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1.  Malleable ribonucleoprotein machine: protein intrinsic disorder in the Saccharomyces cerevisiae spliceosome 
PeerJ  2013;1:e2.
Recent studies revealed that a significant fraction of any given proteome is presented by proteins that do not have unique 3D structures as a whole or in significant parts. These intrinsically disordered proteins possess dramatic structural and functional variability, being especially enriched in signaling and regulatory functions since their lack of fixed structure defines their ability to be involved in interaction with several proteins and allows them to be re-used in multiple pathways. Among recognized disorder-based protein functions are interactions with nucleic acids and multi-target binding; i.e., the functions ascribed to many spliceosomal proteins. Therefore, the spliceosome, a multimegadalton ribonucleoprotein machine catalyzing the excision of introns from eukaryotic pre-mRNAs, represents an attractive target for the focused analysis of the abundance and functionality of intrinsic disorder in its proteinaceous components. In yeast cells, spliceosome consists of five small nuclear RNAs (U1, U2, U4, U5, and U6) and a range of associated proteins. Some of these proteins constitute cores of the corresponding snRNA-protein complexes known as small nuclear ribonucleoproteins (snRNPs). Other spliceosomal proteins have various auxiliary functions. To gain better understanding of the functional roles of intrinsic disorder, we have studied the prevalence of intrinsically disordered proteins in the yeast spliceosome using a wide array of bioinformatics methods. Our study revealed that similar to the proteins associated with human spliceosomes (Korneta & Bujnicki, 2012), proteins found in the yeast spliceosome are enriched in intrinsic disorder.
PMCID: PMC3628832  PMID: 23638354
Spliceosome; Intrinsically disordered protein; Protein structure; RNA–protein complex; Protein–protein interaction; Intrinsic disorder; Protein–RNA interaction; Protein hub; Splicing; Protein function
2.  Sm/Lsm Genes Provide a Glimpse into the Early Evolution of the Spliceosome 
PLoS Computational Biology  2009;5(3):e1000315.
The spliceosome, a sophisticated molecular machine involved in the removal of intervening sequences from the coding sections of eukaryotic genes, appeared and subsequently evolved rapidly during the early stages of eukaryotic evolution. The last eukaryotic common ancestor (LECA) had both complex spliceosomal machinery and some spliceosomal introns, yet little is known about the early stages of evolution of the spliceosomal apparatus. The Sm/Lsm family of proteins has been suggested as one of the earliest components of the emerging spliceosome and hence provides a first in-depth glimpse into the evolving spliceosomal apparatus. An analysis of 335 Sm and Sm-like genes from 80 species across all three kingdoms of life reveals two significant observations. First, the eukaryotic Sm/Lsm family underwent two rapid waves of duplication with subsequent divergence resulting in 14 distinct genes. Each wave resulted in a more sophisticated spliceosome, reflecting a possible jump in the complexity of the evolving eukaryotic cell. Second, an unusually high degree of conservation in intron positions is observed within individual orthologous Sm/Lsm genes and between some of the Sm/Lsm paralogs. This suggests that functional spliceosomal introns existed before the emergence of the complete Sm/Lsm family of proteins; hence, spliceosomal machinery with considerably fewer components than today's spliceosome was already functional.
Author Summary
The spliceosome is a complex molecular machine that removes intervening sequences (introns) from mRNAs. It is unique to eukaryotes. Although prokaryotes have self-splicing introns, they completely lack spliceosomal introns and the spliceosome itself. Yet even the simplest eukaryotic organisms have introns and a rather complex spliceosomal apparatus. Little is known about how this amazing machine rapidly evolved in early eukaryotes. Here, we attempt to reconstruct a part of this evolutionary process using one of the most fundamental components of the spliceosome—the Sm and Lsm family of proteins. Using sequence and structure analysis as well as the analysis of the intron positions in Sm and Lsm genes in conjunction with a wealth of published data, we propose a plausible scenario for some aspects of spliceosomal evolution. In particular, we suggest that the Lsm family of genes could have been the first and the most essential component that allowed rudimentary splicing of early spliceosomal introns. Extensive duplications of Lsm genes and the later rise of the Sm gene family likely reflect a gradual increase in complexity of the spliceosome.
PMCID: PMC2650416  PMID: 19282982
3.  Structural and Functional Characterization of the N Terminus of Schizosaccharomyces pombe Cwf10 
Eukaryotic Cell  2013;12(11):1472-1489.
