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.

Identification of RBPMS as a mammalian smooth muscle master splicing regulator via proximity of its gene with super-enhancers.

Alternative splicing (AS) programs are primarily controlled by regulatory RNA-binding proteins (RBPs). It has been proposed that a small number of master splicing regulators might control cell-specific splicing networks and that these RBPs could be identified by proximity of their genes to transcriptional super-enhancers. Using this approach we identified RBPMS as a critical splicing regulator in differentiated vascular smooth muscle cells (SMCs). RBPMS is highly down-regulated during phenotypic switching of SMCs from a contractile to a motile and proliferative phenotype and is responsible for 20% of the AS changes during this transition. RBPMS directly regulates AS of numerous components of the actin cytoskeleton and focal adhesion machineries whose activity is critical for SMC function in both phenotypes. RBPMS also regulates splicing of other splicing, post-transcriptional and transcription regulators including the key SMC transcription factor Myocardin, thereby matching many of the criteria of a master regulator of AS in SMCs.

All the cells in our body contain the same genetic information, but they only switch on the genes that they need to fulfill their specific role in the organism. Genetic sequences known as enhancers can turn on the genes that are required by a particular cell to perform its tasks. Once a gene is activated, its sequence is faithfully copied into a molecule of RNA which contains segments that code for a protein. A molecular machine then processes the RNA molecule and splices together the coding segments. RNA binding proteins can also regulate this mechanism, and help to splice the coding sections in different ways depending on the type of cell. The process, known as alternative RNA splicing, therefore creates different RNA templates from the same gene. This gives rise to related but different proteins, each suited to the needs of the particular cell in which they are made. However, in some cell types, exactly how this happens has not yet been well documented. For example, in cells that line blood vessels ­ known as vascular smooth muscle cells ­ the RNA binding proteins that drive alternative splicing have not been identified. To find these proteins, Nakagaki-Silva et al. used catalogs of DNA regions called super-enhancers as clues. These sequences strongly activate certain genes in a tissue-specific manner, effectively acting as labels for genes important for a given cell type. In vascular smooth muscle cells, if a super-enhancer switches on a gene that codes for a RNA-binding protein, this protein is probably crucial for the cell to work properly. The approach highlighted a protein called RBPMS, and showed that it controlled alternative RNA splicing of many genes important in smooth muscle cells. This may suggest that when RBPMS regulation is disrupted, certain diseases of the heart and blood vessels could emerge. Finally, the results by Nakagaki-Silva et al. demonstrate that super-enhancers can signpost genes important in regulating splicing or other key processes in particular cell types.

Solution and crystal structures of a C-terminal fragment of the neuronal isoform of the polypyrimidine tract binding protein (nPTB).

The eukaryotic polypyrimidine tract binding protein (PTB) serves primarily as a regulator of alternative splicing of messenger RNA, but is also co-opted to other roles such as RNA localisation and translation initiation from internal ribosome entry sites. The neuronal paralogue of PTB (nPTB) is 75% identical in amino acid sequence with PTB. Although the two proteins have broadly similar RNA binding specificities and effects on RNA splicing, differential expression of PTB and nPTB can lead to the generation of alternatively spliced mRNAs. RNA binding by PTB and nPTB is mediated by four RNA recognition motifs (RRMs). We present here the crystal and solution structures of the C-terminal domain of nPTB (nPTB34) which contains RRMs 3 and 4. As expected the structures are similar to each other and to the solution structure of the equivalent fragment from PTB (PTB34). The result confirms that, as found for PTB, RRMs 3 and 4 of nPTB interact with one another to form a stable unit that presents the RNA-binding surfaces of the component RRMs on opposite sides that face away from each other. The major differences between PTB34 and nPTB34 arise from amino acid side chain substitutions on the exposed ß-sheet surfaces and adjoining loops of each RRM, which are likely to modulate interactions with RNA.