Abstract
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.
Highlights
Alternative splicing (AS) is a key contributor to remodeling the transcriptomes of cells during development and differentiation
We used mouse aorta and bladder as sources of smooth muscle cells (SMCs). These two tissues showed the strongest signal in transgenic mice carrying a splice-sensitive fluorescent reporter based on Tpm1 SMC-specific AS [38], and provide contrasting examples of tonic and phasic smooth muscle tissues [5]
Triplicate RNA samples were purified from the intact medial layer of mouse aortas and the detrusor muscle of bladder, and from SMCs that had been isolated from the two tissues and cultured for 4–7 days without further passaging
Summary
Alternative splicing (AS) is a key contributor to remodeling the transcriptomes of cells during development and differentiation. Numerous analyses have indicated the functional importance of AS, and highlighted the fact that AS and transcriptional control tend to operate on different sets of genes [1,2]. Much has been learned about the cis and transacting components of ‘splicing codes’ that give rise to regulated splicing programs characteristic of various adult tissues. Splicing programs have been well characterized in cardiac and skeletal muscle (reviewed in [4]). By comparison, despite their biomedical importance, knowledge of the regulated splicing programs of smooth muscle cells (SMCs) is more rudimentary
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