Abstract
BackgroundAlternative 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.ResultsRecently, 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.ConclusionThis 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.
Highlights
Alternative splicing is often subjected to complex regulatory control that involves many protein factors and cis-acting Ribonucleic acid (RNA) sequence elements
The intronic sequence upstream of neurofibromatosis type 1 (NF1) exon 23a is UG-rich and contains two potential MBNL protein binding sites We previously demonstrated that the CUG-binding protein (CUG-BP) and ELAV-Like family (CELF) proteins act as negative regulators of NF1 exon 23a inclusion, and that these proteins bind to the UG-rich sequences located in the intronic region upstream of exon 23a (Figure 1B and [27])
Our studies show that all three of the MBNL protein family members function redundantly as positive regulators of NF1 exon 23a when over-expressed in CA77 cells
Summary
Alternative splicing is often subjected to complex regulatory control that involves many protein factors and cis-acting RNA sequence elements. Alternative splicing allows more than one protein product to be generated from a single gene by selectively including or excluding particular exons in the mature mRNA transcripts. This is a prevalent mechanism of gene regulation with as many as 94% of human genes predicted to undergo the process [1,2]. Alternative splicing is a tightly regulated process involving cis-sequences on the RNA and protein factors that can either promote the inclusion or the skipping of a particular alternative exon in the mature mRNA. More recently other means of splicing regulation have been demonstrated including chromatin remodeling and involvement of the C-terminal domain of RNA Polymerase II as a staging platform for splicing factors during coupled transcription and splicing [16,17,18]
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