Identification of MBNL1 and MBNL3 domains required for splicing activation and repression

Abstract

Muscleblind-like 1 (MBNL1) is a splicing regulator that controls developmentally regulated alternative splicing of a large number of exons including exon 11 of the Insulin Receptor (IR) gene and exon 5 of the cardiac Troponin T (cTNT) gene. There are three paralogs of MBNL in humans, all of which promote IR exon 11 inclusion and cTNT exon 5 skipping. Here, we identify a cluster of three binding sequences located downstream of IR exon 11 that constitute the MBNL1 response element and a weaker response element in the upstream intron. In addition, we used sequential deletions to define the functional domains of MBNL1 and MBNL3. We demonstrate that the regions required for splicing regulation are separate from the two pairs of zinc-finger RNA-binding domains. MBNL1 and MBNL3 contain core regulatory regions for both activation and repression located within an 80-amino-acid segment located downstream of the N-terminal zinc-finger pair. Deletions of these regions abolished regulation without preventing RNA binding. These domains have common features with the CUG-BP and ETR3-like Factor (CELF) family of splicing regulators. These results have identified protein domains required for splicing repression and activation and provide insight into the mechanism of splicing regulation by MBNL proteins.

Combinatorial Mutagenesis of MBNL1 Zinc Fingers Elucidates Distinct Classes of Regulatory Events

“…An intriguing possible explanation for this observation is that some MBNL1-mediated splicing events are partially mediated by protein-protein interactions that are important for recruiting MBNL1 to the transcript. Regions outside the ZFs that may be important for mediating such interactions have been recently defined (14). Using RIMs as sensitized backgrounds, we demonstrated that the Q-rich region downstream of ZF2 contributes to ZF3-4 function only in class I events.…”

“…Regions outside the ZFs have recently been hypothesized to be important for MBNL1 splicing activity. Specifically, deletion analysis defined a glutamine-rich region on the C=-terminal end of ZF2 that has been hypothesized to be important for both splicing activation and repression activities (14). The key glutamine (Q) and methionine (M) residues within this region were mutated to alanines to create the MBNL1 Q-mutant (i.e., WT-Q) (Fig.…”

“…Various studies from multiple groups have defined the minimal consensus binding site of MBNL1 as 5=-YGCY-3= (7,13,18). A few functional binding sites within MBNL1-regulated transcripts have been defined, including human cardiac troponin T type 2 (TNNT2, or cTNT) (18,50), human sarcoplasmic/endoplasmic reticulum Ca 2ϩ -ATPase 1 (ATP2A1, or SERCA1) (13,17), human insulin receptor (INSR) (14,42), mouse nuclear factor I/X (Nfix) (7), and mouse verylow-density lipoprotein receptor (Vldlr) (7), as well as sites within the human MBNL1 pre-mRNA (MBNL1, the RNA, will be italicized throughout the text to differentiate from the MBNL1 protein) (12). Yet within these characterized transcripts, no obvious rules for MBNL1 function have been gleamed from the architecture of the MBNL1 binding sites.…”

Rbfox proteins regulate tissue-specific alternative splicing of Mef2D required for muscle differentiation

“…S3). By contrast, the biological activity (Grammatikakis et al, 2011;Hall et al, 2013) and the expression pattern of Ptb family, as well as Mbnl3, suggest that they are putative repressors of Mef2D a2 exon inclusion in myoblasts (supplementary material Fig. S4).…”

Aberrant splicing and expression of the non muscle myosin heavy-chain gene MYH14 in DM1 muscle tissues

“…We then searched specific sequences for splicing regulation around MYH14 exon 6 and found that there are 3 "YGCU(U/G)Y" motifs and 6 "YGCY" sequences in intron 5 and 6 of the gene. These motifs have been shown to directly interact with the MBNL1 protein and mediate the c-TNT and IR gene alternative splicing (Grammatikakis et al, 2010). MBNL1 could therefore regulate the alternative splicing of the MYH14 gene leading to the missplicing observed in DM1 muscle.…”