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1.  The In Vivo Kinetics of RNA Polymerase II Elongation during Co-Transcriptional Splicing 
PLoS Biology  2011;9(1):e1000573.
Kinetic analysis shows that RNA polymerase elongation kinetics are not modulated by co-transcriptional splicing and that post-transcriptional splicing can proceed at the site of transcription without the presence of the polymerase.
RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3′ processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end.
Author Summary
The pre-mRNA emerging from RNA polymerase II during eukaryotic transcription undergoes a series of processing events. These include 5′-capping, intron excision and exon ligation during splicing, 3′-end processing, and polyadenylation. Processing events occur co-transcriptionally, meaning that a variety of enzymes assemble on the pre-mRNA while the polymerase is still engaged in transcription. The concept of co-transcriptional mRNA processing raises questions about the possible coupling between the transcribing polymerase and the processing machineries. Here we examine how the co-transcriptional assembly of the splicing machinery (the spliceosome) might affect the elongation kinetics of the RNA polymerase. Using live-cell microscopy, we followed the kinetics of transcription of genes containing increasing numbers of introns and measured the recruitment of transcription and splicing factors. Surprisingly, a sub-set of splicing factors was recruited to an intronless gene, implying that there is a polymerase-coupled scanning mechanism for intronic sequences. There was no difference in polymerase elongation rates on genes with or without introns, suggesting that the spliceosome does not modulate elongation kinetics. Experiments including inhibition of splicing or transcription, together with stochastic computational simulation, demonstrated that pre-mRNAs can be retained on the gene when polymerase termination precedes completion of splicing. Altogether we show that polymerase elongation kinetics are not affected by splicing events on the emerging pre-mRNA, that increased splicing leads to more splicing factors being recruited to the mRNA, and that post-transcriptional splicing can proceed at the site of transcription in the absence of the polymerase.
PMCID: PMC3019111  PMID: 21264352
2.  The Yeast SR-Like Protein Npl3 Links Chromatin Modification to mRNA Processing 
PLoS Genetics  2012;8(11):e1003101.
Eukaryotic gene expression involves tight coordination between transcription and pre–mRNA splicing; however, factors responsible for this coordination remain incompletely defined. Here, we explored the genetic, functional, and biochemical interactions of a likely coordinator, Npl3, an SR-like protein in Saccharomyces cerevisiae that we recently showed is required for efficient co-transcriptional recruitment of the splicing machinery. We surveyed the NPL3 genetic interaction space and observed a significant enrichment for genes involved in histone modification and chromatin remodeling. Specifically, we found that Npl3 genetically interacts with both Bre1, which mono-ubiquitinates histone H2B as part of the RAD6 Complex, and Ubp8, the de-ubiquitinase of the SAGA Complex. In support of these genetic data, we show that Bre1 physically interacts with Npl3 in an RNA–independent manner. Furthermore, using a genome-wide splicing microarray, we found that the known splicing defect of a strain lacking Npl3 is exacerbated by deletion of BRE1 or UBP8, a phenomenon phenocopied by a point mutation in H2B that abrogates ubiquitination. Intriguingly, even in the presence of wild-type NPL3, deletion of BRE1 exhibits a mild splicing defect and elicits a growth defect in combination with deletions of early and late splicing factors. Taken together, our data reveal a connection between Npl3 and an extensive array of chromatin factors and describe an unanticipated functional link between histone H2B ubiquitination and pre–mRNA splicing.
Author Summary
Pre-messenger RNA splicing is the process by which an intron is identified and removed from a transcript and the protein-coding exons are ligated together. It is carried out by the spliceosome, a large and dynamic molecular machine that catalyzes the splicing reaction. It is now apparent that most splicing occurs while the transcript is still engaged with RNA polymerase, implying that the biologically relevant splicing substrate is chromatin-associated. Here, we used a genetic approach to understand which factors participate in the coordination of transcription and splicing. Having recently shown that the Npl3 protein is involved in the recruitment of splicing factors to chromatin-associated transcripts, we performed a systematic screen for genetically interacting factors. Interestingly, we identified factors that influence the ubiquitin modification of histone H2B, a mark involved in transcription initiation and elongation. We show that disruption of the H2B ubiquitination/de-ubiquitination cycle results in defects in splicing, particularly in the absence of Npl3. Furthermore, the ubiquitin ligase, Bre1, shows genetic interactions with other, more canonical spliceosomal factors. Taken together with the myriad Npl3 interaction partners we found, our data suggest an extensive cross-talk between the spliceosome and chromatin.
PMCID: PMC3510044  PMID: 23209445
3.  Transcript Specificity in Yeast Pre-mRNA Splicing Revealed by Mutations in Core Spliceosomal Components 
PLoS Biology  2007;5(4):e90.
Appropriate expression of most eukaryotic genes requires the removal of introns from their pre–messenger RNAs (pre-mRNAs), a process catalyzed by the spliceosome. In higher eukaryotes a large family of auxiliary factors known as SR proteins can improve the splicing efficiency of transcripts containing suboptimal splice sites by interacting with distinct sequences present in those pre-mRNAs. The yeast Saccharomyces cerevisiae lacks functional equivalents of most of these factors; thus, it has been unclear whether the spliceosome could effectively distinguish among transcripts. To address this question, we have used a microarray-based approach to examine the effects of mutations in 18 highly conserved core components of the spliceosomal machinery. The kinetic profiles reveal clear differences in the splicing defects of particular pre-mRNA substrates. Most notably, the behaviors of ribosomal protein gene transcripts are generally distinct from other intron-containing transcripts in response to several spliceosomal mutations. However, dramatically different behaviors can be seen for some pairs of transcripts encoding ribosomal protein gene paralogs, suggesting that the spliceosome can readily distinguish between otherwise highly similar pre-mRNAs. The ability of the spliceosome to distinguish among its different substrates may therefore offer an important opportunity for yeast to regulate gene expression in a transcript-dependent fashion. Given the high level of conservation of core spliceosomal components across eukaryotes, we expect that these results will significantly impact our understanding of how regulated splicing is controlled in higher eukaryotes as well.
Author Summary
The spliceosome is a large RNA-protein machine responsible for removing the noncoding (intron) sequences that interrupt eukaryotic genes. Nearly everything known about the behavior of this machine has been based on the analysis of only a handful of genes, despite the fact that individual introns vary greatly in both size and sequence. Here we have utilized a microarray-based platform that allows us to simultaneously examine the behavior of all intron-containing genes in the budding yeast S. cerevisiae. By systematically examining the effects of individual mutants in the spliceosome on the splicing of all substrates, we have uncovered a surprisingly complex relationship between the spliceosome and its full complement of substrates. Contrary to the idea that the spliceosome engages in “generic” interactions with all intron-containing substrates in the cell, our results show that the identity of the transcript can differentially affect splicing efficiency when the machine is subtly perturbed. We propose that the wild-type spliceosome can also distinguish among its many substrates as external conditions warrant to function as a specific regulator of gene expression.
Many eukaryotic gene transcripts are spliced; here the authors show that components of the splicing complex can distinguish between different introns in highly homologous transcripts.
PMCID: PMC1831718  PMID: 17388687
4.  Kinetic competition during the transcription cycle results in stochastic RNA processing 
eLife  2014;3:e03939.
