Search

The GGGGCC hexanucleotide expansion in C9orf72 (C9) is the most frequent known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet a clear understanding of how C9 fits into the broader context of ALS/FTD pathology has remained lacking. The repetitive RNA derived from the C9 repeat is known to sequester hnRNPH, a splicing regulator, into insoluble aggregates, resulting in aberrant alternative splicing. Furthermore, hnRNPH insolubility and altered splicing of a robust set of targets have been observed to correlate in C9 and sporadic ALS/FTD patients alike, suggesting that changes along this axis are a core feature of disease pathogenesis. Here, we characterize previously uncategorized RNA splicing defects involving widespread intron retention affecting almost 2000 transcripts in C9ALS/FTD brains exhibiting a high amount of sequestered, insoluble hnRNPH. These intron retention events appear not to alter overall expression levels of the affected transcripts but rather the protein-coding regions. These retained introns affect transcripts in multiple cellular pathways predicted to be involved in C9 as well as sporadic ALS/FTD etiology, including the proteasomal and autophagy systems. The retained intron pre-mRNAs display a number of characteristics, including enrichment of hnRNPH-bound splicing enhancer motifs and a propensity for G-quadruplex (G-Q) formation, linking the defective splicing directly to high amounts of sequestered hnRNPH. Together, our results reveal previously undetected splicing defects in high insoluble hnRNPH-associated C9ALS brains, suggesting a feedback between effective RNA-binding protein dosage and protein quality control in C9, and perhaps all, ALS/FTD.

Burkitt lymphoma (BL) is an aggressive B-cell lymphoma characterized by translocation and deregulation of the proto-oncogene c-MYC. Transcription factor 3 (TCF3) has also been shown to be involved in BL pathogenesis. In BL, TCF3 is constitutively active, and/or expression of its transcriptional targets are altered as a result of BL-associated mutations. Here, we found that BL-related TCF3 mutations affect TCF3 alternative splicing, in part by reducing binding of the splicing regulator hnRNPH1 to exon 18b. This leads to greater exon 18b inclusion, thereby generating more of the mutated E47 isoform of TCF3. Interestingly, upregulation of E47 dysregulates the expression of TCF3 targets PTPN6, and perhaps CCND3, which are known to be involved in BL pathogenesis. Our findings thus reveal a mechanism by which TCF3 somatic mutations affect multilayered gene regulation underlying BL pathogenesis.

The gene encoding the core spliceosomal protein SF3B1 is the most frequently mutated gene encoding a splicing factor in a variety of hematologic malignancies and solid tumors. SF3B1 mutations induce use of cryptic 3' splice sites (3'ss), and these splicing errors contribute to tumorigenesis. However, it is unclear how widespread this type of cryptic 3'ss usage is in cancers and what is the full spectrum of genetic mutations that cause such missplicing. To address this issue, we performed an unbiased pan-cancer analysis to identify genetic alterations that lead to the same aberrant splicing as observed with SF3B1 mutations. This analysis identified multiple mutations in another spliceosomal gene, SUGP1, that correlated with significant usage of cryptic 3'ss known to be utilized in mutant SF3B1 expressing cells. Remarkably, this is consistent with recent biochemical studies that identified a defective interaction between mutant SF3B1 and SUGP1 as the molecular defect responsible for cryptic 3'ss usage. Experimental validation revealed that five different SUGP1 mutations completely or partially recapitulated the 3'ss defects. Our analysis suggests that SUGP1 mutations in cancers can induce missplicing identical or similar to that observed in mutant SF3B1 cancers.

SF3B1 is the most frequently mutated RNA splicing factor in multiple neoplasms, including ~25% of myelodysplastic syndromes (MDS) patients. Mortality in MDS frequently results from severe anemia, but the underlying mechanism is largely unknown. Here we elucidate the detailed, elusive pathway by which SF3B1 mutations cause anemia. We demonstrate, in CRISPR-edited cell models, normal human primary cells, and MDS patient cells, that mutant SF3B1 induces a splicing error in transcripts encoding the kinase MAP3K7, resulting in reduced MAP3K7 protein levels and deactivation of downstream target p38 MAPK. We show that disruption of this MAP3K7-p38 MAPK pathway leads to premature downregulation of GATA1, a master regulator of erythroid differentiation, and that this is sufficient to trigger accelerated differentiation and apoptosis. As a result, the overproduced, late staged erythroblasts undergo apoptosis and are unable to mature in the bone marrow. Our findings provide a detailed mechanism explaining the origins of anemia in MDS patients harboring SF3B1 mutations.

The gene encoding the core spliceosomal protein SF3B1 is the most frequently mutated gene encoding a splicing factor in a variety of hematologic malignancies and solid tumors. SF3B1 mutations induce use of cryptic 3′ splice sites (3′ss), and these splicing errors contribute to tumorigenesis. However, it is unclear how widespread this type of cryptic 3′ss usage is in cancers and what is the full spectrum of genetic mutations that cause such missplicing. To address this issue, we performed an unbiased pan-cancer analysis to identify genetic alterations that lead to the same aberrant splicing as observed with SF3B1 mutations. This analysis identified multiple mutations in another spliceosomal gene, SUGP1, that correlated with significant usage of cryptic 3′ss known to be utilized in mutant SF3B1 expressing cells. Remarkably, this is consistent with recent biochemical studies that identified a defective interaction between mutant SF3B1 and SUGP1 as the molecular defect responsible for cryptic 3′ss usage. Experimental validation revealed that five different SUGP1 mutations completely or partially recapitulated the 3′ss defects. Our analysis suggests that SUGP1 mutations in cancers can induce missplicing identical or similar to that observed in mutant SF3B1 cancers.

