Heteromeric RNP Assembly at LINEs Controls Lineage-Specific RNA Processing.

Long mammalian introns make it challenging for the RNA processing machinery to identify exons accurately. We find that LINE-derived sequences (LINEs) contribute to this selection by recruiting dozens of RNA-binding proteins (RBPs) to introns. This includes MATR3, which promotes binding of PTBP1 to multivalent binding sites within LINEs. Both RBPs repress splicing and 3' end processing within and around LINEs. Notably, repressive RBPs preferentially bind to evolutionarily young LINEs, which are located far from exons. These RBPs insulate the LINEs and the surrounding intronic regions from RNA processing. Upon evolutionary divergence, changes in RNA motifs within LINEs lead to gradual loss of their insulation. Hence, older LINEs are located closer to exons, are a common source of tissue-specific exons, and increasingly bind to RBPs that enhance RNA processing. Thus, LINEs are hubs for the assembly of repressive RBPs and also contribute to the evolution of new, lineage-specific transcripts in mammals. VIDEO ABSTRACT.

Cell-type specific regulator RBPMS switches alternative splicing via higher-order oligomerization and heterotypic interactions with other splicing regulators.

Alternative pre-mRNA splicing decisions are regulated by RNA binding proteins (RBPs) that can activate or repress regulated splice sites. Repressive RBPs typically harness multivalent interactions to bind stably to target RNAs. Multivalency can be achieved by homomeric oligomerization and heteromeric interactions with other RBPs, often mediated by intrinsically disordered regions (IDRs), and by possessing multiple RNA binding domains. Cell-specific splicing decisions often involve the action of widely expressed RBPs, which are able to bind multivalently around target exons, but without effect in the absence of a cell-specific regulator. To address how cell-specific regulators can collaborate with constitutive RBPs in alternative splicing regulation, we used the smooth-muscle specific regulator RBPMS. Recombinant RBPMS is sufficient to confer smooth muscle cell specific alternative splicing of Tpm1 exon 3 in cell-free assays by preventing assembly of ATP-dependent splicing complexes. This activity depends upon a C-terminal IDR that facilitates dynamic higher-order self-assembly, cooperative binding to multivalent RNA and interactions with widely expressed splicing co-regulators, including MBNL1 and RBFOX2, allowing cooperative assembly of stable cell-specific regulatory complexes.

Phosphorylation of the smooth muscle master splicing regulator RBPMS regulates its splicing activity.

We previously identified RBPMS as a master regulator of alternative splicing in differentiated smooth muscle cells (SMCs). RBPMS is transcriptionally downregulated during SMC dedifferentiation, but we hypothesized that RBPMS protein activity might be acutely downregulated by post-translational modifications. Publicly available phosphoproteomic datasets reveal that Thr113 and Thr118 immediately adjacent to the RRM domain are commonly both phosphorylated. An RBPMS T113/118 phosphomimetic T/E mutant showed decreased splicing regulatory activity both in transfected cells and in a cell-free in vitro assay, while a non-phosphorylatable T/A mutant retained full activity. Loss of splicing activity was associated with a modest reduction in RNA affinity but significantly reduced RNA binding in nuclear extract. A lower degree of oligomerization of the T/E mutant might cause lower avidity of multivalent RNA binding. However, NMR analysis also revealed that the T113/118E peptide acts as an RNA mimic which can loop back and antagonize RNA-binding by the RRM domain. Finally, we identified ERK2 as the most likely kinase responsible for phosphorylation at Thr113 and Thr118. Collectively, our data identify a potential mechanism for rapid modulation of the SMC splicing program in response to external signals during the vascular injury response and atherogenesis.

Polypyrimidine tract binding protein 1 regulates the activation of mouse CD8 T cells.