Synthesis of mRNA in eukaryotes involves the coordinated action of many enzymatic processes, including initiation, elongation, splicing, and cleavage. Kinetic competition between these processes has been proposed to determine RNA fate, yet such coupling has never been observed in vivo on single transcripts. In this study, we use dual-color single-molecule RNA imaging in living human cells to construct a complete kinetic profile of transcription and splicing of the β-globin gene. We find that kinetic competition results in multiple competing pathways for pre-mRNA splicing. Splicing of the terminal intron occurs stochastically both before and after transcript release, indicating there is not a strict quality control checkpoint. The majority of pre-mRNAs are spliced after release, while diffusing away from the site of transcription. A single missense point mutation (S34F) in the essential splicing factor U2AF1 which occurs in human cancers perturbs this kinetic balance and defers splicing to occur entirely post-release.
eLife digest
To make a protein, part of a DNA sequence is copied to make a messenger RNA (or mRNA) molecule in a process known as transcription. The enzyme that builds an mRNA molecule first binds to a start point on a DNA strand, and then uses the DNA sequence to build a ‘pre-mRNA’ molecule until a stop signal is reached.
To make the final mRNA molecule, sections called introns are removed from the pre-mRNA molecules, and the parts left behind—known as exons—are then joined together. This process is called splicing. However, it is not fully understood how the splicing process is coordinated with the other stages of transcription. For example, does splicing occur after the pre-mRNA molecule is completed or while it is still being built? And what controls the order in which these processes occur?
One theory about how the different mRNA-making processes are coordinated is called kinetic competition. This theory states that the fastest process is the most likely to occur, even if the other processes use less energy and so might be expected to be preferred. Alternatively, the different steps may be started and stopped by ‘checkpoints’ that cause the different processes to follow on from each other in a set order.
Coulon et al. used fluorescence microscopy to investigate how mRNA molecules are made during the transcription of a human gene that makes a hemoglobin protein. To make the RNA visible, two different fluorescent markers were introduced into the pre-mRNA that cause different regions of the mRNA to glow in different colors. Coulon et al. made the introns fluoresce red and the exons glow green. Unspliced pre-mRNA molecules contain both introns and exons and so fluoresce in both colors, whereas spliced mRNA molecules contain only exons and so only glow with a green color.
By looking at both the red and green fluorescence signals at the same time, Coulon et al. could see when an intron was spliced out of the pre-mRNA. This revealed that in normal cells, splicing can occur either before or after the RNA is released from where it is transcribed. Thus, splicing and transcription does not follow a set pattern, suggesting that checkpoints do not control the sequence of events. Instead, the fact that a spliced mRNA molecule can be formed in different ways suggests kinetic competition controls the process.
In some cancer cells, there are defects in the cellular machinery that controls splicing. When looking at cells with such a defect, Coulon et al. found that splicing only occurred after transcription was completed. This study thus provides insight into the complex workings of mRNA synthesis and establishes a blueprint for understanding how splicing is impaired in diseases such as cancer.
PMCID: PMC4210818  PMID: 25271374
transcription; RNA processing; splicing; single-molecule imaging; fluctuation analysis; human
5.  Analysis of a Splice Array Experiment Elucidates Roles of Chromatin Elongation Factor Spt4–5 in Splicing 
PLoS Computational Biology  2005;1(4):e39.
Splicing is an important process for regulation of gene expression in eukaryotes, and it has important functional links to other steps of gene expression. Two examples of these linkages include Ceg1, a component of the mRNA capping enzyme, and the chromatin elongation factors Spt4–5, both of which have recently been shown to play a role in the normal splicing of several genes in the yeast Saccharomyces cerevisiae. Using a genomic approach to characterize the roles of Spt4–5 in splicing, we used splicing-sensitive DNA microarrays to identify specific sets of genes that are mis-spliced in ceg1, spt4, and spt5 mutants. In the context of a complex, nested, experimental design featuring 22 dye-swap array hybridizations, comprising both biological and technical replicates, we applied five appropriate statistical models for assessing differential expression between wild-type and the mutants. To refine selection of differential expression genes, we then used a robust model-synthesizing approach, Differential Expression via Distance Synthesis, to integrate all five models. The resultant list of differentially expressed genes was then further analyzed with regard to select attributes: we found that highly transcribed genes with long introns were most sensitive to spt mutations. QPCR confirmation of differential expression was established for the limited number of genes evaluated. In this paper, we showcase splicing array technology, as well as powerful, yet general, statistical methodology for assessing differential expression, in the context of a real, complex experimental design. Our results suggest that the Spt4–Spt5 complex may help coordinate splicing with transcription under conditions that present kinetic challenges to spliceosome assembly or function.
Splicing is a key process for the regulation of gene expression in eukaryotes and is credited as being the main reason for the extraordinary complexity of the human proteome relative to the human genome. Accurate splicing is crucial for normal protein function; aberrant transcripts due to splicing mutations are known causes for 15% of genetic diseases. Therefore, elucidation of splicing mechanisms will not only help in understanding the complexity and diversity of higher organisms, but also potentially aid in new therapeutic strategies for treatments of splicing-related genetic disorders. It has been previously shown that splicing has important links to other steps involved with gene expression. In this study, the authors pursue a genome-wide approach, using yeast-based, splicing-sensitive, DNA microarrays in order to further characterize the roles of select splicing factors. They devise novel statistical and computational methods that enable identification of specific sets of genes that are mis-spliced in the chosen splicing factors. Follow-up investigation of known attributes of the genes so elicited indicates that these factors may help coordinate splicing and transcription in situations where additional energy is required to effect splicing.
PMCID: PMC1214541  PMID: 16172632
6.  Genetic Interaction Mapping Reveals a Role for the SWI/SNF Nucleosome Remodeler in Spliceosome Activation in Fission Yeast 
PLoS Genetics  2015;11(3):e1005074.
Although numerous regulatory connections between pre-mRNA splicing and chromatin have been demonstrated, the precise mechanisms by which chromatin factors influence spliceosome assembly and/or catalysis remain unclear. To probe the genetic network of pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, we constructed an epistatic mini-array profile (E-MAP) and discovered many new connections between chromatin and splicing. Notably, the nucleosome remodeler SWI/SNF had strong genetic interactions with components of the U2 snRNP SF3 complex. Overexpression of SF3 components in ΔSWI/SNF cells led to inefficient splicing of many fission yeast introns, predominantly those with non-consensus splice sites. Deletion of SWI/SNF decreased recruitment of the splicing ATPase Prp2, suggesting that SWI/SNF promotes co-transcriptional spliceosome assembly prior to first step catalysis. Importantly, defects in SWI/SNF as well as SF3 overexpression each altered nucleosome occupancy along intron-containing genes, illustrating that the chromatin landscape both affects—and is affected by—co-transcriptional splicing.