Alternative splicing (AS) plays important roles in embryonic stem cell (ESC) differentiation. In this study, we first identified transcripts that display specific AS patterns in pluripotent human ESCs (hESCs) relative to differentiated cells. One of these encodes transcription factor 3 (TCF3), a transcription factor that plays important roles in ESC differentiation. AS creates two TCF3 isoforms, E12 and E47, and we identified two related splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNPs) H1 and F (hnRNP H/F), that regulate TCF3 splicing. We found that hnRNP H/F levels are high in hESCs, leading to high E12 expression, but decrease during differentiation, switching splicing to produce elevated E47 levels. Importantly, hnRNP H/F knockdown not only recapitulated the switch in TCF3 AS but also destabilized hESC colonies and induced differentiation. Providing an explanation for this, we show that expression of known TCF3 target E-cadherin, critical for maintaining ESC pluripotency, is repressed by E47 but not by E12.

Amyotrophic lateral sclerosis (ALS) is caused by mutations in a number of genes, including the gene encoding the RNA/DNA-binding protein translocated in liposarcoma or fused in sarcoma (TLS/FUS or FUS). Previously, we identified a number of FUS target genes, among them MECP2. To investigate how ALS mutations in FUS might impact target gene expression, we examined the effects of several FUS derivatives harboring ALS mutations, such as R521C (FUSC), on MECP2 expression in transfected human U87 cells. Strikingly, FUSC and other mutants not only altered MECP2 alternative splicing but also markedly increased mRNA abundance, which we show resulted from sharply elevated stability. Paradoxically, however, MeCP2 protein levels were significantly reduced in cells expressing ALS mutant derivatives. Providing a parsimonious explanation for these results, biochemical fractionation and in vivo localization studies revealed that MECP2 mRNA colocalized with cytoplasmic FUSC in insoluble aggregates, which are characteristic of ALS mutant proteins. Together, our results establish that ALS mutations in FUS can strongly impact target gene expression, reflecting a dominant effect of FUS-containing aggregates.

SF3B1, which encodes an essential spliceosomal protein, is frequently mutated in myelodysplastic syndromes (MDS) and many cancers. However, the defect of mutant SF3B1 is unknown. Here, we analyzed RNA-sequencing data from MDS patients and confirmed that SF3B1 mutants use aberrant 3′ splice sites. To elucidate the underlying mechanism, we purified complexes containing either wild-type or the hotspot K700E mutant SF3B1, and found that levels of a poorly studied spliceosomal protein, SUGP1, were reduced in mutant spliceosomes. Strikingly, SUGP1 knockdown completely recapitulated the splicing errors, whereas SUGP1 overexpression drove the protein, which our data suggests plays an important role in branchsite recognition, into the mutant spliceosome and partially rescued splicing. Other hotspot SF3B1 mutants showed similar altered splicing and diminished interaction with SUGP1. Our study demonstrates that SUGP1 loss is a common defect of spliceosomes with disease-causing SF3B1 mutations and, since this defect can be rescued, suggests possibilities for therapeutic intervention.

SUMO has gained prominence as a regulator in a number of cellular processes. The roles of sumoylation in RNA metabolism, however, while considerable, remain less well understood. In this chapter we have assembled data from proteomic analyses, localization studies and key functional studies to extend SUMO’s role to the area of mRNA processing and metabolism. Proteomic analyses have identified multiple putative sumoylation targets in complexes functioning in almost all aspects of mRNA metabolism, including capping, splicing and polyadenylation of mRNA precursors. Possible regulatory roles for SUMO have emerged in pre-mRNA 3′ processing, where SUMO influences the functions of polyadenylation factors and activity of the entire complex. SUMO is also involved in regulating RNA editing and RNA binding by hnRNP proteins, and recent reports have suggested the involvement of the SUMO pathway in mRNA export. Together, these reports suggest that SUMO is involved in regulation of many aspects of mRNA metabolism and hold the promise for exciting future studies.

An expanded GGGGCC hexanucleotide in C9ORF72 (C9) is the most frequent known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). It has been proposed that expanded transcripts adopt G-quadruplex (G-Q) structures and associate with proteins, but whether this occurs and contributes to disease is unknown. Here we show first that the protein that predominantly associates with GGGGCC repeat RNA in vitro is the splicing factor hnRNP H, and that this interaction is linked to G-Q formation. We then show that G-Q RNA foci are more abundant in C9 ALS patient fibroblasts and astrocytes compared to those without the expansion, and more frequently colocalize with hnRNP H. Importantly, we demonstrate dysregulated splicing of multiple known hnRNP H-target transcripts in C9 patient brains, which correlates with elevated insoluble hnRNP H/G-Q aggregates. Together, our data implicate C9 expansion-mediated sequestration of hnRNP H as a significant contributor to neurodegeneration in C9 ALS/FTD. DOI: http://dx.doi.org/10.7554/eLife.17820.001

Expression of the mammalian pyruvate kinase M (PKM) gene provides an important example of mutually exclusive splicing. We showed previously that the hnRNP proteins A1, A2 and PTB play a critical role in this process. Here we provide evidence that concentration-dependent interactions involving a network of these proteins are sufficient to determine the outcome of PKM splicing. At high concentrations, such as found in most cancer cells, hnRNP A1 binding to two sites in the upstream regulated exon (exon 9) orchestrates cooperative interactions leading to exon 9 exclusion. At lower concentrations, binding shifts to downstream intronic sites such that exon 9 is included and exon 10 largely excluded, with any mRNA including both exons degraded by nonsense-mediated decay. Together our results provide a mechanism by which a small number of general factors control alternative splicing of a widely expressed transcript.

Gene regulation in response to environmental stress is critical for the survival of all organisms. From Saccharomyces cerevisiae to humans, it has been observed that splicing of mRNA precursors is repressed upon heat shock. However, a mild heat pretreatment often prevents splicing inhibition in response to a subsequent and more severe heat shock, a phenomenon called splicing thermotolerance. We have shown previously that the splicing regulator SRSF10 (formerly SRp38) is specifically dephosphorylated by the phosphatase PP1 in response to heat shock and that dephosphorylated SRSF10 is responsible for splicing repression caused by heat shock. Here we report that a mild heat shock protects SRSF10 from dephosphorylation during a second and more severe heat shock. Furthermore, this “thermotolerance” of SRSF10 phosphorylation, like that of splicing, requires de novo protein synthesis, specifically the synthesis of heat shock proteins. Indeed, overexpression of one of these proteins, Hsp27, inhibits SRSF10 dephosphorylation in response to heat shock and does so by interaction with SRSF10. Our data thus provide evidence that splicing thermotolerance is acquired through maintenance of SRSF10 phosphorylation and that this is mediated at least in part by Hsp27.