The spliceosome is a dynamic macromolecular machine that catalyzes the removal of introns from pre-mRNA, yielding mature message. Schizosaccharomyces pombe Cwf10 (homolog of Saccharomyces cerevisiae Snu114 and human U5-116K), an integral member of the U5 snRNP, is a GTPase that has multiple roles within the splicing cycle. Cwf10/Snu114 family members are highly homologous to eukaryotic translation elongation factor EF2, and they contain a conserved N-terminal extension (NTE) to the EF2-like portion, predicted to be an intrinsically unfolded domain. Using S. pombe as a model system, we show that the NTE is not essential, but cells lacking this domain are defective in pre-mRNA splicing. Genetic interactions between cwf10-ΔNTE and other pre-mRNA splicing mutants are consistent with a role for the NTE in spliceosome activation and second-step catalysis. Characterization of Cwf10-NTE by various biophysical techniques shows that in solution the NTE contains regions of both structure and disorder. The first 23 highly conserved amino acids of the NTE are essential for its role in splicing but when overexpressed are not sufficient to restore pre-mRNA splicing to wild-type levels in cwf10-ΔNTE cells. When the entire NTE is overexpressed in the cwf10-ΔNTE background, it can complement the truncated Cwf10 protein in trans, and it immunoprecipitates a complex similar in composition to the late-stage U5.U2/U6 spliceosome. These data show that the structurally flexible NTE is capable of independently incorporating into the spliceosome and improving splicing function, possibly indicating a role for the NTE in stabilizing conformational rearrangements during a splice cycle.
PMCID: PMC3837936  PMID: 24014766
4.  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
5.  The spliceosomal proteome: at the heart of the largest cellular ribonucleoprotein machine 
Proteomics  2010;10(22):4128-4141.
Almost all primary transcripts in higher eukaryotes undergo several splicing events and alternative splicing is a major factor in generating proteomic diversity. Thus, the spliceosome, the ribonucleoprotein assembly that performs splicing, is a highly critical cellular machine and as expected, a very complex one. Indeed, the spliceosome is one of the largest, if not the largest, molecular machine in the cell with over 150 different components in human. A large fraction of the spliceosomal proteome is organized into ribonucleoprotein particles (snRNPs) by associating with one of the small nuclear RNAs (snRNAs), and the function of many spliceosomal proteins revolve around their association or interaction with the spliceosomal RNAs or the substrate pre-messenger RNAs. In addition to the complex web of protein-RNA interactions, an equally complex network of protein-protein interactions exists in the spliceosome which includes a number of large, conserved proteins with critical functions in the spliceosomal catalytic core. These include the largest conserved nuclear protein, Prp8, which plays a critical role in spliceosomal function in a hitherto unknown manner. Taken together, the large spliceosomal proteome and its dynamic nature has made it a highly challenging system to study, and at the same time, provides an exciting example of the evolution of a proteome around a backbone of primordial RNAs likely dating from the RNA World.
PMCID: PMC3575088  PMID: 21080498
Proteome; Prp8; RNA helicases; Spliceosome; Splicing
6.  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
7.  Structural bioinformatics of the human spliceosomal proteome 
Nucleic Acids Research  2012;40(15):7046-7065.
In this work, we describe the results of a comprehensive structural bioinformatics analysis of the spliceosomal proteome. We used fold recognition analysis to complement prior data on the ordered domains of 252 human splicing proteins. Examples of newly identified domains include a PWI domain in the U5 snRNP protein 200K (hBrr2, residues 258–338), while examples of previously known domains with a newly determined fold include the DUF1115 domain of the U4/U6 di-snRNP protein 90K (hPrp3, residues 540–683). We also established a non-redundant set of experimental models of spliceosomal proteins, as well as constructed in silico models for regions without an experimental structure. The combined set of structural models is available for download. Altogether, over 90% of the ordered regions of the spliceosomal proteome can be represented structurally with a high degree of confidence. We analyzed the reduced spliceosomal proteome of the intron-poor organism Giardia lamblia, and as a result, we proposed a candidate set of ordered structural regions necessary for a functional spliceosome. The results of this work will aid experimental and structural analyses of the spliceosomal proteins and complexes, and can serve as a starting point for multiscale modeling of the structure of the entire spliceosome.
PMCID: PMC3424538  PMID: 22573172
8.  Minor introns are embedded molecular switches regulated by highly unstable U6atac snRNA 
eLife  2013;2:e00780.