Author Summary
It has recently become apparent that most introns are removed from pre-mRNA while the transcript is still engaged with RNA polymerase II (RNAPII). To gain insight into possible roles for chromatin in co-transcriptional splicing, we generated a genome-wide genetic interaction map in fission yeast and uncovered numerous connections between splicing and chromatin. The SWI/SNF remodeling complex is typically thought to activate gene expression by relieving barriers to polymerase elongation imposed by nucleosomes. Here we show that this remodeler is important for an early step in splicing in which Prp2, an RNA-dependent ATPase, is recruited to the assembling spliceosome to promote catalytic activation. Interestingly, introns with sub-optimal splice sites are particularly dependent on SWI/SNF, suggesting the impact of nucleosome dynamics on the kinetics of spliceosome assembly and catalysis. By monitoring nucleosome occupancy, we show significant alterations in nucleosome density in particular splicing and chromatin mutants, which generally paralleled the levels of RNAPII. Taken together, our findings challenge the notion that nucleosomes simply act as barriers to elongation; rather, we suggest that polymerase pausing at nucleosomes can activate gene expression by allowing more time for co-transcriptional splicing.
PMCID: PMC4380400  PMID: 25825871
7.  The SPF27 Homologue Num1 Connects Splicing and Kinesin 1-Dependent Cytoplasmic Trafficking in Ustilago maydis  
PLoS Genetics  2014;10(1):e1004046.
The conserved NineTeen protein complex (NTC) is an integral subunit of the spliceosome and required for intron removal during pre-mRNA splicing. The complex associates with the spliceosome and participates in the regulation of conformational changes of core spliceosomal components, stabilizing RNA-RNA- as well as RNA-protein interactions. In addition, the NTC is involved in cell cycle checkpoint control, response to DNA damage, as well as formation and export of mRNP-particles. We have identified the Num1 protein as the homologue of SPF27, one of NTC core components, in the basidiomycetous fungus Ustilago maydis. Num1 is required for polarized growth of the fungal hyphae, and, in line with the described NTC functions, the num1 mutation affects the cell cycle and cell division. The num1 deletion influences splicing in U. maydis on a global scale, as RNA-Seq analysis revealed increased intron retention rates. Surprisingly, we identified in a screen for Num1 interacting proteins not only NTC core components as Prp19 and Cef1, but several proteins with putative functions during vesicle-mediated transport processes. Among others, Num1 interacts with the motor protein Kin1 in the cytoplasm. Similar phenotypes with respect to filamentous and polar growth, vacuolar morphology, as well as the motility of early endosomes corroborate the genetic interaction between Num1 and Kin1. Our data implicate a previously unidentified connection between a component of the splicing machinery and cytoplasmic transport processes. As the num1 deletion also affects cytoplasmic mRNA transport, the protein may constitute a novel functional interconnection between the two disparate processes of splicing and trafficking.
Author Summary
In eukaryotic cells, nascent mRNA is processed by splicing to remove introns and to join the exon sequences. The processed mRNA is then transported out of the nucleus and employed by ribosomes to synthesize proteins. Splicing is achieved by the spliceosome and associated protein complexes, among them the so-called NineTeen complex (NTC). We have identified the Num1 protein as one of the core components of the NTC in the fungus Ustilago maydis, and could show that it is required for polarized growth of the filamentous fungal cells. Consistent with the NTC function, cells with a num1-deletion show reduced splicing of mRNA. Moreover, we uncover a novel cytoplasmic function of the Num1 protein: It physically interacts with the microtubule-associated Kinesin 1 motor protein, and phenotypic analyses corroborate that both proteins are functionally connected. Our findings reveal a yet unidentified role of a global splicing factor during intracellular trafficking processes. A possible connection between these disparate mechanisms presumably resides in mRNA-export out of the nucleus and/or the transport of mRNA within the cytoplasm.
PMCID: PMC3879195  PMID: 24391515
8.  Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors 
Nucleic Acids Research  2007;35(12):3928-3944.
Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5′ end binding factors, 3′ end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.
PMCID: PMC1919476  PMID: 17537823
9.  The Supraspliceosome — A Multi-Task Machine for Regulated Pre-mRNA Processing in the Cell Nucleus 
Pre-mRNA splicing of Pol II transcripts is executed in the mammalian cell nucleus within a huge (21 MDa) and highly dynamic RNP machine — the supraspliceosome. It is composed of four splicing active native spliceosomes, each resembling an in vitro assembled spliceosome, which are connected by the pre-mRNA. Supraspliceosomes harbor protein splicing factors and all the five-spliceosomal U snRNPs. Recent analysis of specific supraspliceosomes at defined splicing stages revealed that they harbor all five spliceosomal U snRNAs at all splicing stages. Supraspliceosomes harbor additional pre-mRNA processing components, such as the 5′-end and 3′-end processing components, and the RNA editing enzymes ADAR1 and ADAR2. The structure of the native spliceosome, at a resolution of 20 Å, was determined by cryo-EM. A unique spatial arrangement of the spliceosomal U snRNPs within the native spliceosome emerged from in-silico studies, localizing the five U snRNPs mostly within its large subunit, and sheltering the active core components deep within the spliceosomal cavity. The supraspliceosome provides a platform for coordinating the numerous processing steps that the pre-mRNA undergoes: 5′ and 3′-end processing activities, RNA editing, constitutive and alternative splicing, and processing of intronic microRNAs. It also harbors a quality control mechanism termed suppression of splicing (SOS) that, under normal growth conditions, suppresses splicing at abundant intronic latent 5′ splice sites in a reading frame-dependent fashion. Notably, changes in these regulatory processing activities are associated with human disease and cancer. These findings emphasize the supraspliceosome as a multi-task master regulator of pre-mRNA processing in the cell nucleus.
PMCID: PMC4232567  PMID: 25408845
Pre-mRNA splicing; Riponucleoproteins (RNPs); U snRNPs; Alternative splicing; Intronic microRNA biogenesis; Suppression of splicing
10.  The Hierarchy of Exon-Junction Complex Assembly by the Spliceosome Explains Key Features of Mammalian Nonsense-Mediated mRNA Decay 
PLoS Biology  2009;7(5):e1000120.
Protein complexes deposited on messenger RNAs during their maturation are able to recruit components of a cellular RNA surveillance pathway, thereby linking RNA maturation to subsequent steps in RNA quality control.
Exon junction complexes (EJCs) link nuclear splicing to key features of mRNA function including mRNA stability, translation, and localization. We analyzed the formation of EJCs by the spliceosome, the physiological EJC assembly machinery. We studied a comprehensive set of eIF4A3, MAGOH, and BTZ mutants in complete or C-complex–arrested splicing reactions and identified essential interactions of EJC proteins during and after EJC assembly. These data establish that EJC deposition proceeds through a defined intermediate, the pre-EJC, as an ordered, sequential process that is coordinated by splicing. The pre-EJC consists of eIF4A3 and MAGOH-Y14, is formed before exon ligation, and provides a binding platform for peripheral EJC components that join after release from the spliceosome and connect the core structure with function. Specifically, we identified BTZ to bridge the EJC to the nonsense-mediated messenger RNA (mRNA) decay protein UPF1, uncovering a critical link between mRNP architecture and mRNA stability. Based on this systematic analysis of EJC assembly by the spliceosome, we propose a model of how a functional EJC is assembled in a strictly sequential and hierarchical fashion, including nuclear splicing-dependent and cytoplasmic steps.