Translocated in liposarcoma, Ewing's sarcoma and TATA-binding protein-associated factor 15 constitute an interesting and important family of proteins known as the TET proteins. The proteins function in several aspects of cell growth control, including multiple different steps in gene expression, and they are also found mutated in a number of specific diseases. For example, all contain domains for binding nucleic acids and have been shown to function in both RNA polymerase II-mediated transcription and pre-mRNA splicing, possibly connecting these two processes. Chromosomal translocations in human sarcomas result in a fusion of the amino terminus of these proteins, which contains a transcription activation domain, to the DNA-binding domain of a transcription factor. Although the fusion proteins have been characterized in a clinical environment, the function of the cognate full-length protein in normal cells is a more recent topic of study. The first part of this review will describe the TET proteins, followed by detailed descriptions of their multiple roles in cells. The final sections will examine changes that occur in gene regulation in cells expressing the fusion proteins. The clinical implications and treatment of sarcomas will not be addressed but have recently been reviewed.

Since direct analysis of many aspects of spliceosomal function is greatly hindered by the daunting complexity of the spliceosome, the development of functionally validated simple model systems can be of great value. The critical role played by a base-paired complex of U6 and U2 snRNAs in splicing in vivo suggests that this complex could be a suitable starting point for the development of such a simple model system. However, several criteria must be satisfied before such a snRNA-based in vitro system can be considered a valid model for the spliceosomal catalytic core, including similarities at the level of reaction chemistry and cationic and sequence requirements. Previous functional analyses of in vitro assembled base-paired complexes of human U2 and U6 snRNAs have been promising, providing insight into catalysis. Furthermore, they strongly suggest that with further optimization, these RNAs might indeed be able to recapitulate the function of the spliceosomal catalytic core, thus opening the door to several lines of study not previously possible.

SRp38 is an atypical SR protein that functions as a general splicing repressor when dephosphorylated. We now show that phosphorylated SRp38 functions as a sequence-specific splicing activator. Unlike characterized splicing activators, SRp38 functions in the absence of other SR proteins but requires a cofactor for activity. SRp38 was able to induce formation of splicing complex A in the absence of the cofactor, but this factor was necessary for progression to complexes B and C. Mechanistically, SRp38 strengthens the ability of the U1 and U2 small nuclear ribonucleoproteins to stably recognize the pre-mRNA. Extending these findings, analysis of alternative splicing of pre-mRNA encoding the glutamate receptor B revealed that SRp38 alters its splicing pattern in a sequence-specific manner. Together, our data demonstrate that SRp38, in addition to its role as a splicing repressor, can function as an unusual sequence-specific splicing activator.

Complex multicellular organisms require a diverse set of proteins to set the form and function of specialized cell types. The availability of complete genomic sequences has revealed that instead of a large increase in the number of protein coding genes compared with unicellular organisms, more complex eukaryotes instead obtain more diversity out of a relatively limited number of genes through the process of alternative splicing (AS). AS results in the cell type-, developmental stage-, sex-, or signal-regulated changes in composition of an mRNA produced from a given gene, brought about by changes in splice site choice (Black 2003; Matlin et al. 2005). There are many different types of AS events, ranging from the tissue-specific inclusion of a cassette exon to the Dscam gene in Drosophila, which contains four clusters of exons containing 12, 48, 33, and 2 mutually exclusive variants, an extreme example of AS complexity (Bharadwaj and Kolodkin 2006). Additionally, it is clear from the relatively small number of AS events that have been studied extensively at a mechanistic level that regulation of AS takes many forms, as will be discussed in more detail later in this article. Lastly, the functional outcomes of AS vary greatly, from effectively turning off a gene (the result of including an exon containing a premature stop codon, for example), to a subtle change in a protein’s function. In spite of this complexity, the goal of understanding the changes in AS patterns in terms of changes in expression and regulation of factors that regulate AS across cell types appears achievable. This is in part because recent technical advances allow us, starting with an individual splicing factor, to determine its genome-wide role in AS regulation. However, the ambitious goal of determining a cellular code for AS will be impossible to realize without a complete list of AS regulators, and currently there is no reason to believe we are anywhere near completing such a list in any metazoan. Kuroyanagi et al. (2006) have developed recently a system in Caenorhabditis elegans, which allows for the straightforward visualization of individual AS events in vivo, which in turn allows for the identification of mutants defective in regulating the event. Using this approach, Kuroyanagi and colleagues (Kuroyanagi et al. 2006, 2007; Ohno et al. 2008) have identified four proteins that regulate two different AS events, a feat that would be laborious using in vitro approaches. Three of these proteins had not been shown previously to participate in regulation of AS, indicating that we may be far from a complete list of AS regulators. Their data also highlight the fact that regulators of AS are often highly conserved throughout metazoans, meaning that filling the list of splicing regulators in C. elegans will likely contribute to filling our own.

Tri-methylation of histone H3 on lysine 4 (H3K4me3) localizes near the 5′ region of genes and is tightly associated with active loci. Several proteins, such as CHD1, BPTF, JMJD2A, and the ING tumor suppressor family, directly recognize this lysine methyl mark. However, how H3K4me3 recognition participates in active transcription remains poorly characterized. Here we identify specific CHD1-interacting proteins via H3K4me3 affinity purification, including numerous factors mediating post-initiation events. Conventional biochemical purification revealed a stable complex between CHD1 and components of the spliceosome. Depletion of CHD1 in extracts dramatically reduced splicing efficiency in vitro, indicating a functional link between CHD1 and the spliceosome. Knockdown of CHD1 and H3K4me3 levels by siRNA reduced association of U2 snRNP components with chromatin, and more importantly, altered the efficiency of pre-mRNA splicing on active genes in vivo. These findings suggest that methylated H3K4 serves to facilitate the competency of pre-mRNA maturation through the bridging of spliceosomal components to H3K4me3 via CHD1.

