Splice-sensitive array profiling suggests a role for STAR proteins and PTB in control of smooth muscle cell alternative splicing

SELINA XIAO WANG; MIRIAM LLORIAN; NICOLAS BELLORA; CHRISTOPHER SMITH

Davos

Congreso; 18th Annual Meeting of the RNA Society; 2013

International RNA Society

Tissue-specific alternative splicing has been extensively investigated in striated muscles (heart and skeletal muscle), and a large amount is known about the relevant RNA sequence elements and RNA binding proteins involved. By contrast, the regulation of alternative splicing in smooth muscle cells (SMCs) has been relatively neglected. Vascular SMCs show phenotypic plasticity and can interconvert between a differentiated ?contractile? phenotype and a more proliferative ?synthetic? phenotype marked by increased synthesis of extracellular matrix proteins. This phenotypic modulation process plays a significant pathophysiological role in various cardiovascular diseases. While the transcriptional changes during phenotypic modulation have been well investigated, knowledge about the global changes in alternative splicing has been very limited. We have used splice-sensitive microarrays to interrogate global changes in both transcript levels and alternative splicing during phenotypic modulation of mouse aorta and bladder smooth muscle. Genes affected by alternative splicing showed distinct functional enrichments from those that were transcriptionally up or down-regulated. Splicing particularly affected cytoskeletal proteins, while ion channels and receptors were transcriptionally down-regulated and receptor binding and extracellular matrix proteins were upregulated. Nucleic acid binding proteins were significantly depleted among the transcriptionally regulated genes. We identified sets of cassette exons that were substantially up or down-regulated during phenotypic modulation. Computational analysis showed that exons that are included in contractile SMCs are associated with PTB-binding motifs on the upstream side, where PTB represses splicing. Downstream of these exons there was a substantial enrichment of motifs resembling the binding sites for members of the signal transduction and activation of RNA (STAR) protein family (UUAAC, UAACC, ACUAA, CUAAC), which have not previously been associated with regulation of splicing in SMCs. Candidate STAR-regulated exons with potential binding sites in downstream intron were manually identified from 50 top-ranked events and the splicing pattern changes of 7 events (Ncam1 exon 2, Atp2b4 exon 20, Cacna2d1 exon 23, Ppp4r1 exon 3, Sfrs10 exon 2, and Bnip2 exon 10, Ppp1r12a exon 24) were validated by RT-PCR. In preliminary experiments using an Ncam1 exon 2 minigene reporter in proliferative rat PAC1 cells, overexpression of Sam68, SLM1 and SF1 significantly increased Ncam1 exon 2 inclusion, with Sam68 being the strongest regulator. These initial observations suggest that STAR proteins might act widely to promote inclusion of cassette exons in contractile SMCs, while many of the same exons might be repressed by PTB in de-differentiated cells.