RRM
2010 Rustbelt RNA Meeting
RRM
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Poster abstracts
Abstract:
The Neurofibromatosis Type I (NF1) pre-mRNA undergoes alternative splicing. NF1 exon 23a is an alternative exon that shows differential expression in brain versus other tissue types. Exon 23a, which is skipped in neuronal tissues and included in other tissue types falls within the best-characterized domain of the protein, the GAP-related domain. Through its GAP-related domain, NF1 negatively regulates Ras signaling, thus controlling cell growth and proliferation. The type II isoform, which contains exon 23a, is ten times weaker in regulating Ras than the type I isoform, in which exon 23a is skipped. Mice in which the balance of the two NF1 isoforms has been disrupted show learning disabilities.
In our lab, we have identified several factors that regulate the NF1 exon 23a inclusion. We have shown that the Hu proteins and the CUG-BP and ETR-3 like factors (CELF proteins) promote NF1 exon 23a skipping, while TIA-1 and TIAR promote exon 23a inclusion (ref. 1 & 2). The Muscleblind-like (MBNL) proteins are additional candidates for NF1 splicing regulation, and we hypothesize that they promote NF1 exon 23a inclusion. MBNL proteins are known to act as antagonists to the CELF proteins in at least six pre-mRNA targets, although the two families also regulate distinct targets. MBNL proteins bind to the YGCU(U/G)Y motif (Y, pyrimidine) with a preference for stem-loop RNA secondary structures.
There are two potential MBNL binding motifs in the intronic sequence upstream to NF1 exon 23a, and this RNA sequence is predicted to form a stem-loop structure. Point mutations that disrupt the potential MBNL binding motifs in NF1 led to decreased inclusion of exon 23a from an NF1 splicing reporter. Over-expression of MBNL1 promoted NF1 exon 23a inclusion, in a low MBNL expressing cell line, suggesting that they are positive regulators. Interestingly, over-expression of MBNL proteins partially rescues the increased skipping of NF1 exon 23a that results from the over-expression of CELF proteins in HeLa cells. Binding of recombinant MBNL1 protein to the NF1 pre-mRNA was confirmed by UV crosslinking. Future work will focus on the regulation of this event by MBNL and how MBNL and CELF proteins function as antagonists in this system. In addition the secondary structure of this region will be probed using in vitro RNA structure probing methods.
References:
1. Zhu, Hui et al., 2008. Regulation of Neuron Specific Alternative Splicing of Neurofibromatosis Type I Pre-mRNA. Mol Cell Biol. 28: 1240-1251.
2. Barron, Victoria A. et al., 2010. The Neurofibromatosis Type I Pre-mRNA is a Novel Target of CELF Protein-Mediated Splicing Regulation. 38: 253-264.
Keywords: Muscleblind Proteins, Alternative Splicing, Neurofibromatosis Type I