Formation of transcription-induced R-loops poses a critical threat to genomic integrity throughout evolution. We have recently shown that the SR protein ASF/SF2 prevents R-loop formation in vertebrates by cotranscriptionally binding to nascent mRNA precursors to prevent their reassociation with template DNA. Here, we identify another RNA binding protein, RNPS1, that when overexpressed strongly suppresses the high molecular weight (HMW) DNA fragmentation, hypermutation, and G2 cell cycle arrest phenotypes of ASF/SF2-depleted cells. Furthermore, ablation of RNPS1 by RNA interference in HeLa cells leads to accumulation of HMW DNA fragments. As ASF/SF2 depletion does not influence RNPS1 expression, and RNPS1 cannot compensate for ASF/SF2 function in splicing, our data suggest that RNPS1 is able to function together with ASF/SF2 to form RNP complexes on nascent transcripts, and thereby prevent formation of transcriptional R-loops.

Splicing of introns from mRNA precursors is a two-step reaction performed by the spliceosome, an immense cellular machine consisting of over 200 different proteins and five small RNAs (snRNAs). We previously demonstrated that fragments of two of these RNAs, U6 and U2, can catalyze by themselves a splicing-related reaction, involving one of the two substrates of the first step of splicing, the branch site substrate. Here we show that these same RNAs can catalyze a reaction between RNA sequences that resemble the 5′ splice site and the branch site, the two reactants of the first step of splicing. The reaction is dependent on the sequence of the 5′ splice site consensus sequence and the catalytically essential domains of U6, and thus it resembles the authentic splicing reaction. Our results demonstrate the ability of protein-free snRNAs to recognize the sequences involved in the first splicing step and to perform splicing-related catalysis between these two pre-mRNA-like substrates.

The neurodegenerative disease spinal muscular atrophy is caused by mutation of the survival motor neuron 1 ( SMN1 ) gene. SMN2 is a nearly identical copy of SMN1 that is unable to prevent disease, because most SMN2 transcripts lack exon 7 and thus produce a nonfunctional protein. A key cause of inefficient SMN2 exon 7 splicing is a single nucleotide difference between SMN1 and SMN2 within exon 7. We previously provided evidence that this base change suppresses exon 7 splicing by creating an inhibitory element, a heterogeneous nuclear ribonucleoprotein (hnRNP) A1-dependent exonic splicing silencer. We now find that another rare nucleotide difference between SMN1 and SMN2 , in intron 7, potentially creates a second SMN2 -specific hnRNP A1 binding site. Remarkably, this single base change does indeed create a high-affinity hnRNP A1 binding site, and base substitutions that disrupt it restore exon 7 inclusion in vivo and prevent hnRNP A1 binding in vitro . We propose that interactions between hnRNP A1 molecules bound to the exonic and intronic sites cooperate to exclude exon 7 and discuss the significance of this exclusion with respect to SMN expression and splicing control more generally.

Serine/arginine-rich splicing factor 2 (SRSF2) is an RNA-binding protein that plays important roles in splicing of mRNA precursors. SRSF2 mutations are frequently found in patients with myelodysplastic syndromes and certain leukemias, but how these mutations affect SRSF2 function has only begun to be examined. We used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease to introduce the P95H mutation to SRSF2 in K562 leukemia cells, generating an isogenic model so that splicing alterations can be attributed solely to mutant SRSF2. We found that SRSF2 (P95H) misregulates 548 splicing events (

Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors., Author Summary A gene can express multiple isoforms varying in the 3’ end, a phenomenon called alternative cleavage and polyadenylation, or APA. Previous studies have indicated that most eukaryotic genes display APA and the APA profile changes under different physiological and pathological conditions. However, how APA is regulated in the cell is unclear. Here using gene knockdown and high throughput sequencing we examine how APA is regulated by factors in the machinery responsible for cleavage and polyadenylation as well as factors that play essential roles in splicing. We identify several factors that play significant roles in APA in the last exon, including CFI-25/68, PABPN1, PABPC1, Fip1 and Pcf11. We also elucidate how cleavage and polyadenylation events are regulated in introns and near the transcription start site. We uncover a group of APA events that are highly regulated by core factors as well as in cell differentiation and development. We present an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the transcription start site, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core cleavage and polyadenylation factors.

The cellular response to stresses such as heat shock involves changes in gene expression. It is well known that the splicing of messenger RNA precursors is generally repressed on heat shock, but the factors responsible have not been identified. SRp38 is an SR protein splicing factor that functions as a general repressor of splicing. It is activated by dephosphorylation and required for splicing repression in M-phase cells. Here we show that SRp38 is also dephosphorylated on heat shock and that this dephosphorylation correlates with splicing inhibition. Notably, depletion of SRp38 from heat-shocked cell extracts derepresses splicing, and adding back dephosphorylated SRp38 specifically restores inhibition. We further show that dephosphorylated SRp38 interacts with a U1 small nuclear ribonucleoprotein particle (snRNP) protein, and that this interaction interferes with 5'-splice-site recognition by the U1 snRNP. Finally, SRp38-deficient DT40 cells show an altered cell-cycle profile consistent with a mitotic defect; they are also temperature sensitive and defective in recovery after heat shock. SRp38 thus plays a crucial role in cell survival under stress conditions by inhibiting the splicing machinery.

