Nuclear matrix protein Matrin3 regulates alternative splicing and forms overlapping regulatory networks with PTB.

Matrin3 is an RNA- and DNA-binding nuclear matrix protein found to be associated with neural and muscular degenerative diseases. A number of possible functions of Matrin3 have been suggested, but no widespread role in RNA metabolism has yet been clearly demonstrated. We identified Matrin3 by its interaction with the second RRM domain of the splicing regulator PTB. Using a combination of RNAi knockdown, transcriptome profiling and iCLIP, we find that Matrin3 is a regulator of hundreds of alternative splicing events, principally acting as a splicing repressor with only a small proportion of targeted events being co-regulated by PTB. In contrast to other splicing regulators, Matrin3 binds to an extended region within repressed exons and flanking introns with no sharply defined peaks. The identification of this clear molecular function of Matrin3 should help to clarify the molecular pathology of ALS and other diseases caused by mutations of Matrin3.

Functional interactions between polypyrimidine tract binding protein and PRI peptide ligand containing proteins.

Polypyrimidine tract binding protein (PTBP1) is a heterogeneous nuclear ribonucleoprotein (hnRNP) that plays roles in most stages of the life-cycle of pre-mRNA and mRNAs in the nucleus and cytoplasm. PTBP1 has four RNA binding domains of the RNA recognition motif (RRM) family, each of which can bind to pyrimidine motifs. In addition, RRM2 can interact via its dorsal surface with proteins containing short peptide ligands known as PTB RRM2 interacting (PRI) motifs, originally found in the protein Raver1. Here we review our recent progress in understanding the interactions of PTB with RNA and with various proteins containing PRI ligands.

MBNL1 and PTB cooperate to repress splicing of Tpm1 exon 3.

Exon 3 of the rat α-tropomyosin (Tpm1) gene is repressed in smooth muscle cells, allowing inclusion of the mutually exclusive partner exon 2. Two key types of elements affect repression of exon 3 splicing: binding sites for polypyrimidine tract-binding protein (PTB) and additional negative regulatory elements consisting of clusters of UGC or CUG motifs. Here, we show that the UGC clusters are bound by muscleblind-like proteins (MBNL), which act as repressors of Tpm1 exon 3. We show that the N-terminal region of MBNL1, containing its four CCCH zinc-finger domains, is sufficient to mediate repression. The same region of MBNL1 can make a direct protein-to-protein interaction with PTB, and RNA binding by MBNL promotes this interaction, apparently by inducing a conformational change in MBNL. Moreover, single molecule analysis showed that MBNL-binding sites increase the binding of PTB to its own sites. Our data suggest that the smooth muscle splicing of Tpm1 is mediated by allosteric assembly of an RNA-protein complex minimally comprising PTB, MBNL and their cognate RNA-binding sites.

Stoichiometry of a regulatory splicing complex revealed by single-molecule analyses.

Splicing is regulated by complex interactions of numerous RNA-binding proteins. The molecular mechanisms involved remain elusive, in large part because of ignorance regarding the numbers of proteins in regulatory complexes. Polypyrimidine tract-binding protein (PTB), which regulates tissue-specific splicing, represses exon 3 of alpha-tropomyosin through distant pyrimidine-rich tracts in the flanking introns. Current models for repression involve either PTB-mediated looping or the propagation of complexes between tracts. To test these models, we used single-molecule approaches to count the number of bound PTB molecules both by counting the number of bleaching steps of GFP molecules linked to PTB within complexes and by analysing their total emissions. Both approaches showed that five or six PTB molecules assemble. Given the domain structures, this suggests that the molecules occupy primarily multiple overlapping potential sites in the polypyrimidine tracts, excluding propagation models. As an alternative to direct looping, we propose that repression involves a multistep process in which PTB binding forms small local loops, creating a platform for recruitment of other proteins that bring these loops into close proximity.

In vivo requirement of the small subunit of U2AF for recognition of a weak 3' splice site.

The U2 snRNP auxiliary factor (U2AF) is an essential splicing factor composed of two subunits, a large, 65-kDa subunit (U2AF(65)) and a small subunit, U2AF(35). U2AF(65) binds to the polypyrimidine tract upstream from the 3' splice site and promotes U2 snRNP binding to the pre-mRNA. Based on in vitro studies, it has been proposed that U2AF(35) plays a role in assisting U2AF(65) recruitment to nonconsensus polypyrimidine tracts. Here we have analyzed in vivo the roles of the two subunits of U2AF in the selection between alternative 3' splice sites associated with polypyrimidine tracts of different strengths. Our results reveal a feedback mechanism by which RNA interference (RNAi)-mediated depletion of U2AF(65) triggers the downregulation of U2AF(35). We further show that the knockdown of each U2AF subunit inhibits weak 3' splice site recognition, while overexpression of U2AF(65) alone is sufficient to activate the selection of this splice site. A variant of U2AF(65) lacking the interaction domain with U2AF(35) shows a reduced ability to promote this splicing event, suggesting that recognition of the weak 3' splice site involves the U2AF heterodimer. Furthermore, our data suggest that, rather than being required for splicing of all pre-mRNA substrates containing a weak polypyrimidine tract, U2AF(35) regulates the selection of weak 3' splice sites in a specific subset of cellular transcripts.

Matrin3: connecting gene expression with the nuclear matrix.

As indicated by its name, Matrin3 was discovered as a component of the nuclear matrix, an insoluble fibrogranular network that structurally organizes the nucleus. Matrin3 possesses both DNA- and RNA-binding domains and, consistent with this, has been shown to function at a number of stages in the life cycle of messenger RNAs. These numerous activities indicate that Matrin3, and indeed the nuclear matrix, do not just provide a structural framework for nuclear activities but also play direct functional roles in these activities. Here, we review the structure, functions, and molecular interactions of Matrin3 and of Matrin3-related proteins, and the pathologies that can arise upon mutation of Matrin3. WIREs RNA 2016, 7:303-315. doi: 10.1002/wrna.1336 For further resources related to this article, please visit the WIREs website.

Regulation of alternative pre-mRNA splicing.

Alternative splicing plays a prevalent role in generating functionally diversified proteomes from genomes with a more limited repertoire of protein-coding genes. Alternative splicing is frequently regulated with cell type or developmental specificity and in response to signaling pathways, and its mis-regulation can lead to disease. Co-regulated programs of alternative splicing involve interplay between a host of cis-acting transcript features and trans-acting RNA-binding proteins. Here, we review the current state of understanding of the logic and mechanism of regulated alternative splicing and indicate how this understanding can be exploited to manipulate splicing for therapeutic purposes.

Crystallographic analysis of polypyrimidine tract-binding protein-Raver1 interactions involved in regulation of alternative splicing.

The polypyrimidine tract-binding protein (PTB) is an important regulator of alternative splicing. PTB-regulated splicing of α-tropomyosin is enhanced by Raver1, a protein with four PTB-Raver1 interacting motifs (PRIs) that bind to the helical face of the second RNA recognition motif (RRM2) in PTB. We present the crystal structures of RRM2 in complex with PRI3 and PRI4 from Raver1, which--along with structure-based mutagenesis--reveal the molecular basis of their differential binding. High-affinity binding by Raver1 PRI3 involves shape-matched apolar contacts complemented by specific hydrogen bonds, a new variant of an established mode of peptide-RRM interaction. Our results refine the sequence of the PRI motif and place important structural constraints on functional models of PTB-Raver1 interactions. Our analysis indicates that the observed Raver1-PTB interaction is a general mode of binding that applies to Raver1 complexes with PTB paralogues such as nPTB and to complexes of Raver2 with PTB.