The RNA-binding protein polypyrimidine tract binding protein 1 (PTBP1) has been found to have roles in CD4 T-cell activation, but its function in CD8 T cells remains untested. We show it is dispensable for the development of naïve mouse CD8 T cells, but is necessary for the optimal expansion and production of effector molecules by antigen-specific CD8 T cells in vivo. PTBP1 has an essential role in regulating the early events following activation of the naïve CD8 T cell leading to IL-2 and TNF production. It is also required to protect activated CD8 T cells from apoptosis. PTBP1 controls alternative splicing of over 400 genes in naïve CD8 T cells in addition to regulating the abundance of ∼200 mRNAs. PTBP1 is required for the nuclear accumulation of c-Fos, NFATc2, and NFATc3, but not NFATc1. This selective effect on NFAT proteins correlates with PTBP1-promoted expression of the shorter Aß1 isoform and exon 13 skipped Aß2 isoform of the catalytic A-subunit of calcineurin phosphatase. These findings reveal a crucial role for PTBP1 in regulating CD8 T-cell activation.

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.

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.

Dissecting domains necessary for activation and repression of splicing by Muscleblind-like protein 1.

BACKGROUND: Alternative splicing contributes to the diversity of the proteome, and provides the cell with an important additional layer of regulation of gene expression. Among the many RNA binding proteins that regulate alternative splicing pathways are the Muscleblind-like (MBNL) proteins. MBNL proteins bind YGCY motifs in RNA via four CCCH zinc fingers arranged in two tandem arrays, and play a crucial role in the transition from embryonic to adult muscle splicing patterns, deregulation of which leads to Myotonic Dystrophy. Like many other RNA binding proteins, MBNL proteins can act as both activators or repressors of different splicing events. RESULTS: We used targeted point mutations to interfere with the RNA binding of MBNL1 zinc fingers individually and in combination. The effects of the mutations were tested in assays for splicing repression and activation, including overexpression, complementation of siRNA-mediated knockdown, and artificial tethering using MS2 coat protein. Mutations were tested in the context of both full length MBNL1 as well as a series of truncation mutants. Individual mutations within full length MBNL1 had little effect, but mutations in ZF1 and 2 combined were more detrimental than those in ZF 3 and 4, upon splicing activation, repression and RNA binding. Activation and repression both required linker sequences between ZF2 and 3, but activation was more sensitive to loss of linker sequences. CONCLUSIONS: Our results highlight the importance of RNA binding by MBNL ZF domains 1 and 2 for splicing regulatory activity, even when the protein is artificially recruited to its regulatory location on target RNAs. However, RNA binding is not sufficient for activity; additional regions between ZF 2 and 3 are also essential. Activation and repression show differential sensitivity to truncation of this linker region, suggesting interactions with different sets of cofactors for the two types of activity.

Defining the roles and interactions of PTB.

PTB (polypyrimidine tract-binding protein) is an abundant and widely expressed RNA-binding protein with four RRM (RNA recognition motif) domains. PTB is involved in numerous post-transcriptional steps in gene expression in both the nucleus and cytoplasm, but has been best characterized as a regulatory repressor of some ASEs (alternative splicing events), and as an activator of translation driven by IRESs (internal ribosome entry segments). We have used a variety of approaches to characterize the activities of PTB and its molecular interactions with RNA substrates and protein partners. Using splice-sensitive microarrays we found that PTB acts not only as a splicing repressor but also as an activator, and that these two activities are determined by the location at which PTB binds relative to target exons. We have identified minimal splicing repressor and activator domains, and have determined high resolution structures of the second RRM domain of PTB binding to peptide motifs from the co-repressor protein Raver1. Using single-molecule techniques we have determined the stoichiometry of PTB binding to a regulated splicing substrate in whole nuclear extracts. Finally, we have used tethered hydroxyl radical probing to determine the locations on viral IRESs at which each of the four RRM domains bind. We are now combining tethered probing with single molecule analyses to gain a detailed understanding of how PTB interacts with pre-mRNA substrates to effect either repression or activation of splicing.

A peptide motif in Raver1 mediates splicing repression by interaction with the PTB RRM2 domain.