Removal of introns from pre-messenger RNAs in eukaryotes is carried out by the spliceosome, an assembly of a large number of proteins and five small nuclear RNAs (snRNAs). We showed previously that an in vitro transcribed and assembled base-paired complex of U2 and U6 snRNA segments catalyzes a reaction that resembles the first step of splicing. Upon incubation with a short RNA oligonucleotide containing the consensus sequence of the pre-mRNA branch site, the U2/U6 complex catalyzed a reaction between the 2′ OH of a bulged adenosine and a phosphate in the catalytically important AGC triad of U6, leading to the formation of an X-shaped product, RNA X, apparently linked by an unusual phosphotriester bond. Here we characterize this splicing-related reaction further, showing that RNA X formation is an equilibrium reaction, and that the low yield of the reaction likely reflects an unfavorable equilibrium coefficient. Consistent with a phosphotriester linkage, RNA X is highly alkali-sensitive, but only mildly acid-sensitive. We also show that mutations in the AGC sequence of U6 can have significant effects on RNA X formation, further extending the similarities between splicing and RNA X formation. We also demonstrate that pseudouridylation of U2 enhances RNA X formation, and that U6 snRNA purified from nuclear extracts is capable of forming RNA X. Our data suggest that the ability to form RNA X might be an intrinsic property of spliceosomal snRNAs.

SR proteins constitute a family of splicing factors that play key roles in both constitutive and regulated splicing in metazoan organisms. The proteins are extensively phosphorylated, and kinases capable of phosphorylating them have been identified. However, little is known about how these kinases function, for example, whether they target specific SR proteins or whether the kinases themselves are regulated. Here we describe properties of one such kinase, Clk/Sty, the founding member of the Clk/Sty family of dual-specificity kinases. Clk/Sty is autophosphorylated on both Ser/Thr and Thr residues, and using both direct kinase assays and SR protein-dependent splicing assays, we have analyzed the effects of each type of modification. We find not only that the pattern of phosphorylation on a specific SR protein substrate, ASF/SF2, is modulated by autophosphorylation but also that the ability of Clk/Sty to recognize different SR proteins is influenced by the extent and nature of autophosphorylation. Strikingly, phosphorylation of ASF/SF2 is sensitive to changes in Tyr, but not Ser/Thr, autophosphorylation while that of SC35 displays the opposite pattern. In contrast, phosphorylation of a third SR protein, SRp40, is unaffected by autophosphorylation. We also present biochemical data indicating that as expected for a factor directly involved in splicing control (but in contrast to recent reports), Clk/Sty is found in the nucleus of several different cell types.

A novel SR protein-specific kinase (SRPK) from the SRPK family was identified for the first time in a protozoan organism. The primary structure of the protein, named TcSRPK, presents a significant degree of identity with other metazoan members of the family. In vitro phosphorylation experiments showed that TcSRPK has the same substrate specificity relative to other SRPKs. TcSRPK was able to generate a mAb104-recognized phosphoepitope, a SRPK landmark. Expression of TcSRPK in different Schizosaccharomyces pombe strains lead to conserved phenotypes, indicating that TcSRPK is a functional homologue of metazoan SRPKs. In functional alternative splicing assays in vivo in HeLa cells, TcSRPK enhanced SR protein-dependent inclusion of the EDI exon of the fibronectin minigene. When tested in vitro, it inhibited splicing either on nuclear extracts or on splicing-deficient S100 extracts complemented with ASF/SF2. This inhibition was similar to that observed with human SRPK1. This work constitutes the first report of a member of this family of proteins and the existence of an SR-network in a protozoan organism. The implications in the origins and control of splicing are discussed.

SR proteins constitute a family of pre-mRNA splicing factors that play important roles in both constitutive and regulated splicing. Here, we describe one member of the family, which we call SRp38, with unexpected properties. Unlike other SR proteins, SRp38 cannot activate splicing and is essentially inactive in splicing assays. However, dephosphorylation converts SRp38 to a potent, general repressor that inhibits splicing at an early step. To investigate the cellular function of SRp38, we examined its possible role in cell cycle control. We show first that splicing, like other steps in gene expression, is inhibited in extracts of mitotic cells. Strikingly, SRp38 was found to be dephosphorylated specifically in mitotic cells, and we show that dephosphorylated SRp38 is required for the observed splicing repression.

The 3′ ends of most eukaryotic messenger RNAs must undergo a maturation step that includes an endonuc-leolytic cleavage followed by addition of a polyadenylate tail. While this reaction is catalyzed by the action of only two enzymes it is supported by an unexpectedly large number of proteins. This complexity reflects the necessity of coordinating this process with other nuclear events, and growing evidence indicates that even more factors than previously thought are necessary to connect 3′ processing to additional cellular pathways. In this review we summarize the current understanding of the molecular machinery involved in this step of mRNA maturation, focusing on new core and auxiliary proteins that connect polyadenylation to splicing, DNA damage, transcription and cancer.

Removal of intervening sequences from eukaryotic messenger RNA precursors is carried out by the spliceosome, a complex assembly of five small nuclear RNAs (snRNAs) and a large number of proteins. Although it has been suggested that the spliceosome might be an RNA enzyme, direct evidence for this has been lacking, and the identity of the catalytic domain of the spliceosome is unknown. Here we show that a protein-free complex of two snRNAs, U2 and U6, can bind and position a small RNA containing the sequence of the intron branch site, and activate the branch adenosine to attack a catalytically critical domain of U6 in a reaction that is related to the first step of splicing. Our data provide direct evidence for the catalytic potential of spliceosomal snRNAs.

In human papillomaviruses, expression of the late genes L1 and L2, encoding the capsid proteins, is restricted to the upper layers of the infected epithelium. A 79-nt GU-rich negative regulatory element (NRE) located at the 3′ untranslated region of the human papillomavirus 16 L1 gene was identified previously as key to the posttranscriptional control of late gene expression. Here, we demonstrate that in epithelial cells, the NRE can directly bind the U2 auxiliary splicing factor 65-kDa subunit, the cleavage stimulation factor 64-kDa subunit, and the Elav-like HuR protein. On induction of epithelial cell differentiation, levels of the U2 auxiliary splicing factor 65-kDa subunit decrease, levels of the cleavage stimulation factor 64-kDa subunit increase, and the levels of HuR remain unchanged, although redistribution of the HuR from the nucleus to the cytoplasm is observed. Late gene transcripts, which appear to be fully processed, are detected in undifferentiated W12 cells, but are confined in the nucleus. We propose that repression of late gene expression in basal epithelial cells may be caused by nuclear retention or cytoplasmic instability of NRE-containing late gene transcripts.

