Concerted alterations in non-productive alternative splicing of core spliceosome components and splicing factors during smooth muscle cell phenotypic modulation

ADRIAN BUCKROYD; NICOLAS BELLORA; CATHY SHANAHAN; CHRIS SMITH

Cold Spring Harbor Labs.

Congreso; Eukariotic mRNA Processing 2013; 2013

University of Colorado, Boulder; University of Pennsylvania; Max-Planck-Institute-CBG, Germany

Unproductive splicing of splicing factor mRNAs is thought to form an auto-regulatory mechanism to prevent overexpression of these factors, often by NMD mediated mechanisms. siRNA mediated knockdown of splicing factor genes that are able to undergo unproductive splicing can lead to alterations in splicing patterns, but large-scale regulation of such events in a physiological setting has not been reported in the literature. In the course of profiling splicing changes that occur during phenotypic modulation of mouse smooth muscle cells between a contractile phenotype and a synthetic/proliferative phenotype, we observed a significant number of changes in alternative splicing of mRNAs encoding splicing snRNP components (e.g. Snrnp70, SnrnpA1, Sf3b1, Sf3b3) as well as splicing activators (Srsf1, Srsf2, Srsf6, Srsf7, Tra2beta). In every case, the splicing pattern in the differentiated phenotype was predicted to be non-functional, due to intron retention, inclusion of poison cassette exons or exon skipping. Strikingly, two intron retention events in the Srsf1 gene changed in opposite directions, but both are predicted to functionally collaborate to reduce expression in the contractile cells. Immunofluorescence analysis of the same splicing factors in rat aorta smooth muscle cells showed marked increases in expression during phenotypic modulation, while levels of RNA polymerase and other auxiliary splicing regulators (e.g. PTB and Mbnl) remained relatively constant. Thus the concerted non-productive splicing events appear to be associated with lower levels of the splicing machinery, rather than resulting from feedback responses to excessively high levels. Our data suggest that the alternative splicing programme in contractile smooth muscle cells is controlled not only by a range of auxiliary RNA binding proteins, but also by concerted down-regulation of the core splicing machinery.