Eukaryotes have two types of spliceosomes, comprised of either major (U1, U2, U4, U5, U6) or minor (U11, U12, U4atac, U6atac; <1%) snRNPs. The high conservation of minor introns, typically one amidst many major introns in several hundred genes, despite their poor splicing, has been a long-standing enigma. Here, we discovered that the low abundance minor spliceosome’s catalytic snRNP, U6atac, is strikingly unstable (t½<2 hr). We show that U6atac level depends on both RNA polymerases II and III and can be rapidly increased by cell stress-activated kinase p38MAPK, which stabilizes it, enhancing mRNA expression of hundreds of minor intron-containing genes that are otherwise suppressed by limiting U6atac. Furthermore, p38MAPK-dependent U6atac modulation can control minor intron-containing tumor suppressor PTEN expression and cytokine production. We propose that minor introns are embedded molecular switches regulated by U6atac abundance, providing a novel post-transcriptional gene expression mechanism and a rationale for the minor spliceosome’s evolutionary conservation.
eLife digest
The central dogma of biology states that genetic material, DNA, is transcribed into RNA, which is then translated into proteins. However, the genes of many organisms have stretches of non-coding DNA that interrupt the sequences that code for protein. These non-coding sequences, which are called introns, must be removed, and the remaining sequences—which are called exons—must then be joined together to produce a messenger RNA (mRNA) transcript that is ready to be translated into protein.
The process of removing the introns and joining the exons is called splicing, and it is carried out by a molecular machine called the spliceosome. However, in addition to containing typical (‘major’) introns, several hundred human genes also contain a single ‘minor’ intron, and a minor spliceosome is needed to remove it. Minor introns occur in many highly conserved genes, but they are often inefficiently spliced. This means that the resulting mRNA transcripts may not be translated into proteins—which is puzzling given that these proteins perform important roles within the cell.
The major and minor spliceosomes are composed of proteins and small non-coding RNA molecules (which, as their name suggests, are never translated in cells). Now Younis et al. shed new light on the minor spliceosome by showing that a small non-coding RNA molecule known as U6atac, which catalyzes the removal of introns by the minor spliceosome, is highly unstable in human cells. This means that U6atac is a limiting factor for the splicing of minor introns—a process that is already limited by the very low abundance of the minor spliceosome under normal conditions. However, Younis et al. found that this bottleneck could be relieved by halting the degradation of U6atac. Experiments showed that U6atac can be stabilized by a key signaling molecule, a protein kinase (called p38MAPK), which is activated in response to stress. The resulting higher levels of U6atac promoted splicing of the introns in its target mRNA transcripts, and also modulated various signaling pathways in the cells.
Together, these results imply that the minor spliceosome is used as a valve that can help cells to adapt to stress and other changes. Moreover, by helping to translate mRNA transcripts that are already present in cells, it enables proteins to be produced rapidly in response to stress, bypassing the need for a fresh round of transcription.
PMCID: PMC3728624  PMID: 23908766
snRNA; U6atac; splicing; gene regulation; Human
9.  The Spliceosome-Activating Complex: Molecular Mechanisms Underlying the Function of a Pleiotropic Regulator 
Correct interpretation of the coding capacity of RNA polymerase II transcribed eukaryotic genes is determined by the recognition and removal of intronic sequences of pre-mRNAs by the spliceosome. Our current knowledge on dynamic assembly and subunit interactions of the spliceosome mostly derived from the characterization of yeast, Drosophila, and human spliceosomal complexes formed on model pre-mRNA templates in cell extracts. In addition to sequential structural rearrangements catalyzed by ATP-dependent DExH/D-box RNA helicases, catalytic activation of the spliceosome is critically dependent on its association with the NineTeen Complex (NTC) named after its core E3 ubiquitin ligase subunit PRP19. NTC, isolated recently from Arabidopsis, occurs in a complex with the essential RNA helicase and GTPase subunits of the U5 small nuclear RNA particle that are required for both transesterification reactions of splicing. A compilation of mass spectrometry data available on the composition of NTC and spliceosome complexes purified from different organisms indicates that about half of their conserved homologs are encoded by duplicated genes in Arabidopsis. Thus, while mutations of single genes encoding essential spliceosome and NTC components lead to cell death in other organisms, differential regulation of some of their functionally redundant Arabidopsis homologs permits the isolation of partial loss of function mutations. Non-lethal pleiotropic defects of these mutations provide a unique means for studying the roles of NTC in co-transcriptional assembly of the spliceosome and its crosstalk with DNA repair and cell death signaling pathways.
