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.

The RNA-binding protein PTBP1 is necessary for B cell selection in germinal centers.

Antibody affinity maturation occurs in germinal centers (GCs), where B cells cycle between the light zone (LZ) and the dark zone. In the LZ, GC B cells bearing immunoglobulins with the highest affinity for antigen receive positive selection signals from helper T cells, which promotes their rapid proliferation. Here we found that the RNA-binding protein PTBP1 was needed for the progression of GC B cells through late S phase of the cell cycle and for affinity maturation. PTBP1 was required for proper expression of the c-MYC-dependent gene program induced in GC B cells receiving T cell help and directly regulated the alternative splicing and abundance of transcripts that are increased during positive selection to promote proliferation.

Alternative splicing as a source of phenotypic diversity.

A major goal of evolutionary genetics is to understand the genetic processes that give rise to phenotypic diversity in multicellular organisms. Alternative splicing generates multiple transcripts from a single gene, enriching the diversity of proteins and phenotypic traits. It is well established that alternative splicing contributes to key innovations over long evolutionary timescales, such as brain development in bilaterians. However, recent developments in long-read sequencing and the generation of high-quality genome assemblies for diverse organisms has facilitated comparisons of splicing profiles between closely related species, providing insights into how alternative splicing evolves over shorter timescales. Although most splicing variants are probably non-functional, alternative splicing is nonetheless emerging as a dynamic, evolutionarily labile process that can facilitate adaptation and contribute to species divergence.

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.

Myocardin regulates exon usage in smooth muscle cells through induction of splicing regulatory factors.

Differentiation of smooth muscle cells (SMCs) depends on serum response factor (SRF) and its co-activator myocardin (MYOCD). The role of MYOCD for the SMC program of gene transcription is well established. In contrast, the role of MYOCD in control of SMC-specific alternative exon usage, including exon splicing, has not been explored. In the current work we identified four splicing factors (MBNL1, RBPMS, RBPMS2, and RBFOX2) that correlate with MYOCD across human SMC tissues. Forced expression of MYOCD family members in human coronary artery SMCs in vitro upregulated expression of these splicing factors. For global profiling of transcript diversity, we performed RNA-sequencing after MYOCD transduction. We analyzed alternative transcripts with three different methods. Exon-based analysis identified 1637 features with differential exon usage. For example, usage of 3´ exons in MYLK that encode telokin increased relative to 5´ exons, as did the 17 kDa telokin to 130 kDa MYLK protein ratio. Dedicated event-based analysis identified 239 MYOCD-driven splicing events. Events involving MBNL1, MCAM, and ACTN1 were among the most prominent, and this was confirmed using variant-specific PCR analyses. In support of a role for RBPMS and RBFOX2 in MYOCD-driven splicing we found enrichment of their binding motifs around differentially spliced exons. Moreover, knockdown of either RBPMS or RBFOX2 antagonized splicing events stimulated by MYOCD, including those involving ACTN1, VCL, and MBNL1. Supporting an in vivo role of MYOCD-SRF-driven splicing, we demonstrate altered Rbpms expression and splicing in inducible and SMC-specific Srf knockout mice. We conclude that MYOCD-SRF, in part via RBPMS and RBFOX2, induce a program of differential exon usage and alternative splicing as part of the broader program of SMC differentiation.

Essential requirement for polypyrimidine tract binding proteins 1 and 3 in the maturation and maintenance of mature B cells in mice.

The maturation of immature B cells and the survival of mature B cells is stringently controlled to maintain a diverse repertoire of antibody specificities while avoiding self-reactivity. At the molecular level this is regulated by signaling from membrane Ig and the BAFF-receptor that sustain a pro-survival program of gene expression. Whether and how posttranscriptional mechanisms contribute to B cell maturation and survival remains poorly understood. Here, we show that the polypyrimidine tract binding proteins (PTBP) PTBP1 and PTBP3 bind to a large and overlapping set of transcripts in B cells. Both PTBP1 and PTBP3 bind to introns and exons where they are predicted to regulate alternative splicing. Moreover, they also show high-density of binding to 3' untranslated regions suggesting they influence the transcriptome in diverse ways. We show that PTBP1 and PTBP3 are required in B cells beyond the immature cell stage to sustain transitional B cells and the B1, marginal zone and follicular B cell lineages. Therefore, PTBP1 and PTBP3 promote the maturation of quiescent B cells by regulating gene expression at the posttranscriptional level.