Polypyrimidine tract-binding protein (PTB) is a regulatory splicing repressor. Raver1 acts as a PTB corepressor for splicing of alpha-tropomyosin (Tpm1) exon 3. Here we define a minimal region of Raver1 that acts as a repressor domain when recruited to RNA. A conserved [S/G][I/L]LGxxP motif is essential for splicing repressor activity and sufficient for interaction with PTB. An adjacent proline-rich region is also essential for repressor activity but not for PTB interaction. NMR analysis shows that LLGxxP peptides interact with a hydrophobic groove on the dorsal surface of the RRM2 domain of PTB, which constitutes part of the minimal repressor region of PTB. The requirement for the PTB-Raver1 interaction that we have characterized may serve to bring the additional repressor regions of both proteins into a configuration that allows them to synergistically effect exon skipping.

A drug-repositioning screen using splicing-sensitive fluorescent reporters identifies novel modulators of VEGF-A splicing with anti-angiogenic properties.

Alternative splicing of the vascular endothelial growth factor A (VEGF-A) terminal exon generates two protein families with differing functions. Pro-angiogenic VEGF-Axxxa isoforms are produced via selection of the proximal 3' splice site of the terminal exon. Use of an alternative distal splice site generates the anti-angiogenic VEGF-Axxxb proteins. A bichromatic splicing-sensitive reporter was designed to mimic VEGF-A alternative splicing and was used as a molecular tool to further investigate this alternative splicing event. Part of VEGF-A's terminal exon and preceding intron were inserted into a minigene construct followed by the coding sequences for two fluorescent proteins. A different fluorescent protein is expressed depending on which 3' splice site of the exon is used during splicing (dsRED denotes VEGF-Axxxa and EGFP denotes VEGF-Axxxb). The fluorescent output can be used to follow splicing decisions in vitro and in vivo. Following successful reporter validation in different cell lines and altering splicing using known modulators, a screen was performed using the LOPAC library of small molecules. Alterations to reporter splicing were measured using a fluorescent plate reader to detect dsRED and EGFP expression. Compounds of interest were further validated using flow cytometry and assessed for effects on endogenous VEGF-A alternative splicing at the mRNA and protein level. Ex vivo and in vitro angiogenesis assays were used to demonstrate the anti-angiogenic effect of the compounds. Furthermore, anti-angiogenic activity was investigated in a Matrigel in vivo model. To conclude, we have identified a set of compounds that have anti-angiogenic activity through modulation of VEGF-A terminal exon splicing.

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.

Tropomyosin exons as models for alternative splicing.

Three of the four mammalian tropomyosin (Tm) genes are alternatively spliced, most commonly by mutually exclusive selection from pairs of internal or 3' end exons. Alternative splicing events in the TPM1, 2 and 3 genes have been analysed experimentally in various levels ofdetail. In particular, mutually exclusive exon pairs in the betaTm (TPM2) and alphaTm (TPM1) genes are among the most intensively studied models for striated and smooth muscle specific alternative splicing, respectively. Analysis of these model systems has provided important insights into general mechanisms and strategies of splicing regulation.

Repression of alpha-actinin SM exon splicing by assisted binding of PTB to the polypyrimidine tract.

Polypyrimidine tract binding protein (PTB) acts as a regulatory repressor of a large number of alternatively spliced exons, often requiring multiple binding sites in order to repress splicing. In one case, cooperative binding of PTB has been shown to accompany repression. The SM exon of the alpha-actinin pre-mRNA is also repressed by PTB, leading to inclusion of the alternative upstream NM exon. The SM exon has a distant branch point located 386 nt upstream of the exon with an adjacent 26 nucleotide pyrimidine tract. Here we have analyzed PTB binding to the NM and SM exon region of the alpha-actinin pre-mRNA. We find that three regions of the intron bind PTB, including the 3' end of the polypyrimidine tract (PPT) and two additional regions between the PPT and the SM exon. The downstream PTB binding sites are essential for full repression and promote binding of PTB to the PPT with a consequent reduction in U2AF(65) binding. Our results are consistent with a repressive mechanism in which cooperative binding of PTB to the PPT competes with binding of U2AF(65), thereby specifically blocking splicing of the SM exon.

A class of human exons with predicted distant branch points revealed by analysis of AG dinucleotide exclusion zones.