U2 and U6 small nuclear RNAs are thought to play critical roles in pre-mRNA splicing catalysis. Genetic evidence suggests they form an extensively base-paired structure within the spliceosome that is required for catalysis. Especially in light of significant similarities with group II self-splicing introns, we wished to investigate whether the purified RNAs might by themselves be able to form a complex similar to that which appears to exist in the spliceosome. To this end, we synthesized and purified large segments of human U2 and U6 snRNAs. Upon annealing, the two RNAs efficiently formed a stable and apparently extensively base-paired (Tm = 50-60 degrees C in the presence of 20 mM Mg2+) complex. To investigate possible tertiary interactions, we subjected the annealed complex to UV irradiation, and two crosslinked species were identified and characterized. The major one links the second G in the highly conserved and critical ACAGAGA sequence in U6 with an A in U2 just 5' to U2-U6 helix Ia and opposite the invariant AGC in U6. Remarkably, this crosslink indicates a tertiary interaction essentially identical to one detected previously by genetic covariation in yeast. Together our results suggest that purified U2 and U6 snRNAs can anneal and fold to form a structure resembling that likely to exist in the catalytically active spliceosome.

The SR (Ser-Arg) proteins are a family of nuclear factors that play multiple important roles in splicing of mRNA precursors in metazoan organisms, functioning in both constitutive and regulated splicing. Certain of these functions are redundant, such that any single SR proteins will suffice, but other functions are unique and are specific to a given family member. A number of studies during the past year have investigated the basis for SR protein specificity.

Alternative pre-mRNA splicing is a powerful mechanism of gene expression control in eukaryotic cells that can influence cell function, cellular differentiation, and development. In some instances, as exemplified by the sex-specific regulation of transformer splicing by the Sex-lethal (Sxl) gene product in Drosophila, it may be used as an on/off switch of gene expression, mimicking transcriptional control (1). Other examples, like the cell-specific splicing of the calcitonin/CGRP pre-mRNA (2) or the sex-specific splicing of the Drosophila doublesex pre-mRNA (1), illustrate that alternative splicing provides an economical way to switch between mutually exclusive gene functions in a cell- or stage-specific manner. Its extraordinary versatility in the regulation of gene function is amply demonstrated by recent findings suggesting that electrical tuning of the hair cells in the inner ear of the chick is accomplished in part through variations in the splicing of the cSlo primary transcript, which has the potent...

U6 snRNA is the most conserved of all the snRNAs involved in pre-mRNA splicing, and likely plays an important role in splicing catalysis. Using a U6 snRNA fragment encompassing residues 25-99, we have identified a strong, UV-sensitive tertiary intramolecular interaction. A 5' deletion that removed sequences up to nt 37 only slightly reduced crosslinking, but further deletion of 11 bases, eliminating the nearly invariant ACAGAGA sequence, essentially abolished crosslinking, as did deletion of sequences 3' of 82A. The crosslinked residues were mapped to 44G in the ACAGAGA sequence and to 81C, the nucleotide at the base of the U6 intramolecular helix, opposite the G of the invariant AGC trinucleotide. This interaction is striking in that it has the potential to juxtapose invariant regions of U6 believed to play critical roles in splicing catalysis.

The RNA-binding protein Tra2 is an important regulator of sex determination in Drosophila. Recently, two mammalian Tra2 homologs of unknown function have been described. Here, we show that human Tra2 proteins are present in HeLa cell nuclear extracts and that they bind efficiently and specifically to a previously characterized pre-mRNA splicing enhancer element. Indeed, both purified proteins bound preferentially to RNA sequences containing GAA repeats, characteristic of many enhancer elements. Neither Tra2 protein functioned in constitutive splicing in vitro, but both activated enhancer-dependent splicing in a sequence-specific manner and restored it after inhibition with competitor RNA. Our findings indicate that mammalian Tra2 proteins are sequence-specific splicing activators that likely participate in the control of cell-specific splicing patterns.

Splicing factor SRSF10 is known to function as a sequence-specific splicing activator. Here, we used RNA-seq coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Motif analysis revealed that SRSF10 binding to cassette exons was associated with exon inclusion, whereas the binding of SRSF10 within downstream constitutive exons was associated with exon exclusion. This positional effect was further demonstrated by the mutagenesis of potential SRSF10 binding motifs in two minigene constructs. Functionally, many of SRSF10-verified alternative exons are linked to pathways of stress and apoptosis. Consistent with this observation, cells depleted of SRSF10 expression were far more susceptible to endoplasmic reticulum stress-induced apoptosis than control cells. Importantly, reconstituted SRSF10 in knockout cells recovered wild-type splicing patterns and considerably rescued the stress-related defects. Together, our results provide mechanistic insight into SRSF10-regulated alternative splicing events in vivo and demonstrate that SRSF10 plays a crucial role in cell survival under stress conditions.

We showed previously that ASF/SF2, a member of the SR protein family of splicing factors, can activate a splicing enhancer element composed of high-affinity ASF/SF2 binding sites. To determine whether other SR proteins can behave similarly, we selected a high-affinity RNA-binding site (B1) for the SR protein SRp40. Strikingly, the success of this selection was completely dependent on phosphorylation of the RS domain, as unphosphorylated SRp40 failed to select specific sequences. We show that three copies of B1 function as a strong splicing enhancer, activating an intron with suboptimal splicing signals in nuclear extracts. Enhancer activity in S100 extracts (which lack SR proteins) required SRp40 and a nuclear fraction previously found to be required for ASF/SF2-dependent splicing. Importantly, enhancer activity was lost when SRp40 was replaced by ASF/SF2 or SC35, and SRp40 was the only classical SR protein found to be associated with the enhancer. Together, our results indicate that phosphorylation-dependent, sequence-specific RNA binding can impart unique activities to individual SR proteins.