Impact of spliceosome mutations on RNA splicing in myelodysplasia: dysregulated genes/pathways and clinical associations.

SF3B1, SRSF2, and U2AF1 are the most frequently mutated splicing factor genes in the myelodysplastic syndromes (MDS). We have performed a comprehensive and systematic analysis to determine the effect of these commonly mutated splicing factors on pre-mRNA splicing in the bone marrow stem/progenitor cells and in the erythroid and myeloid precursors in splicing factor mutant MDS. Using RNA-seq, we determined the aberrantly spliced genes and dysregulated pathways in CD34+ cells of 84 patients with MDS. Splicing factor mutations result in different alterations in splicing and largely affect different genes, but these converge in common dysregulated pathways and cellular processes, focused on RNA splicing, protein synthesis, and mitochondrial dysfunction, suggesting common mechanisms of action in MDS. Many of these dysregulated pathways and cellular processes can be linked to the known disease pathophysiology associated with splicing factor mutations in MDS, whereas several others have not been previously associated with MDS, such as sirtuin signaling. We identified aberrantly spliced events associated with clinical variables, and isoforms that independently predict survival in MDS and implicate dysregulation of focal adhesion and extracellular exosomes as drivers of poor survival. Aberrantly spliced genes and dysregulated pathways were identified in the MDS-affected lineages in splicing factor mutant MDS. Functional studies demonstrated that knockdown of the mitosis regulators SEPT2 and AKAP8, aberrantly spliced target genes of SF3B1 and SRSF2 mutations, respectively, led to impaired erythroid cell growth and differentiation. This study illuminates the effect of the common spliceosome mutations on the MDS phenotype and provides novel insights into disease pathophysiology.

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.

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.

Intron retention as a component of regulated gene expression programs.

Intron retention has long been an exemplar of regulated splicing with case studies of individual events serving as models that provided key mechanistic insights into the process of splicing control. In organisms such as plants and budding yeast, intron retention is well understood as a major mechanism of gene expression regulation. In contrast, in mammalian systems, the extent and functional significance of intron retention have, until recently, remained greatly underappreciated. Technical challenges to the global detection and quantitation of transcripts with retained introns have often led to intron retention being overlooked or dismissed as "noise". Now, however, with the wealth of information available from high-throughput deep sequencing, combined with focused computational and statistical analyses, we are able to distinguish clear intron retention patterns in various physiological and pathological contexts. Several recent studies have demonstrated intron retention as a central component of gene expression programs during normal development as well as in response to stress and disease. Furthermore, these studies revealed various ways in which intron retention regulates protein isoform production, RNA stability and translation efficiency, and rapid induction of expression via post-transcriptional splicing of retained introns. In this review, we highlight critical findings from these transcriptomic studies and discuss commonalties in the patterns prevalent in intron retention networks at the functional and regulatory levels.

Generation of functionally distinct isoforms of PTBP3 by alternative splicing and translation initiation.

Polypyrimidine tract binding protein (PTBP1) is a widely expressed RNA binding protein that acts as a regulator of alternative splicing and of cytoplasmic mRNA functions. Vertebrates contain two closely-related paralogs with >75% amino acid sequence identity. Early replacement of PTBP1 by PTBP2 during neuronal differentiation causes a concerted set of splicing changes. By comparison, very little is known about the molecular functions or physiological roles of PTBP3, although its expression and conservation throughout the vertebrates suggest a role in haematopoietic cells. To begin to understand its functions we have characterized the mRNA and protein isoform repertoire of PTBP3. Combinatorial alternative splicing events at the 5' end of the gene allow for the generation of eight mRNA and three major protein isoforms. Individual mRNAs generate up to three protein isoforms via alternative translation initiation by re-initiation and leaky scanning using downstream AUG codons. The N-terminally truncated PTBP3 isoforms lack nuclear localization signals and/or most of the RRM1 domain and vary in their RNA binding properties and nuclear/cytoplasmic distribution, suggesting that PTBP3 may have major post-transcriptional cytoplasmic roles. Our findings set the stage for understanding the non-redundant physiological roles of PTBP3.

