RRM
2007Rustbelt RNA Meeting
RRM
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Talk abstracts
Abstract:
RNA-binding proteins Fox-1/Fox-2 were identified as the novel major neuron-specific negative regulators that mediate the neuron-specific calcitonin gene-related peptide (CGRP) processing pattern (Zhou et el, Mol Cell Biol 27, 830). Although it is clear that the regulatory effect of Fox proteins depends on two UGCAUG silencer RNA sequence motifs, it is not known how these proteins block inclusion of the calcitonin/CGRP of exon 4, which is an alternative 3’-terminal exon flanked by a 3’ splice site and a polyadenylation site on either end.
Here, we provide results of biochemical analysis to demonstrate that Fox-1/Fox-2 proteins directly inhibit splicing of calcitonin-specific exon 4 and blocks formation of the prespliceosomal early (E) complex by preventing the assembly of U2 snRNP auxiliary factor (U2AF) on the splicing acceptor site upstream of calcitonin-specific exon 4. Furthermore, using an MS2-MBP affinity purification approach, we demonstrated the mechanism whereby Fox proteins inhibit binding of U2AF. First, Fox proteins interfere with binding of the splicing factor 1 (SF1) to the branchpoint sequence through interacting with the -34 UGCAUG silencer element, which is immediately adjacent to the branchpoint. Second, binding of two SR proteins (Tra2-beta and SRp55) to exonic splicing enhancer elements is antagonized by the Fox proteins that interact with +45 UGCAUG silencer element. In HeLa cells, over-expression of Tra2-beta or SRp55 proteins can reverse the Fox-mediated reduction in calcitonin-specific exon 4 splicing pattern.
These results give the first mechanistic insights into how Fox proteins inhibit splicing, which remained unknown although alternative splicing of several pre-mRNAs has been shown to be regulated by these proteins.
Keywords: Fox protein, Calcitonin, splicing