BACKGROUND: The three consensus elements at the 3' end of human introns--the branch point sequence, the polypyrimidine tract, and the 3' splice site AG dinucleotide--are usually closely spaced within the final 40 nucleotides of the intron. However, the branch point sequence and polypyrimidine tract of a few known alternatively spliced exons lie up to 400 nucleotides upstream of the 3' splice site. The extended regions between the distant branch points (dBPs) and their 3' splice site are marked by the absence of other AG dinucleotides. In many cases alternative splicing regulatory elements are located within this region. RESULTS: We have applied a simple algorithm, based on AG dinucleotide exclusion zones (AGEZ), to a large data set of verified human exons. We found a substantial number of exons with large AGEZs, which represent candidate dBP exons. We verified the importance of the predicted dBPs for splicing of some of these exons. This group of exons exhibits a higher than average prevalence of observed alternative splicing, and many of the exons are in genes with some human disease association. CONCLUSION: The group of identified probable dBP exons are interesting first because they are likely to be alternatively spliced. Second, they are expected to be vulnerable to mutations within the entire extended AGEZ. Disruption of splicing of such exons, for example by mutations that lead to insertion of a new AG dinucleotide between the dBP and 3' splice site, could be readily understood even though the causative mutation might be remote from the conventional locations of splice site sequences.

A novel polypyrimidine tract-binding protein paralog expressed in smooth muscle cells.

Polypyrimidine tract-binding protein (PTB) is an abundant widespread RNA-binding protein with roles in regulation of pre-mRNA alternative splicing and 3'-end processing, internal ribosomal entry site-driven translation, and mRNA localization. Tissue-restricted paralogs of PTB have previously been reported in neuronal and hematopoietic cells. These proteins are thought to replace many general functions of PTB, but to have some distinct activities, e.g. in the tissue-specific regulation of some alternative splicing events. We report the identification and characterization of a fourth rodent PTB paralog (smPTB) that is expressed at high levels in a number of smooth muscle tissues. Recombinant smPTB localized to the nucleus, bound to RNA, and was able to regulate alternative splicing. We suggest that replacement of PTB by smPTB might be important in controlling some pre-mRNA alternative splicing events.

Autoregulation of polypyrimidine tract binding protein by alternative splicing leading to nonsense-mediated decay.

Polypyrimdine tract binding protein (PTB) is a regulator of alternative splicing, mRNA 3' end formation, mRNA stability and localization, and IRES-mediated translation. Transient overexpression of PTB can influence alternative splicing, sometimes resulting in nonphysiological splicing patterns. Here, we show that alternative skipping of PTB exon 11 leads to an mRNA that is removed by NMD and that this pathway consumes at least 20% of the PTB mRNA in HeLa cells. We also show that exon 11 skipping is itself promoted by PTB in a negative feedback loop. This autoregulation may serve both to prevent disruptively high levels of PTB expression and to restore nuclear levels when PTB is mobilized to the cytoplasm. Our findings suggest that alternative splicing can act not only to generate protein isoform diversity but also to quantitatively control gene expression and complement recent bioinformatic analyses, indicating a high prevalence of human alternative splicing leading to NMD.

The PTB interacting protein raver1 regulates alpha-tropomyosin alternative splicing.

Regulated switching of the mutually exclusive exons 2 and 3 of alpha-tropomyosin (TM) involves repression of exon 3 in smooth muscle cells. Polypyrimidine tract-binding protein (PTB) is necessary but not sufficient for regulation of TM splicing. Raver1 was identified in two-hybrid screens by its interactions with the cytoskeletal proteins actinin and vinculin, and was also found to interact with PTB. Consistent with these interactions raver1 can be localized in either the nucleus or cytoplasm. Here we show that raver1 is able to promote the smooth muscle-specific alternative splicing of TM by enhancing PTB-mediated repression of exon 3. This activity of raver1 is dependent upon characterized PTB-binding regulatory elements and upon a region of raver1 necessary for interaction with PTB. Heterologous recruitment of raver1, or just its C-terminus, induced very high levels of exon 3 skipping, bypassing the usual need for PTB binding sites downstream of exon 3. This suggests a novel mechanism for PTB-mediated splicing repression involving recruitment of raver1 as a potent splicing co-repressor.