Role for Fox-1/Fox-2 in mediating the neuronal pathway of calcitonin/calcitonin gene-related peptide alternative RNA processing.
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 U2AF(65) 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.
Zhou
Hua-Lin
HL
Department of Genetics, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106, USA. hxl47@case.edu
Baraniak
Andrew P
AP
Lou
Hua
H
D017398:Alternative Splicing / Q000235:genetics*; D020029:Base Pairing; D001483:Base Sequence; D015740:Calcitonin Gene-Related Peptide / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D005091:Exons / Q000235:genetics; D006367:HeLa Cells; D051542:Hepatocyte Nuclear Factor 3-beta / Q000378:metabolism*; D006801:Humans; D007438:Introns / Q000235:genetics; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D034622:RNA Interference; D022821:RNA Splice Sites / Q000235:genetics; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism; D000072668:Splicing Factor U2AF
C496502:FOXA2 protein, human; D009687:Nuclear Proteins; C455479:RBFOX1 protein, human; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; D051542:Hepatocyte Nuclear Factor 3-beta; D015740:Calcitonin Gene-Related Peptide