Role for Fox-1/Fox-2 in Mediating the Neuronal Pathway of Calcitonin/Calcitonin Gene-Related Peptide Alternative RNA Processing

Abstract

Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3′ splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF⁶⁵ binding to the 3′ splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.

We thank the following individuals for providing antibody and plasmids: Doug Black at UCLA (anti-Fox-1 and anti-Fox-2 antibodies), Sachiyo Kawamoto at the NIH (F011, F411, F402, A016, and A713 Fox expression plasmids), and Andrew Lieberman at the University of Michigan (Fox-1 cDNA plasmid). We thank Helen Salz and Jo Ann Wise for critical reading of the manuscript.

This study was supported by an NIH grant to H.L. (NS-049103-01). A.P.B. was supported by NIH grant RO1 GM63090 to M. A. Garcia-Blanco.