Author Summary
The first step in the expression of eukaryotic protein-coding genes is transcription into a messenger RNA (mRNA) precursor in the nucleus. These precursor mRNAs then undergo maturation through the removal of introns in a process termed splicing. During splicing, the splicing machinery or “spliceosome” deposits a complex of proteins onto the mRNA that accompanies it during post-transcriptional steps in gene expression, including the regulation of mRNA stability, transport out of the nucleus, cellular localisation, and translation. This complex, the exon junction complex (EJC), represents a molecular memory of the splicing process. Understanding the biogenesis of EJCs and their downstream effects helps reveal the basic principles by which the primary steps of mRNA synthesis are coupled to the regulation of gene expression. Here we show that EJCs are assembled in a strictly splicing-dependent manner through an unexpected, coordinated, and hierarchical assembly pathway. Importantly, we show that the EJC recruits the cytoplasmic protein BTZ, which then bridges the complex to an mRNA quality-control machinery called the nonsense-mediated decay pathway that degrades mRNAs containing premature stop codons. This finding suggests that the EJC and bridging by BTZ help determine the stability of mRNA and thus are essential for proper cellular surveillance of mRNA quality.
PMCID: PMC2682485  PMID: 19478851
11.  PPS, a Large Multidomain Protein, Functions with Sex-Lethal to Regulate Alternative Splicing in Drosophila 
PLoS Genetics  2010;6(3):e1000872.
Alternative splicing controls the expression of many genes, including the Drosophila sex determination gene Sex-lethal (Sxl). Sxl expression is controlled via a negative regulatory mechanism where inclusion of the translation-terminating male exon is blocked in females. Previous studies have shown that the mechanism leading to exon skipping is autoregulatory and requires the SXL protein to antagonize exon inclusion by interacting with core spliceosomal proteins, including the U1 snRNP protein Sans-fille (SNF). In studies begun by screening for proteins that interact with SNF, we identified PPS, a previously uncharacterized protein, as a novel component of the machinery required for Sxl male exon skipping. PPS encodes a large protein with four signature motifs, PHD, BRK, TFS2M, and SPOC, typically found in proteins involved in transcription. We demonstrate that PPS has a direct role in Sxl male exon skipping by showing first that loss of function mutations have phenotypes indicative of Sxl misregulation and second that the PPS protein forms a complex with SXL and the unspliced Sxl RNA. In addition, we mapped the recruitment of PPS, SXL, and SNF along the Sxl gene using chromatin immunoprecipitation (ChIP), which revealed that, like many other splicing factors, these proteins bind their RNA targets while in close proximity to the DNA. Interestingly, while SNF and SXL are specifically recruited to their predicted binding sites, PPS has a distinct pattern of accumulation along the Sxl gene, associating with a region that includes, but is not limited to, the SxlPm promoter. Together, these data indicate that PPS is different from other splicing factors involved in male-exon skipping and suggest, for the first time, a functional link between transcription and SXL–mediated alternative splicing. Loss of zygotic PPS function, however, is lethal to both sexes, indicating that its role may be of broad significance.
Author Summary
In Drosophila the sex-specific ON/OFF regulation of Sex-lethal (Sxl) is controlled by an autoregulatory splicing mechanism that depends on the SXL protein interacting with general splicing factors. Here we identify PPS as a novel component of the machinery required for Sxl splicing autoregulation by showing that the lack of pps function interferes with Sxl expression and that the PPS protein is physically linked to the Sxl pre–mRNA, the SXL protein and components of the general splicing machinery. PPS, however, stands apart from all other proteins known to control Sxl splicing because it is not a general splicing factor. Furthermore, PPS has a distinct pattern of accumulation along the Sxl transcription unit that suggests PPS is loaded onto the RNA at the promoter. Together with the observation that the PPS protein contains four signature motifs typically found in proteins that function in transcriptional regulation, our data suggest that linking transcription to splicing regulation is important for controlling Sxl expression. This idea is especially intriguing because it indicates that the coupling of transcription and splicing seen in vitro and in cell culture studies is likely to be pertinent to developmentally controlled patterns of gene expression in the living animal.
PMCID: PMC2832672  PMID: 20221253
12.  Feed-Forward Microprocessing and Splicing Activities at a MicroRNA–Containing Intron 
PLoS Genetics  2011;7(10):e1002330.
The majority of mammalian microRNA (miRNA) genes reside within introns of protein-encoding and non-coding genes, yet the mechanisms coordinating primary transcript processing into both mature miRNA and spliced mRNA are poorly understood. Analysis of melanoma invasion suppressor miR-211 expressed from intron 6 of melastatin revealed that microprocessing of miR-211 promotes splicing of the exon 6–exon 7 junction of melastatin by a mechanism requiring the RNase III activity of Drosha. Additionally, mutations in the 5′ splice site (5′SS), but not in the 3′SS, branch point, or polypyrimidine tract of intron 6 reduced miR-211 biogenesis and Drosha recruitment to intron 6, indicating that 5′SS recognition by the spliceosome promotes microprocessing of miR-211. Globally, knockdown of U1 splicing factors reduced intronic miRNA expression. Our data demonstrate novel mutually-cooperative microprocessing and splicing activities at an intronic miRNA locus and suggest that the initiation of spliceosome assembly may promote microprocessing of intronic miRNAs.
Author Summary
MicroRNA (miRNA) genes are transcribed as long primary RNAs containing local hairpins that are excised by the Microprocessor complex minimally composed of Drosha and DGCR8. Most mammalian miRNAs reside in introns of protein-encoding and non-coding genes, but it is unclear how microprocessing of an intronic miRNA and splicing at the host gene intron affect each other. We recently reported that in melanoma, a miRNA expressed from intron 6 of melastatin (miR-211) assumes the tumor suppressive function of its host gene. In our current work, we detected elevated melastatin exon 6–exon 7 junctions relative to other exon-exon junctions that lack intronic miRNAs, suggesting that microprocessing promotes splicing. We show that microprocessing of miR-211 precedes completion of splicing of the exon 6–exon 7 junctions and that Drosha's endonuclease activity is required to facilitate exon 6–exon 7 junction formation. Additionally, we found that the first step of spliceosome assembly, recognition of the 5′ splice site by the U1 snRNP complex, promotes microprocessing of miR-211 and other intronic but not intergenic miRNAs. Our findings reveal a mutually cooperative, physical, and functional coupling of intronic miRNA biogenesis and splicing at the host intron, and they suggest a global positive effect of spliceosome assembly on intronic miRNA microprocessing.
PMCID: PMC3197686  PMID: 22028668
13.  Discovery and Analysis of Evolutionarily Conserved Intronic Splicing Regulatory Elements 
PLoS Genetics  2007;3(5):e85.
Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs). Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5′ and 94% altered 3′ splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%–45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%–50% of ISREs were enriched near alternatively spliced (AS) exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events.
Author Summary
During RNA splicing, sequences (introns) in a pre-mRNA are excised and discarded, and the remaining sequences (exons) are joined to form the mature RNA. Splicing is regulated not only by the binding of the basic splicing machinery to splice sites located at the exon–intron boundaries, but also by the combined effects of various other splicing factors that bind to a multitude of sequence elements located both in the exons as well as the flanking introns. Instances of alternative splicing, where usage of splice site(s) is incomplete or different between tissues, cell types, or lineages, can be created by the interaction of sequence elements and tissue, cell type, and stage-specific splicing factors. To better understand constitutive and alternative pre-mRNA splicing, the authors describe a comparative genomics approach, using available mammalian genomes, to systematically identify splicing regulatory elements located in the introns proximal to exons. A quarter of the elements were tested experimentally, and most of them altered splicing in human cells. The authors also showed that that the intronic elements are close to tissue-specific alternative exons and are more likely to be located in specific positions in the introns, suggestive of potential regulatory function. These elements are also frequently found in tissue-specific genes, suggesting a coupling between expression and alternative splicing of these genes. Finally, the authors propose a strategy using the elements to identify the binding sites of several splicing factors.