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.

The organization of RNA contacts by PTB for regulation of FAS splicing.

Post-transcriptional steps of gene expression are regulated by RNA binding proteins. Major progress has been made in characterizing RNA-protein interactions, from high resolution structures to transcriptome-wide profiling. Due to the inherent technical challenges, less attention has been paid to the way in which proteins with multiple RNA binding domains engage with target RNAs. We have investigated how the four RNA recognition motif (RRM) domains of Polypyrimidine tract binding (PTB) protein, a major splicing regulator, interact with FAS pre-mRNA under conditions in which PTB represses FAS exon 6 splicing. A combination of tethered hydroxyl radical probing, targeted inactivation of individual RRMs and single molecule analyses revealed an unequal division of labour between the four RRMs of PTB. RNA binding by RRM4 is the most important for function despite the low intrinsic binding specificity and the complete lack of effect of disrupting individual RRM4 contact points on the RNA. The ordered RRM3-4 di-domain packing provides an extended binding surface for RNA interacting at RRM4, via basic residues in the preceding linker. Our results illustrate how multiple alternative low-specificity binding configurations of RRM4 are consistent with repressor function as long as the overall ribonucleoprotein architecture provided by appropriate di-domain packing is maintained.

A pathway involving HDAC5, cFLIP and caspases regulates expression of the splicing regulator polypyrimidine tract binding protein in the heart.

Polypyrimidine tract binding protein (PTB) regulates pre-mRNA splicing, having special relevance for determining gene expression in the differentiating muscle. We have previously shown that PTB protein abundance is progressively reduced during heart development without reduction of its own transcript. Simultaneous reduction of histone deacetylase (HDAC) expression prompted us to investigate the potential link between these events. HDAC5-deficient mice have reduced cardiac PTB protein abundance, and HDAC inhibition in myocytes causes a reduction in endogenous expression of cellular FLICE-like inhibitory protein (cFLIP) and caspase-dependent cleavage of PTB. In agreement with this, cardiac PTB expression is abnormally high in mice with cardiac-specific executioner caspase deficiency, and cFLIP overexpression prevents PTB cleavage in vitro. Caspase-dependent cleavage triggers further fragmentation of PTB, and these fragments accumulate in the presence of proteasome inhibitors. Experimental modification of the above processes in vivo and in vitro results in coherent changes in the alternative splicing of genes encoding tropomyosin-1 (TPM1), tropomyosin-2 (TPM2) and myocyte enhancer factor-2 (MEF2). Thus, we report a pathway connecting HDAC, cFLIP and caspases regulating the progressive disappearance of PTB, which enables the expression of the adult variants of proteins involved in the regulation of contraction and transcription during cardiac muscle development.

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.

Alternative splicing resulting in nonsense-mediated mRNA decay: what is the meaning of nonsense?

Alternative splicing (AS) strongly affects gene expression by generating protein isoform diversity. However, up to one-third of human AS events create a premature termination codon that would cause the resulting mRNA to be degraded by nonsense-mediated mRNA decay (NMD). The extent to which such events represent functionally selected post-transcriptional gene control, as opposed to noise in the splicing process, has been a contentious issue. Recent analyses indicate that many splicing regulatory proteins are themselves regulated by AS-NMD. Intriguingly, many of these AS-NMD events are coincident with ultraconserved genomic elements, which indicates their importance to vertebrate biology. Examination of these highly conserved events has led to new insights into the functions of AS-NMD and the role it can have in physiological circumstances.