PMCID: PMC3355604  PMID: 22639636
spliceosome; NineTeen complex; co-transcriptional splicing; DNA repair; cell death signaling; Arabidopsis
10.  Spliceosome Database: a tool for tracking components of the spliceosome 
Nucleic Acids Research  2012;41(Database issue):D132-D141.
The spliceosome is the extremely complex macromolecular machine responsible for pre-mRNA splicing. It assembles from five U-rich small nuclear RNAs (snRNAs) and over 200 proteins in a highly dynamic fashion. One important challenge to studying the spliceosome is simply keeping track of all these proteins, a situation further complicated by the variety of names and identifiers that exist in the literature for them. To facilitate studies of the spliceosome and its components, we created a database of spliceosome-associated proteins and snRNAs, which is available at and can be queried through a simple browser interface. In the database, we cataloged the various names, orthologs and gene identifiers of spliceosome proteins to navigate the complex nomenclature of spliceosome proteins. We also provide links to gene and protein records for the spliceosome components in other databases. To navigate spliceosome assembly dynamics, we created tools to compare the association of spliceosome proteins with complexes that form at specific stages of spliceosome assembly based on a compendium of mass spectrometry experiments that identified proteins in purified splicing complexes. Together, the information in the database provides an easy reference for spliceosome components and will support future modeling of spliceosome structure and dynamics.
PMCID: PMC3531166  PMID: 23118483
11.  Spliceosomal Proteomics in Trypanosoma brucei Reveal New RNA Splicing Factors▿ †  
Eukaryotic Cell  2009;8(7):990-1000.
In trypanosomatid parasites, spliced leader (SL) trans splicing is an essential nuclear mRNA maturation step which caps mRNAs posttranscriptionally and, in conjunction with polyadenylation, resolves individual mRNAs from polycistronic precursors. While all trypanosomatid mRNAs are trans spliced, intron removal by cis splicing is extremely rare and predicted to occur in only four pre-mRNAs. trans- and cis-splicing reactions are carried out by the spliceosome, which consists of U-rich small nuclear ribonucleoprotein particles (U snRNPs) and of non-snRNP factors. Mammalian and yeast spliceosome complexes are well characterized and found to be associated with up to 170 proteins. Despite the central importance of trans splicing in trypanosomatid gene expression, only the core RNP proteins and a few snRNP-specific proteins are known. To characterize the trypanosome spliceosomal protein repertoire, we conducted a proteomic analysis by tagging and tandem affinity-purifying the canonical core RNP protein SmD1 in Trypanosoma brucei and by identifying copurified proteins by mass spectrometry. The set of 47 identified proteins harbored nearly all spliceosomal snRNP factors characterized in trypanosomes thus far and 21 proteins lacking a specific annotation. A bioinformatic analysis combined with protein pull-down assays and immunofluorescence microscopy identified 10 divergent orthologues of known splicing factors, including the missing U1-specific protein U1A. In addition, a novel U5-specific, and, as we show, an essential splicing factor was identified that shares a short, highly conserved N-terminal domain with the yeast protein Cwc21p and was thus tentatively named U5-Cwc21. Together, these data strongly indicate that most of the identified proteins are components of the spliceosome.
PMCID: PMC2708463  PMID: 19429779
12.  RSR-2, the Caenorhabditis elegans Ortholog of Human Spliceosomal Component SRm300/SRRM2, Regulates Development by Influencing the Transcriptional Machinery 
PLoS Genetics  2013;9(6):e1003543.
Protein components of the spliceosome are highly conserved in eukaryotes and can influence several steps of the gene expression process. RSR-2, the Caenorhabditis elegans ortholog of the human spliceosomal protein SRm300/SRRM2, is essential for viability, in contrast to the yeast ortholog Cwc21p. We took advantage of mutants and RNA interference (RNAi) to study rsr-2 functions in C. elegans, and through genetic epistasis analysis found that rsr-2 is within the germline sex determination pathway. Intriguingly, transcriptome analyses of rsr-2(RNAi) animals did not reveal appreciable splicing defects but instead a slight global decrease in transcript levels. We further investigated this effect in transcription and observed that RSR-2 colocalizes with DNA in germline nuclei and coprecipitates with chromatin, displaying a ChIP-Seq profile similar to that obtained for the RNA Polymerase II (RNAPII). Consistent with a novel transcription function we demonstrate that the recruitment of RSR-2 to chromatin is splicing-independent and that RSR-2 interacts with RNAPII and affects RNAPII phosphorylation states. Proteomic analyses identified proteins associated with RSR-2 that are involved in different gene expression steps, including RNA metabolism and transcription with PRP-8 and PRP-19 being the strongest interacting partners. PRP-8 is a core component of the spliceosome and PRP-19 is the core component of the PRP19 complex, which interacts with RNAPII and is necessary for full transcriptional activity. Taken together, our study proposes that RSR-2 is a multifunctional protein whose role in transcription influences C. elegans development.
