Muscleblind proteins regulate alternative splicing

Abstract

Although the muscleblind (MBNL) protein family has been implicated in myotonic dystrophy (DM), a specific function for these proteins has not been reported. A key feature of the RNA-mediated pathogenesis model for DM is the disrupted splicing of specific pre-mRNA targets. Here we demonstrate that MBNL proteins regulate alternative splicing of two pre-mRNAs that are misregulated in DM, cardiac troponin T (cTNT) and insulin receptor (IR). Alternative cTNT and IR exons are also regulated by CELF proteins, which were previously implicated in DM pathogenesis. MBNL proteins promote opposite splicing patterns for cTNT and IR alternative exons, both of which are antagonized by CELF proteins. CELF- and MBNL-binding sites are distinct and regulation by MBNL does not require the CELF-binding site. The results are consistent with a mechanism for DM pathogenesis in which expanded repeats cause a loss of MBNL and/or gain of CELF activities, leading to misregulation of alternative splicing of specific pre-mRNA targets.

Figure 1

MBNL1, 2 and 3 regulate splicing of cTNT and IR alternative exons. Human and chicken cTNT and human IR minigenes were expressed with or without each of the three GFP–MBNL fusion proteins or with GFP alone. Duplicate transfections were used for extraction of RNA and protein. Inclusion of cTNT exon 5 or IR exon 11 was assayed by RT–PCR. Percent exon inclusion is calculated as ((mRNA+exon)/(mRNA−exon+mRNA+exon)) × 100. Results are derived from at least three independent experiments. Expression of GFP–MBNL1 (∼72 kDa), GFP–MBNL2 (∼58 kDa), GFP–MBNL3 (∼70 kDa) and EGFP (∼27 kDa) was detected by Western blot analysis using an anti-GFP monoclonal antibody. All three MBNL proteins promote exon 5 skipping of (A) chicken and (B) human cTNT exon 5 in primary skeletal muscle cultures. (C) All three MBNL proteins promote exon 11 inclusion in a human IR minigene in HEK293 cells. (D) MBNL proteins have minimal effects on splicing of exon EN in a clathrin light-chain B minigene in primary skeletal muscle cultures.

Figure 2

Endogenous MBNL1 regulates the splicing of human cTNT and IR minigenes. siRNA and minigenes were transfected into HeLa cells. (A) Western blot confirming depletion of endogenous MBNL1 by independent transfection of two different siRNA constructs using the MBNL1 monoclonal (mAb) 3A4, which recognizes two MBNL1 isoforms generated by alternative splicing (∼41 and 42 kDa). GAPDH (∼36 kDa) was used as a loading control. (B) Immunofluorescence using mAb 3A4 to confirm depletion of endogenous protein after independent transfection of each MBNL1 siRNA construct. Scale bar, 10 μm. (C) siRNA-mediated depletion of MBNL1 with two independent constructs reproduces the DM splicing patterns for cTNT and IR minigenes. RT–PCR results are from at least three transfections. GFP siRNA had no effect on splicing of any of the tested minigenes. MBNL1 siRNA had minimal effects on splicing of a rat clathrin light-chain minigene.

Figure 3

MBNL1 binds upstream of exon 5 in human cTNT at a site distinct from the CUG-BP1-binding site. (A) Binding of recombinant GST–MBNL1 to uniformly ³²P-labeled RNA was assayed by UV crosslinking. Scanning mutagenesis was performed by replacing 6 nt blocks with AUAAUA and identified two binding sites 18 and 36 nt upstream of the alternative exon. The MBNL1-binding sites (M) and the CUG-BP1-binding site (C) are located on opposite sides of exon 5. (+) and (−) indicate binding; (•) indicates a putative branch point adenosine. (B) Four nucleotide substitutions significantly reduce binding of recombinant MBNL1 detected by UV crosslinking. Competition of GST–MBNL1 binding to ³²P-labeled RNA G by the indicated picomoles of nonlabeled RNAs G or M shown in A). (C, D) MBNL1-binding site mutations reduce responsiveness to MBNL1, MBNL2 and MBNL3 coexpression but not CUG-BP1 in COSM6 cells. Human cTNT minigenes containing the natural sequence (C) or the four nucleotide substitutions (mutation M in A) in the MBNL1-binding site (D) were coexpressed with GFP or the indicated GFP fusion proteins. Exon inclusion was assayed by RT–PCR.

Figure 4

MBNL1 binds to cis-elements in chicken cTNT intron 5 required for muscle-specific splicing. (A) The chicken cTNT MSE1–4 RNA contains an alternative exon flanked by four MSEs. GST–MBNL1 bound weakly to MSE1 and strongly to MSE4 in UV-crosslinking assays. (B) Competition of GST–MBNL1 binding to labeled chicken cTNT MSE1–4 RNA by nonlabeled MSE RNAs. Picomoles of competitor RNA are indicated. (C) Scanning mutagenesis identified two MBNL1-binding sites within MSE4. (D) Alignment of the four MBNL1-binding motifs in human and chicken cTNT reveals a common motif.

Figure 5

Regulation of human cTNT by MBNL1 is independent of CELF regulation. The (A) wild-type cTNT minigene or a (B) mutant cTNT minigene with point mutations that prevent CUG-BP1 binding and regulation were cotransfected with the indicated siRNA constructs, a plasmid expressing a DMPK minigene with 960 CUG repeats (Philips et al, 1998) or a GFP–MBNL1 expression plasmid in HeLa cells.

Figure 6

Deletion of the human IR CUG-BP1-binding site does not affect regulation by MBNL1. (A) All the three MBNL proteins promote exon 11 inclusion of a mutant human IR minigene lacking the CUG-BP1-binding site in HEK293 cells. (B) RNAi depletion of MBNL1 in HeLa cells using the indicated siRNA constructs promotes exon 11 skipping in a human IR minigene lacking the CUG-BP1-binding site.