PMCID: PMC1877881  PMID: 17530930
14.  Structural basis for the recognition of spliceosomal SmN/B/B’ proteins by the RBM5 OCRE domain in splicing regulation 
eLife  null;5:e14707.
The multi-domain splicing factor RBM5 regulates the balance between antagonistic isoforms of the apoptosis-control genes FAS/CD95, Caspase-2 and AID. An OCRE (OCtamer REpeat of aromatic residues) domain found in RBM5 is important for alternative splicing regulation and mediates interactions with components of the U4/U6.U5 tri-snRNP. We show that the RBM5 OCRE domain adopts a unique β–sheet fold. NMR and biochemical experiments demonstrate that the OCRE domain directly binds to the proline-rich C-terminal tail of the essential snRNP core proteins SmN/B/B’. The NMR structure of an OCRE-SmN peptide complex reveals a specific recognition of poly-proline helical motifs in SmN/B/B’. Mutation of conserved aromatic residues impairs binding to the Sm proteins in vitro and compromises RBM5-mediated alternative splicing regulation of FAS/CD95. Thus, RBM5 OCRE represents a poly-proline recognition domain that mediates critical interactions with the C-terminal tail of the spliceosomal SmN/B/B’ proteins in FAS/CD95 alternative splicing regulation.
eLife digest
The information required to produce proteins is encoded within genes. In the first step of creating a protein, its gene is “transcribed” to form a pre-messenger RNA molecule (called pre-mRNA for short). Both the gene and the pre-mRNA contain regions called exons that code for protein, and regions called introns that do not. The pre-mRNA therefore undergoes a process called splicing to remove the introns and join the exons together into a final mRNA molecule that is “translated” to make the protein.
Many pre-mRNAs can be spliced in several different ways to include different combinations of exons in the final mRNA molecule. This process of “alternative splicing” allows different versions of a protein to be produced from the same gene. Changes that alter the pattern of alternative splicing in a cell affect various cellular and developmental processes and have been linked to diseases such as cancer.
The pre-mRNA transcribed from a gene called FAS can be alternatively spliced so that it either does or does not contain an exon that enables the protein to embed itself in the cell membrane. The protein produced from mRNA that includes this exon generates a cell response that leads to cell death. By contrast, protein produced from mRNA that lacks this exon is released from cells and promotes their survival. A splicing factor called RBM5 promotes the removal of this exon from FAS pre-mRNA.
RBM5 binds to some of the proteins that make up the molecular machine that splices pre-mRNA molecules. Mourão, Bonnal, Soni, Warner et al. have now used a technique called nuclear magnetic resonance spectroscopy to solve the three-dimensional structure formed when RBM5 binds to one of these proteins, called SmN. Further experiments introduced specific mutations to the proteins to investigate their effects in human cells. This revealed that mutations that impaired the association between RBM5 and SmN compromised the activity of RBM5 to regulate the alternative splicing of FAS pre-mRNA molecules.
Future research could examine how RBM5 associates with pre-mRNAs and other components of the splicing machinery, and investigate whether proteins that are closely related to RBM5 act in similar ways.
PMCID: PMC5127646  PMID: 27894420
alternative splicing; protein-protein interactions; poly-proline; OCRE domain; structural biology; NMR-spectroscopy; Human
15.  Modelling Reveals Kinetic Advantages of Co-Transcriptional Splicing 
PLoS Computational Biology  2011;7(10):e1002215.
Messenger RNA splicing is an essential and complex process for the removal of intron sequences. Whereas the composition of the splicing machinery is mostly known, the kinetics of splicing, the catalytic activity of splicing factors and the interdependency of transcription, splicing and mRNA 3′ end formation are less well understood. We propose a stochastic model of splicing kinetics that explains data obtained from high-resolution kinetic analyses of transcription, splicing and 3′ end formation during induction of an intron-containing reporter gene in budding yeast. Modelling reveals co-transcriptional splicing to be the most probable and most efficient splicing pathway for the reporter transcripts, due in part to a positive feedback mechanism for co-transcriptional second step splicing. Model comparison is used to assess the alternative representations of reactions. Modelling also indicates the functional coupling of transcription and splicing, because both the rate of initiation of transcription and the probability that step one of splicing occurs co-transcriptionally are reduced, when the second step of splicing is abolished in a mutant reporter.
Author Summary
The coding information for the synthesis of proteins in mammalian cells is first transcribed from DNA to messenger RNA (mRNA), before being translated from mRNA to protein. Each step is complex, and subject to regulation. Certain sequences of DNA must be skipped in order to generate a functional protein, and these sequences, known as introns, are removed from the mRNA by the process of splicing. Splicing is well understood in terms of the proteins and complexes that are involved, but the rates of reactions, and models for the splicing pathways, have not yet been established. We present a model of splicing in yeast that accounts for the possibilities that splicing may take place while the mRNA is in the process of being created, as well as the possibility that splicing takes place once mRNA transcription is complete. We assign rates to the reactions in the pathway, and show that co-transcriptional splicing is the preferred pathway. In order to reach these conclusions, we compare a number of alternative models by a quantitative computational method. Our analysis relies on the quantitative measurement of messenger RNA in live cells - this is a major challenge in itself that has only recently been addressed.
PMCID: PMC3192812  PMID: 22022255
16.  Single molecule analysis reveals reversible and irreversible steps during spliceosome activation 
eLife  null;5:e14166.
The spliceosome is a complex machine composed of small nuclear ribonucleoproteins (snRNPs) and accessory proteins that excises introns from pre-mRNAs. After assembly the spliceosome is activated for catalysis by rearrangement of subunits to form an active site. How this rearrangement is coordinated is not well-understood. During activation, U4 must be released to allow U6 conformational change, while Prp19 complex (NTC) recruitment is essential for stabilizing the active site. We used multi-wavelength colocalization single molecule spectroscopy to directly observe the key events in Saccharomyces cerevisiae spliceosome activation. Following binding of the U4/U6.U5 tri-snRNP, the spliceosome either reverses assembly by discarding tri-snRNP or proceeds to activation by irreversible U4 loss. The major pathway for NTC recruitment occurs after U4 release. ATP stimulates both the competing U4 release and tri-snRNP discard processes. The data reveal the activation mechanism and show that overall splicing efficiency may be maintained through repeated rounds of disassembly and tri-snRNP reassociation.
eLife digest
The genes in an organism’s DNA may be expressed to form a protein via an intermediate molecule called RNA. In many organisms including humans, gene expression often begins by making a precursor molecule called a pre-mRNA. The pre-mRNA contains regions called exons that code for the protein product and regions called introns that do not. A machine in the cell called the spliceosome has the job of removing the introns in the pre-mRNA and stitching the exons together by a process known as splicing.
