Autoregulated Splicing of muscleblind-like 1 (MBNL1) Pre-mRNA

Devika P Gates,Leslie A Coonrod,J Andrew Berglund

doi:10.1074/jbc.m111.236547

Abstract

Muscleblind-like 1 (MBNL1) is a splicing factor whose improper cellular localization is a central component of myotonic dystrophy. In myotonic dystrophy, the lack of properly localized MBNL1 leads to missplicing of many pre-mRNAs. One of these events is the aberrant inclusion of exon 5 within the MBNL1 pre-mRNA. The region of the MBNL1 gene that includes exon 5 and flanking intronic sequence is highly conserved in vertebrate genomes. The 3'-end of intron 4 is non-canonical in that it contains a predicted branch point that is 141 nucleotides from the 3'-splice site and an AAG 3'-splice site. Using a minigene that includes exon 4, intron 4, exon 5, intron 5, and exon 6 of MBNL1, we showed that MBNL1 regulates inclusion of exon 5. Mapping of the intron 4 branch point confirmed that branching occurs primarily at the predicted distant branch point. Structure probing and footprinting revealed that the highly conserved region between the branch point and 3'-splice site is primarily unstructured and that MBNL1 binds within this region of the pre-mRNA. Deletion of the MBNL1 response element eliminated MBNL1 splicing regulation and led to complete inclusion of exon 5, which is consistent with the suppressive effect of MBNL1 on splicing.

Highlights

Splicing factors and small nuclear ribonucleoproteins (U1, U2, U4, U5, and U6) to form the spliceosome, which catalyzes intron removal
The inclusion of exon 5 could be almost completely blocked (8% inclusion; Fig. 1D) by the overexpression of Muscleblind-like 1 (MBNL1) from a co-transfected plasmid. These results show that protein levels of MBNL1 play a significant role in the regulation of MBNL1 exon 5 and that we can recapitulate MBNL1-regulated splicing in this HeLa cell system
Like MBNL1 and MBNL2, this factor autoregulates the splicing of its own pre-mRNA through the usage of a predicted distant branch point [36]

Summary

Introduction

Splicing factors and small nuclear ribonucleoproteins (U1, U2, U4, U5, and U6) to form the spliceosome, which catalyzes intron removal (for a review, see Ref. 3). Intron 4 of MBNL1 contains a predicted distant branch point sequence (TGAT; Fig. 1B, bold text) that is 141 nucleotides upstream of the 3Ј-ss. We showed that the MBNL1 protein can regulate a non-canonical intron by binding a mostly unstructured 90-nucleotide response element within the AGEZ upstream of exon 5.

Results

Conclusion