ASF/SF2 is a well-studied SR protein that plays important roles in premRNA splicing and other aspects of RNA metabolism. Genetic inactivation experiments have revealed the fundamental roles of ASF/SF2 and other SR proteins in cell viability and animal development. However, the nature of the events triggered by in vivo depletion of ASF/SF2 remained largely elusive. Recently, we have demonstrated a significant function of ASF/SF2 in the maintenance of genome stability by preventing the formation of R loops, which provided new insights into the essential roles of ASF/SF2 in cellular physiology.

Alternative splicing of mRNA precursors enables one gene to produce multiple protein isoforms with differing functions. Under normal conditions, this mechanism is tightly regulated in order for the human genome to generate proteomic diversity sufficient for the functional requirements of complex tissues. When deregulated, however, cancer cells take advantage of this mechanism to produce aberrant proteins with added, deleted, or altered functional domains that contribute to tumorigenesis. Here, we discuss aspects of alternative splicing misregulation in cancer, focusing on splicing events affected by deregulation of regulatory splicing factors and also recent studies identifying mutated components of the splicing machinery. Significance: An increasing body of evidence indicates that aberrant splicing of mRNA precursors leads to production of aberrant proteins that contribute to tumorigenesis. Recent studies show that alterations in cellular concentrations of regulatory splicing factors and mutations in components of the core splicing machinery provide major mechanisms of misregulation of mRNA splicing in cancer. A better understanding of this misregulation will potentially reveal a group of novel drug targets for therapeutic intervention. Cancer Discov; 3(11); 1228–37. ©2013 AACR.

Splicing of mRNA precursors consists of two steps that are almost invariably tightly coupled to facilitate efficient generation of spliced mRNA. However, we described previously a splicing substrate that is completely blocked after the first step. We have now investigated the basis for this unusual second-step inhibition and unexpectedly elucidated two independent mechanisms. One involves a stem–loop structure located downstream of the 3′splice site, and the other involves an exonic splicing silencer (ESS) situated 3′ to the structure. Both elements contribute to the second-step block in vitro and also cause exon skipping in vivo. Importantly, we identified far upstream element-binding protein 1 (FUBP1), a single-stranded DNA- and RNA-binding protein not previously implicated in splicing, as a strong ESS binding protein, and several assays implicate it in ESS function. We demonstrate using depletion/add-back experiments that FUBP1 acts as a second-step repressor in vitro and show by siRNA-mediated knockdown and overexpression assays that it modulates exon inclusion in vivo. Together, our results provide additional insights into splicing control, and identify FUBP1 as a splicing regulator.

Multiple forms of poly(A) polymerase (PAPs I, II, and III) cDNA have previously been isolated from bovine, human, and/or frog cDNA libraries. PAPs I and II are long forms of the enzyme that contain four functional domains: an apparent ribonucleoprotein-type RNA-binding domain, a catalytic region that may be related to the polymerase module, two nuclear localization signals (NLSs I and 2), and a C-terminal Ser/Thr-rich region. PAP III would encode a truncated protein that lacks the NLSs and the S/T-rich region. To investigate further the structure and expression of these forms, we isolated the mouse PAP gene and an intronless pseudogene from a mouse liver genomic library. The structure of the gene indicates that different forms of PAP are produced by alternative splicing (PAPs I and II) or by competition between polyadenylation and splicing (PAP III). The pseudogene appears to reflect yet another form of long PAP, which we call PAP IV. Mouse PAP III and two additional truncated forms, PAPs V and VI, which would be produced by use of poly(A) sites in adjacent introns, were also isolated from a mouse brain cDNA library. RNase protection and reverse transcription-PCR analyses showed that PAP II, V, and VI are expressed in all tissues tested but that PAP I and/or IV and III are tissue specific. However, immunoblot analysis detected only the long forms, raising the possibility that the short-form RNAs are not translated. Purified recombinant baculovirus-expressed PAPs were tested in several in vitro assays, and the short forms were found to be inactive. We discuss the possible significance of this complex expression pattern.

Mammalian Clk/Sty is the prototype for a family of dual specificity kinases (termed LAMMER kinases) that have been conserved in evolution, but whose physiological substrates are unknown. In a yeast two-hybrid screen, the Clk/Sty kinase specifically interacted with RNA binding proteins, particularly members of the serine/arginine-rich (SR) family of splicing factors. Clk/Sty itself has an serine/arginine-rich non-catalytic N-terminal region which is important for its association with SR splicing factors. In vitro, Clk/Sty efficiently phosphorylated the SR family member ASF/SF2 on serine residues located within its serine/arginine-rich region (the RS domain). Tryptic phosphopeptide mapping demonstrated that the sites on ASF/SF2 phosphorylated in vitro overlap with those phosphorylated in vivo. Immunofluorescence studies showed that a catalytically inactive form of Clk/Sty co-localized with SR proteins in nuclear speckles. Overexpression of the active Clk/Sty kinase caused a redistribution of SR proteins within the nucleus. These results suggest that Clk/Sty kinase directly regulates the activity and compartmentalization of SR splicing factors.

The mammalian SR-type splicing factors ASF/SF2 and SC-35 play crucial roles in pre-mRNA splicing and have been shown to shift splice site choice in vitro. We have mapped the ASF/SF2 gene in mice and humans and the SC-35 gene in mice. Somatic cell hybrid mapping of the human ASF/SF2 gene (SFRS1 locus) reveals that it resides on chromosome 17, and fluorescence in situ hybridization refines this localization to 17q21.3-q22. Recombinant inbred mapping of the mouse ASF/SF2 gene (Sfrs1 locus) and the mouse SC-35 gene (Sfrs2 locus) demonstrates that both genes are located in a part of mouse chromosome 11 that is homologous to human chromosome 17. Mapping of Sfrs1 using F1 hybrid backcross mice between the strains C57BL/6 and DDK places Sfrs1 very near the marker D11Mit38 and indicates that the ASF/SF2 gene is closely linked to the Ovum mutant locus.