The spliceosome is made up of dozens of components that assemble on the pre-mRNAs. Before a newly assembled spliceosome can carry out splicing, it must be activated. The activation process involves several steps that are powered by the cell's universal power source (a molecule called ATP), including the release of many components from the spliceosome. Many of the details of the activation process are unclear.
Spliceosomes in the yeast species Saccharomyces cerevisiae are similar to spliceosomes from humans, and so are often studied experimentally. Hoskins et al. have now used a technique called colocalization single molecule fluorescence spectroscopy to follow, in real time, a single yeast spliceosome molecule as it activates. This technique uses a specialized microscope and a number of colored lasers to detect different spliceosome proteins at the same time. Hoskins et al. found that one of the steps during activation is irreversible – once that step occurs, the spliceosome must either perform the next activation steps or start the processes of assembly and activation over again.
Hoskins et al. also discovered that ATP causes some spliceosomes to be discarded during activation and not used for splicing. This indicates that before spliceosomes are allowed to activate, they may undergo 'quality control', which may be important for making sure that gene expression occurs efficiently and correctly. Future studies will investigate how this quality control process works in further detail.
PMCID: PMC4922858  PMID: 27244240
splicing; spliceosome; snRNP; single-molecule; fluorescence; RNA; S. cerevisiae
17.  A Broad Set of Chromatin Factors Influences Splicing 
PLoS Genetics  2016;12(9):e1006318.
Several studies propose an influence of chromatin on pre-mRNA splicing, but it is still unclear how widespread and how direct this phenomenon is. We find here that when assembled in vivo, the U2 snRNP co-purifies with a subset of chromatin-proteins, including histones and remodeling complexes like SWI/SNF. Yet, an unbiased RNAi screen revealed that the outcome of splicing is influenced by a much larger variety of chromatin factors not all associating with the spliceosome. The availability of this broad range of chromatin factors impacting splicing further unveiled their very context specific effect, resulting in either inclusion or skipping, depending on the exon under scrutiny. Finally, a direct assessment of the impact of chromatin on splicing using an in vitro co-transcriptional splicing assay with pre-mRNAs transcribed from a nucleosomal template, demonstrated that chromatin impacts nascent pre-mRNP in their competence for splicing. Altogether, our data show that numerous chromatin factors associated or not with the spliceosome can affect the outcome of splicing, possibly as a function of the local chromatin environment that by default interferes with the efficiency of splicing.
Author Summary
Splicing is an RNA editing step allowing to produce multiple transcripts from a single gene. The gene itself is organized in chromatin, associating DNA and multiple proteins. Some proteins regulating the compaction of the chromatin also affect RNA splicing. Yet, it was unclear whether these chromatin proteins were exceptions or whether chromatin very generally affected the outcome of splicing. Here, we show that a subset of chromatin proteins is physically in interaction with the enzyme responsible for RNA splicing. In addition, several chromatin proteins not found directly associated with the splicing machinery were also able to influence RNA splicing, suggesting that chromatin compaction very globally plays a role in splicing. This finding was confirmed using the first in vitro assay combining transcription and splicing in the context of chromatin; this assay showed that assembling DNA with chromatin proteins influences the efficiency of splicing.
PMCID: PMC5035054  PMID: 27662573
18.  RBM5 Is a Male Germ Cell Splicing Factor and Is Required for Spermatid Differentiation and Male Fertility 
PLoS Genetics  2013;9(7):e1003628.
Alternative splicing of precursor messenger RNA (pre-mRNA) is common in mammalian cells and enables the production of multiple gene products from a single gene, thus increasing transcriptome and proteome diversity. Disturbance of splicing regulation is associated with many human diseases; however, key splicing factors that control tissue-specific alternative splicing remain largely undefined. In an unbiased genetic screen for essential male fertility genes in the mouse, we identified the RNA binding protein RBM5 (RNA binding motif 5) as an essential regulator of haploid male germ cell pre-mRNA splicing and fertility. Mice carrying a missense mutation (R263P) in the second RNA recognition motif (RRM) of RBM5 exhibited spermatid differentiation arrest, germ cell sloughing and apoptosis, which ultimately led to azoospermia (no sperm in the ejaculate) and male sterility. Molecular modelling suggested that the R263P mutation resulted in compromised mRNA binding. Within the adult mouse testis, RBM5 localises to somatic and germ cells including spermatogonia, spermatocytes and round spermatids. Through the use of RNA pull down coupled with microarrays, we identified 11 round spermatid-expressed mRNAs as putative RBM5 targets. Importantly, the R263P mutation affected pre-mRNA splicing and resulted in a shift in the isoform ratios, or the production of novel spliced transcripts, of most targets. Microarray analysis of isolated round spermatids suggests that altered splicing of RBM5 target pre-mRNAs affected expression of genes in several pathways, including those implicated in germ cell adhesion, spermatid head shaping, and acrosome and tail formation. In summary, our findings reveal a critical role for RBM5 as a pre-mRNA splicing regulator in round spermatids and male fertility. Our findings also suggest that the second RRM of RBM5 is pivotal for appropriate pre-mRNA splicing.
Author Summary
The production of functional spermatozoa is an extraordinarily complex process that transforms a conventional round cell into the highly specialised sperm cell. These events require the coordinated activation of thousands of genes. It is likely that this complexity contributes to the large number of idiopathic infertility cases seen in humans. In an effort to improve the field's understanding of male fertility, we used a random mutagenesis screen to produce the Joey mouse line and to conclusively define RBM5 as an essential regulator of male fertility. The Joey line carries a mutation in the Rbm5 gene, which leads to a complete block of spermatid (haploid male germ cell) differentiation and ultimately a total loss of sperm production. Our results reveal a physiological role for RBM5 in the splicing of several spermatid-expressed mRNAs that are critical for the production of spermatozoa. This study is the first to show that RBM5, via its effects on mRNA splicing in the testis, is required for male fertility. These data improve our understanding of the regulatory networks of gene expression that control sperm production and as such may lead to the development of novel approaches to enhance or suppress fertility in men.
PMCID: PMC3723494  PMID: 23935508
19.  Lack of an effect of the efficiency of RNA 3'-end formation on the efficiency of removal of either the final or the penultimate intron in intact cells. 
Molecular and Cellular Biology  1995;15(1):488-496.
Evidence exists from studies using intact cells that intron removal can be influenced by the reactivity of upstream and downstream splice sites and that cleavage and polyadenylation can be influenced by the reactivity of upstream splice sites. These results indicate that sequences within 3'-terminal introns can function in the removal of upstream introns as well as the formation of RNA 3' ends. Evidence from studies using intact cells for an influence of RNA 3'-end formation on intron removal is lacking. We report here that mutations within polyadenylation sequences that either decrease or increase the efficiency of RNA 3'-end formation have no effect on the efficiencies with which either the 3'-terminal or the penultimate intron is removed by splicing. Northern (RNA) blot hybridization, RNase mapping, and an assay that couples reverse transcription and PCR were used to analyze the effects of deletions and a substitution of the polyadenylation sequences within the human gene for triosephosphate isomerase (TPI). TPI pre-mRNA harbors six introns that are constitutively removed by splicing. Relative to normal levels, each of the deletions was found to reduce the nuclear and cytoplasmic levels of TPI mRNA, increase the nuclear level of unprocessed RNA 3' ends, and decrease the nuclear level of processed RNA 3' ends. The simplest interpretation of these data indicates that (i) the rate of 3'-end formation normally limits the amount of mRNA produced and (ii) the deletions decrease and the substitution increases the efficiency of RNA 3'-end formation. While each of the deletions and the substitution altered the absolute levels of intron 6-containing, intron 5-containing, intron 6-free, and intron 5-free RNAs, in no case was there an abnormal ratio of intron-containing to intron-free RNA for either intron. Therefore, at least for TPI RNA, while the efficiency of removal of the 3'-terminal intron influences the efficiency of removal of either the 3'-end formation, the efficiency of RNA 3'-end formation does not influence the efficiency of removal of either the 3'-terminal or penultimate intron. The dependence of TPI RNA 3'-end formation on splicing may reflect the suboptimal strengths of the corresponding regulatory sequences and may function to ensure that TPI pre-mRNA is not released from the chromatin template until it has formed a complex with spliceosomes. If so, then the independence of TPI RNA splicing on 3'-end formation may be rationalized by the lack of a comparable function.
