing - PubMed (original) (raw)

Interaction of muscleblind, CUG-BP1 and hnRNP H proteins in DM1-associated aberrant IR splicing

Sharan Paul et al. EMBO J. 2006.

Abstract

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.

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Figures

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.

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).

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).

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.

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.

Figure 6

Overexpression of MBNL1 and MBNL2 increases recruitment of hnRNP H to DM1 foci. Normal myoblasts expressing GFP-MBNL1 (A–C) and DM1 myoblasts expressing GFP (D–F), GFP-MBNL1 (G–I), GFP-MBNL2 (J–L), GFP-hnRNP H (P–R), or co-expressing GFP-hnRNP H and Flag-MBNL1 (S–U) or GFP-hnRNP H and Flag-MBNL2 (V–X) are shown. Distribution of endogenous hnRNP H (M–O) was studied using anti-hnRNP H polyclonal antibodies conjugated with FITC (green signal). GFP tagged proteins and endogenous hnRNP H are visualized as a green signal (A, D, G, J, M, P, S and V). The mutant DMPK transcripts encoding the expanded CUG tract was detected by hybridization with a (CAG)10-Cy3 probe (red signal: B, E, H, K, N, Q, T and W). Transcripts containing expanded repeat were not observed in the normal myoblasts (B). Merged images (C, F, I, L, O, R, U and X) where super-imposition of green and red signals are observed as yellow signals demonstrate that GFP-MBNL1 (I), GFP-MBNL2 (L) and GFP-hnRNP H co-expressed with Flag-MBNL1 (U) or Flag-MBNL2 (X) co-localize with the mutant DMPK RNA in DM1 myoblasts. However, GFP alone in DM1 myoblasts (F), endogenous hnRNP H (O) and GFP-hnRNP H (R) did not co-localize significantly with the mutant DMPK RNA. The percent of foci that co-localize with each protein are tabulated in Table I. The asterisk (*) represents significant differences from GFP (Student's two-tailed _t_-test; P<0.05).

Figure 7

Overexpression of MBNL1 in conjunction with decreased hnRNP H levels partially rescues aberrant IR splicing in DM1 myoblasts. (A) DM1 myoblasts were transfected with siRNAs directed against hnRNP H or CUG-BP1 or hnRNPH and CUG-BP1 and total RNA isolated 5 days post-transfection was subjected to RT–PCR analysis using IR primers. Levels of IR-B obtained in the experiment shown in (i) are indicated. (ii) Total protein (10 μg) was analyzed by Western blot to measure the silencing achieved for hnRNP H and CUG-BP1 using anti-hnRNP H and anti-CUG-BP1 antibodies, respectively. (B) DM1 myoblasts were transfected with siRNAs directed against hnRNP H. After 3 days, the culture was transduced with recombinant adenoviruses expressing Flag-MBNL1. At 48 h post-transduction, total RNA was isolated and subjected to RT–PCR analysis and the percentage of IR-B was measured. Levels of IR-B obtained in the experiment shown in (i) are indicated. (ii) Total protein (10 μg) was analyzed by Western blot to measure the silencing achieved for hnRNP H and the levels of Flag-MBNL1 expressed using anti-hnRNP H and anti-Flag antibodies, respectively. The blots were re-probed for GAPDH protein as a loading control. In the RT–PCR analyses, GAPDH RNA was amplified as an internal control. (C) The results from three independent experiments are tabulated. The asterisk (*) represents significant differences from the control (Student's two-tailed _t_-test; P<0.05).

Figure 8

MBNL1, hnRNP H and CUG-BP1 bind directly to human IR-B RNA in vitro. (A) Depicts the schematic of the human IR-B minigene (Kosaki et al, 1998). (B) UV crosslinking experiments were carried out using uniformly 32P-labeled IR-B RNA and 700 ng purified GST or His-MBNL1, His-hnRNP H and His-CUG-BP1. After incubation, reaction mixtures were UV irradiated and digested with RNase T1. The samples were analyzed by SDS–PAGE. The position of each protein-RNA complex is shown by an arrow.

Figure 9

Coordinate regulations of IR splicing in normal and DM1 cells. A model for the regulation of IR exon 11 splicing in normal and DM1 myoblasts by MBNL1, MBNL2, hnRNP H and CUG-BP1 is shown in panels A–E.

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Interaction of muscleblind, CUG-BP1 and hnRNP H proteins in DM1-associated aberrant IR splicing - PubMed (original) (raw)