Work over the last two decades has provided a wealth of data indicating that the RNA polymerase II transcriptional machinery can play an important role in facilitating the splicing of its transcripts. In particular, the C-terminal domain of the RNA polymerase II large subunit (CTD) is central in the coupling of transcription and splicing. While this has long been assumed to involve physical interactions between splicing factors and the CTD, few functional connections between the CTD and such factors have been established. We recently used a biochemical approach to identify a splicing factor that interacts directly with the CTD to activate splicing and, in doing so, may play a role in the process of spliceosome assembly.

Pre-mRNA splicing is frequently coupled to transcription by RNA polymerase II (RNAPII). This coupling requires the C-terminal domain of the RNAPII largest subunit (CTD), although the underlying mechanism is poorly understood. Using a biochemical complementation assay, we previously identified an activity that stimulates CTD-dependent splicing in vitro. We purified this activity and found that it consists of a complex of two well-known splicing factors: U2AF65 and the Prp19 complex (PRP19C). We provide evidence that both U2AF65 and PRP19C are required for CTD-dependent splicing activation, that U2AF65 and PRP19C interact both in vitro and in vivo, and that this interaction is required for activation of splicing. Providing the link to the CTD, we show that U2AF65 binds directly to the phosphorylated CTD, and that this interaction results in increased recruitment of U2AF65 and PRP19C to the pre-mRNA. Our results not only provide a mechanism by which the CTD enhances splicing, but also describe unexpected interactions important for splicing and its coupling to transcription.

Publisher Summary This chapter focuses on the regulation and the influence of splicing on two important cellular processes: programmed cell death (apoptosis) and cell cycle control. Apoptosis is a process that removes deleterious or useless cells during animal development and is one of many cellular processes in which alternative splicing plays an important regulatory role. Oligonucleosomal DNA fragmentation is one of the hallmarks of apoptotic cell death. The cell cycle is a collection of highly ordered processes that lead to the duplication of a cell. Specific splicing factors are required for cell cycle progression by modulating splicing of transcripts encoding cell cycle regulators. In metazoan organisms, most transcripts synthesized by RNA polymerase (RNAP) II contain non-coding intervening sequences called introns, which must be accurately and efficiently removed by the process of pre-mRNA splicing to form translatable mRNAs. Alternative splicing is the removal of introns in different combinations. It produces diverse mature mRNAs encoding structurally and functionally distinct protein isoforms from a single gene. Alternative splicing is widely involved in gene expression in higher eukaryotes, especially in vertebrates. Pre-mRNA splicing takes place within a large molecular complex, the spliceosome, that is composed of five small nuclear RNA and a large number of protein factors. Regulation of alternative splicing largely relies on a broad spectrum of interactions between sequence elements in the mRNA precursor and a complex repertoire of protein factors. Shifting the balance between alternatively spliced isoforms of a given pre-mRNA is important in modulating both programmed cell death (apoptosis) and cell cycle control.

We showed previously that a branch site mutation in simian virus 40 early pre-mRNA that prevented small t antigen mRNA splicing could be efficiently suppressed by a compensatory mutation in a coexpressed U2 small nuclear (sn) RNA gene. We have now generated second-site mutations in this suppressor gene to investigate regions of U2 RNA required for function. A number of mutations in a putative stem at the 5' end of the molecule inhibited splicing, indicating that bases in this region are important for activity. However, several lines of evidence suggested that formation of the entire stem is not essential for splicing. Indeed, mutations that strengthen the stem actually inhibited splicing, and evidence that this prevents a required base-pairing interaction with U6 snRNA is presented. These results suggest that the relative stabilities of competing intra- and intermolecular base-pairing interactions play an important role in the splicing reaction. Mutations in a conserved single-stranded region immediately 3' to the branch site recognition sequence all inhibited splicing, indicating that this region is required for U2 function, although its exact role remains unknown. Finally, two mutations in the loop of stem IV at the 3' end of the molecule, which destroy the binding site of U2 sn ribonucleoprotein B", prevented small t splicing; this finding contrasts with previous studies which utilized different assay systems. Analysis of the accumulation and subcellular localization of all of the mutant RNAs showed that they were similar to those of the parental suppressor U2 RNA, indicating that the effects observed indeed reflect defects in splicing.

A protein present in nuclear extracts of Drosophila embryos binds multiple sites in the promoter and genetically defined autoregulatory element of the pair-rule gene even-skipped (eve). We reported here the isolation of a cDNA encoding this binding activity, the sequence of which identifies it as the 69 kDa zinc finger tramtrack (ttk) protein. As ttk was previously implicated in controlling the expression of another pair-rule gene, fushi tarazu (ftz), our findings suggest that ttk plays a role in the regulation of at least two developmentally important genes. An additional ttk-related cDNA clone was isolated which gives rise to an 88 kDa protein with an alternative set of zinc fingers having a DNA binding specificity distinct from that of the 69 kDa protein. Both proteins were shown to be encoded by the ttk gene through alternative splicing, providing the first example of the use of this mechanism to generate related proteins with distinct DNA binding specificities. Whole mount in situ hybridization analysis revealed different patterns of embryonic expression of the two ttk mRNA isoforms.

Alternative splicing of mRNA precursors provides an important means of genetic control and is a crucial step in the expression of most genes. Alternative splicing markedly affects human development, and its misregulation underlies many human diseases. Although the mechanisms of alternative splicing have been studied extensively, until the past few years we had not begun to realize fully the diversity and complexity of alternative splicing regulation by an intricate protein-RNA network. Great progress has been made by studying individual transcripts and through genome-wide approaches, which together provide a better picture of the mechanistic regulation of alternative pre-mRNA splicing.