PMCID: PMC231997  PMID: 7799958
20.  Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast 
G3: Genes|Genomes|Genetics  2016;6(6):1513-1523.
Pre-mRNA splicing is an essential component of eukaryotic gene expression and is highly conserved from unicellular yeasts to humans. Here, we present the development and implementation of a sequencing-based reverse genetic screen designed to identify nonessential genes that impact pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, an organism that shares many of the complex features of splicing in higher eukaryotes. Using a custom-designed barcoding scheme, we simultaneously queried ∼3000 mutant strains for their impact on the splicing efficiency of two endogenous pre-mRNAs. A total of 61 nonessential genes were identified whose deletions resulted in defects in pre-mRNA splicing; enriched among these were factors encoding known or predicted components of the spliceosome. Included among the candidates identified here are genes with well-characterized roles in other RNA-processing pathways, including heterochromatic silencing and 3ʹ end processing. Splicing-sensitive microarrays confirm broad splicing defects for many of these factors, revealing novel functional connections between these pathways.
PMCID: PMC4889648  PMID: 27172183
Schizosaccharomyces pombe; genetic screen; genomics; heterochromatin; pre-mRNA splicing
21.  snRNA 3′ End Processing by a CPSF73-Containing Complex Essential for Development in Arabidopsis 
PLoS Biology  2016;14(10):e1002571.
Uridine-rich small nuclear RNAs (snRNAs) are the basal components of the spliceosome and play essential roles in splicing. The biogenesis of the majority of snRNAs involves 3′ end endonucleolytic cleavage of the nascent transcript from the elongating DNA-dependent RNA ploymerase II. However, the protein factors responsible for this process remain elusive in plants. Here, we show that DEFECTIVE in snRNA PROCESSING 1 (DSP1) is an essential protein for snRNA 3′ end maturation in Arabidopsis. A hypomorphic dsp1-1 mutation causes pleiotropic developmental defects, impairs the 3′ end processing of snRNAs, increases the levels of snRNA primary transcripts (pre-snRNAs), and alters the occupancy of Pol II at snRNA loci. In addition, DSP1 binds snRNA loci and interacts with Pol-II in a DNA/RNA-dependent manner. We further show that DSP1 forms a conserved complex, which contains at least four additional proteins, to catalyze snRNA 3′ end maturation in Arabidopsis. The catalytic component of this complex is likely the cleavage and polyadenylation specificity factor 73 kDa-I (CSPF73-I), which is the nuclease cleaving the pre-mRNA 3′ end. However, the DSP1 complex does not affect pre-mRNA 3′ end cleavage, suggesting that plants may use different CPSF73-I-containing complexes to process snRNAs and pre-mRNAs. This study identifies a complex responsible for the snRNA 3′ end maturation in plants and uncovers a previously unknown function of CPSF73 in snRNA maturation.
This study identifies a protein complex in plants that is responsible for the maturation of the 3′ ends of spliceosomal snRNAs and uncovers a novel function for the mRNA 3′ cleavage nuclease CPSF73.
Author Summary
snRNAs form the RNA components of the spliceosome and are required for spliceosome formation and splicing. The generation of snRNAs involves 3′ end endonucleolytic cleavage of primary snRNA transcripts (pre-snRNAs). The factors responsible for pre-snRNA 3′ end cleavage are known in metazoans, but many of these components are missing in plants. Therefore, the proteins that catalyze pre-snRNA cleavage in plants and the mechanism leading to plant snRNA 3′ maturation are unknown. Here, we show that a DSP1 complex (containing DSP1, DSP2, DSP3, DSP4, and CPFS73-I) is responsible for pre-snRNA 3′ end cleavage in Arabidopsis. We further show that CPSF73-I, which is known to cleave the pre-mRNA 3′ end, is likely the enzyme also catalyzing snRNA 3′ end maturation in plants. Interestingly, plants appear to use two different CPSF73-I-containing complexes to catalyze the maturation of mRNAs and snRNAs. The study thereby identifies an snRNA-processing complex in plants and also elucidates a new role for CPSF73-I in this process.
PMCID: PMC5079582  PMID: 27780203
22.  Recruitment of the Complete hTREX Complex Is Required for Kaposi's Sarcoma–Associated Herpesvirus Intronless mRNA Nuclear Export and Virus Replication 
PLoS Pathogens  2008;4(10):e1000194.
A cellular pre-mRNA undergoes various post-transcriptional processing events, including capping, splicing and polyadenylation prior to nuclear export. Splicing is particularly important for mRNA nuclear export as two distinct multi-protein complexes, known as human TREX (hTREX) and the exon-junction complex (EJC), are recruited to the mRNA in a splicing-dependent manner. In contrast, a number of Kaposi's sarcoma–associated herpesvirus (KSHV) lytic mRNAs lack introns and are exported by the virus-encoded ORF57 protein. Herein we show that ORF57 binds to intronless viral mRNAs and functions to recruit the complete hTREX complex, but not the EJC, in order assemble an export component viral ribonucleoprotein particle (vRNP). The formation of this vRNP is mediated by a direct interaction between ORF57 and the hTREX export adapter protein, Aly. Aly in turn interacts directly with the DEAD-box protein UAP56, which functions as a bridge to recruit the remaining hTREX proteins to the complex. Moreover, we show that a point mutation in ORF57 which disrupts the ORF57-Aly interaction leads to a failure in the ORF57-mediated recruitment of the entire hTREX complex to the intronless viral mRNA and inhibits the mRNAs subsequent nuclear export and virus replication. Furthermore, we have utilised a trans-dominant Aly mutant to prevent the assembly of the complete ORF57-hTREX complex; this results in a vRNP consisting of viral mRNA bound to ORF57, Aly and the nuclear export factor, TAP. Strikingly, although both the export adapter Aly and the export factor TAP were present on the viral mRNP, a dramatic decrease in intronless viral mRNA export and virus replication was observed in the absence of the remaining hTREX components (UAP56 and hTHO-complex). Together, these data provide the first direct evidence that the complete hTREX complex is essential for the export of KSHV intronless mRNAs and infectious virus production.
