Alternative splicing of the neurofibromatosis type 1 pre-mRNA is regulated by the muscleblind-like proteins and the CUG-BP and ELAV-like factors

BACKGROUND: Alternative splicing is often subjected to complex regulatory control that involves many protein factors and cis-acting RNA sequence elements. One major challenge is to identify all of the protein players and define how they control alternative expression of a particular exon in a combinatorial manner. The Muscleblind-like (MBNL) and CUG-BP and ELAV-Like family (CELF) proteins are splicing regulatory proteins, which function as antagonists in the regulation of several alternative exons. Currently only a limited number of common targets of MBNL and CELF are known that are antagonistically regulated by these two groups of proteins. RESULTS: Recently, we identified neurofibromatosis type 1 (NF1) exon 23a as a novel target of negative regulation by CELF proteins. Here we report that MBNL family members are positive regulators of this exon. Overexpression of MBNL proteins promote exon 23a inclusion in a low MBNL-expressing cell line, and simultaneous siRNA-mediated knockdown of MBNL1 and MBNL2 family members in a high MBNL-expressing cell line promotes exon 23a skipping. Importantly, these two groups of proteins antagonize each other in regulating inclusion of exon 23a. Furthermore, we analyzed the binding sites of these proteins in the intronic sequences upstream of exon 23a by UV cross-linking assays. We show that in vitro, in addition to the previously identified preferred binding sequence UGCUGU, the MBNL proteins need the neighboring sequences for optimal binding. CONCLUSION: This study along with our previous work that demonstrated roles for Hu, CELF, and TIA-1 and TIAR proteins in the regulation of NF1 exon 23a establish that this exon is under tight, complex control.