The alternative splicing program of differentiated smooth muscle cells involves concerted non-productive splicing of post-transcriptional regulators.
Alternative splicing (AS) is a key component of gene expression programs that drive cellular differentiation. Smooth muscle cells (SMCs) are important in the function of a number of physiological systems; however, investigation of SMC AS has been restricted to a handful of events. We profiled transcriptome changes in mouse de-differentiating SMCs and observed changes in hundreds of AS events. Exons included in differentiated cells were characterized by particularly weak splice sites and by upstream binding sites for Polypyrimidine Tract Binding protein (PTBP1). Consistent with this, knockdown experiments showed that that PTBP1 represses many smooth muscle specific exons. We also observed coordinated splicing changes predicted to downregulate the expression of core components of U1 and U2 snRNPs, splicing regulators and other post-transcriptional factors in differentiated cells. The levels of cognate proteins were lower or similar in differentiated compared to undifferentiated cells. However, levels of snRNAs did not follow the expression of splicing proteins, and in the case of U1 snRNP we saw reciprocal changes in the levels of U1 snRNA and U1 snRNP proteins. Our results suggest that the AS program in differentiated SMCs is orchestrated by the combined influence of auxiliary RNA binding proteins, such as PTBP1, along with altered activity and stoichiometry of the core splicing machinery.
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK.
Miriam
M
Llorian
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK.
Clare
C
Gooding
Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK Catalan Institute for Research and Advanced Studies (ICREA), E08010 Barcelona, Spain.
Nicolas
N
Bellora
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK Computational Genomics, Universitat Pompeu Fabra, E08003 Barcelona, Spain.
Martina
M
Hallegger
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK.
Adrian
A
Buckroyd
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK.
Xiao
X
Wang
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK.
Dipen
D
Rajgor
INIBIOMA, CONICET-UNComahue, Bariloche 8400 Río Negro, Argentina.
Melis
M
Kayikci
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK.
Jack
J
Feltham
Computational Genomics, Universitat Pompeu Fabra, E08003 Barcelona, Spain.
Jernej
J
Ule
Department of Molecular Neuroscience, UCL Institute of Neurology, Queen Square, London, WC1N 3BG, UK MRC-Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.
Eduardo
E
Eyras
Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QW, UK cwjs1@cam.ac.uk.
Christopher W J
CW
Smith
D017398:Alternative Splicing; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D005091:Exons; D020869:Gene Expression Profiling; D007438:Introns; D051379:Mice; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D059372:Nucleotide Motifs; D012323:RNA Processing, Post-Transcriptional; D000072260:RNA Splicing Factors / Q000378:metabolism; D020871:RNA Stability; D012342:RNA, Small Nuclear / Q000235:genetics; D051381:Rats
D000072260:RNA Splicing Factors; D012342:RNA, Small Nuclear