Author Summary
Following gene expression in the nucleus, newly transcribed messenger RNA (mRNA) is exported to the cytoplasm, where it is translated into protein. In mammals the vast majority of mRNAs contain introns that must be removed by the spliceosome prior to nuclear export. In addition to excising introns, splicing is also essential for the recruitment of a several protein complexes to mRNA, one example being the human transcription/export complex, which is required for mRNA export. Herpesviruses, such as Kaposi's sarcoma–associated herpesvirus, replicate by hijacking components of the host cells biological machinery, including those proteins necessary for mRNA export. An intriguing caveat in herpesvirology is that herpesviruses, such as Kaposi's sarcoma–associated herpesvirus, produce some mRNAs that lack introns and do not undergo splicing. How then are these intronless mRNAs exported to the cytoplasm? The answer lies in a virus protein called ORF57 that is able to bind to the intronless mRNA and then export them to the cytoplasm. ORF57 achieves this function by mimicking splicing and recruiting the human transcription/export complex to the intronless viral mRNA, thus facilitating its export into the cytoplasm.
PMCID: PMC2569588  PMID: 18974867
23.  A Novel Intra-U1 snRNP Cross-Regulation Mechanism: Alternative Splicing Switch Links U1C and U1-70K Expression 
PLoS Genetics  2013;9(10):e1003856.
The U1 small nuclear ribonucleoprotein (snRNP)-specific U1C protein participates in 5′ splice site recognition and regulation of pre-mRNA splicing. Based on an RNA-Seq analysis in HeLa cells after U1C knockdown, we found a conserved, intra-U1 snRNP cross-regulation that links U1C and U1-70K expression through alternative splicing and U1 snRNP assembly. To investigate the underlying regulatory mechanism, we combined mutational minigene analysis, in vivo splice-site blocking by antisense morpholinos, and in vitro binding experiments. Alternative splicing of U1-70K pre-mRNA creates the normal (exons 7–8) and a non-productive mRNA isoform, whose balance is determined by U1C protein levels. The non-productive isoform is generated through a U1C-dependent alternative 3′ splice site, which requires an adjacent cluster of regulatory 5′ splice sites and binding of intact U1 snRNPs. As a result of nonsense-mediated decay (NMD) of the non-productive isoform, U1-70K mRNA and protein levels are down-regulated, and U1C incorporation into the U1 snRNP is impaired. U1-70K/U1C-deficient particles are assembled, shifting the alternative splicing balance back towards productive U1-70K splicing, and restoring assembly of intact U1 snRNPs. Taken together, we established a novel feedback regulation that controls U1-70K/U1C homeostasis and ensures correct U1 snRNP assembly and function.
Author Summary
The accurate removal of intervening sequences (introns) from precursor messenger RNAs (pre-mRNAs) represents an essential step in the expression of most eukaryotic protein-coding genes. Alternative splicing can create from a single primary transcript various mature mRNAs with diverse, sometimes even antagonistic, biological functions. Many human diseases are based on alternative-splicing defects, and most interestingly, certain defects are caused by mutations in general splicing factors that participate in each splicing event. To address the question of how a general splicing factor can regulate alternative splicing events, here we investigated the regulatory role of the U1C protein, a specific component of the U1 small nuclear ribonucleoprotein (snRNP) and important in initial 5′ splice site recognition. Our RNA-Seq analysis demonstrated that U1C affects more than 300 cases of alternative splicing in the human system. One U1C target, U1-70K, appeared to be particularly interesting, because both protein products are components of the U1 snRNP and functionally depend on each other. Analyzing the mechanistic basis of this intra-U1 snRNP cross-regulation, we discovered a U1C-dependent alternative splicing switch in the U1-70K pre-mRNA that regulates U1-70K expression. In sum, this feedback loop controls and links U1C and U1-70K homeostasis to guarantee correct U1 snRNP assembly and function.
PMCID: PMC3798272  PMID: 24146627
24.  BRR2a Affects Flowering Time via FLC Splicing 
PLoS Genetics  2016;12(4):e1005924.
Several pathways control time to flowering in Arabidopsis thaliana through transcriptional and posttranscriptional gene regulation. In recent years, mRNA processing has gained interest as a critical regulator of flowering time control in plants. However, the molecular mechanisms linking RNA splicing to flowering time are not well understood. In a screen for Arabidopsis early flowering mutants we identified an allele of BRR2a. BRR2 proteins are components of the spliceosome and highly conserved in eukaryotes. Arabidopsis BRR2a is ubiquitously expressed in all analyzed tissues and involved in the processing of flowering time gene transcripts, most notably FLC. A missense mutation of threonine 895 in BRR2a caused defects in FLC splicing and greatly reduced FLC transcript levels. Reduced FLC expression increased transcription of FT and SOC1 leading to early flowering in both short and long days. Genome-wide experiments established that only a small set of introns was not correctly spliced in the brr2a mutant. Compared to control introns, retained introns were often shorter and GC-poor, had low H3K4me1 and CG methylation levels, and were often derived from genes with a high-H3K27me3-low-H3K36me3 signature. We propose that BRR2a is specifically needed for efficient splicing of a subset of introns characterized by a combination of factors including intron size, sequence and chromatin, and that FLC is most sensitive to splicing defects.
Author Summary
Timing of flowering has a great effect on reproductive success and fitness. It is controlled by many external signals and internal states involving a large set of genes. Here we report that the Arabidopsis thaliana BRR2a gene is needed for normal flowering. BRR2 proteins are components of the spliceosome and highly conserved in eukaryotes. BRR2a is needed for splicing of a subset of introns, most noticeably in the transcript of the flowering repressor FLC. Reduced FLC expression increased transcription of key floral activators, leading to early flowering in both short and long days. Genome-wide experiments established that full BRR2a activity was required only for a small group of introns. We propose that uncompromised BRR2a activity is most important for efficient splicing of a subset of introns of particular size, sequence and chromatin composition, and that FLC is most sensitive to splicing defects.
PMCID: PMC4839602  PMID: 27100965
25.  Widespread Use of Non-productive Alternative Splice Sites in Saccharomyces cerevisiae  
PLoS Genetics  2014;10(4):e1004249.
Saccharomyces cerevisiae has been used as a model system to investigate the mechanisms of pre-mRNA splicing but only a few examples of alternative splice site usage have been described in this organism. Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many S. cerevisiae intron-containing genes exhibit usage of alternative splice sites, but many transcripts generated by splicing at these sites are non-functional because they introduce premature termination codons, leading to degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3′ splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3′-splice site in GCR1. The use of non-productive alternative splice sites can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results show that alternative splicing is frequent in S. cerevisiae but masked by RNA degradation and that the use of alternative splice sites in this organism is mostly aimed at controlling transcript levels rather than increasing proteome diversity.
Author Summary
Accurate gene expression requires the transfer of gene information from DNA to RNA. When DNA is transcribed into RNA, part of the RNA needs to be removed (spliced) to generate a proper copy of the genetic information. This process needs to be very accurate to preserve the genetic information that will be transferred into proteins. Our study shows that in baker's yeast, the splicing process does not always produce the correctly spliced products, as RNA splicing events frequently utilize incorrect splice sites. However, these deficient RNA molecules are eliminated from cells by a quality control mechanism to preserve the integrity of the genetic information. However, incorrect splicing is not useless, as it can be used to regulate the quantity of RNA that is generated.
PMCID: PMC3983031  PMID: 24722551

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