Author Summary
It is well known that splicing occurs cotranscriptionally but the functional coupling between splicing and transcription has not been studied carefully in the context of a multicellular organism in development. We took advantage of the amenable C. elegans genetics and genomics to demonstrate a functional relationship between RSR-2, whose yeast and human orthologs are components of the spliceosome, and transcription. Although we found that RSR-2 interacts with proteins present in the spliceosome, moderate inhibition of rsr-2 by RNAi did not significantly affect splicing, but rather caused a decrease in transcript levels that was critical for germline sex determination. Our investigation on such a paradox of a spliceosomal component affecting transcription resulted in several lines of evidence linking RSR-2 with transcription: (i) RSR-2 immunoprecipitates chromatin resembling the ChIP-Seq profile of RNAPII, (ii) RSR-2 is present in intronless genes, (iii) rsr-2(RNAi) globally modifies the distribution of RNAPII along genes and its phosphorylation state, (iv) RSR-2 coimmunoprecipitates with RNAPII, and (v) RSR-2 interacts with PRP-19, which is a component of the spliceosome required for efficient transcriptional activity. Our findings raise an intriguing question: to what extent does a moderate alteration in some spliceosome components affect the gene expression process by perturbing splicing or transcription?
PMCID: PMC3675011  PMID: 23754964
13.  Spliceosome Structure and Function 
Pre-mRNA splicing is catalyzed by the spliceosome, a multimegadalton ribonucleoprotein (RNP) complex comprised of five snRNPs and numerous proteins. Intricate RNA-RNA and RNP networks, which serve to align the reactive groups of the pre-mRNA for catalysis, are formed and repeatedly rearranged during spliceosome assembly and catalysis. Both the conformation and composition of the spliceosome are highly dynamic, affording the splicing machinery its accuracy and flexibility, and these remarkable dynamics are largely conserved between yeast and metazoans. Because of its dynamic and complex nature, obtaining structural information about the spliceosome represents a major challenge. Electron microscopy has revealed the general morphology of several spliceosomal complexes and their snRNP subunits, and also the spatial arrangement of some of their components. X-ray and NMR studies have provided high resolution structure information about spliceosomal proteins alone or complexed with one or more binding partners. The extensive interplay of RNA and proteins in aligning the pre-mRNA's reactive groups, and the presence of both RNA and protein at the core of the splicing machinery, suggest that the spliceosome is an RNP enzyme. However, elucidation of the precise nature of the spliceosome's active site, awaits the generation of a high-resolution structure of its RNP core.
Spliceosomes contain five snRNPs and numerous non-snRNP proteins. These continuously rearrange during spliceosome assembly and activation so that the reactive groups in the pre-mRNA substrate are correctly aligned for catalysis.
PMCID: PMC3119917  PMID: 21441581
14.  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
15.  A bird’s-eye view of post-translational modifications in the spliceosome and their roles in spliceosome dynamics† 
Molecular bioSystems  2010;6(11):2093-2102.
Pre-mRNA splicing, the removal of noncoding intron sequences from the pre-mRNA, is a critical reaction in eukaryotic gene expression. Pre-mRNA splicing is carried out by a remarkable macromolecular machine, the spliceosome, which undergoes dynamic rearrangements of its RNA and protein components to assemble its catalytic center. While significant progress has been made in describing the “moving parts” of this machine, the mechanisms by which spliceosomal proteins mediate the ordered rearrangements within the spliceosome remain elusive. Here we explore recent evidence from proteomics studies revealing extensive post-translational modification of splicing factors. While the functional significance of most of these modifications remains to be characterized, we describe recent studies in which the roles of specific post-translational modifications of splicing factors have been characterized. These examples illustrate the importance of post-translational modifications in spliceosome dynamics
PMCID: PMC4065859  PMID: 20672149
16.  The Crystal Structure of PPIL1 Bound to Cyclosporine A Suggests a Binding Mode for a Linear Epitope of the SKIP Protein 
PLoS ONE  2010;5(4):e10013.
The removal of introns from pre-mRNA is carried out by a large macromolecular machine called the spliceosome. The peptidyl-prolyl cis/trans isomerase PPIL1 is a component of the human spliceosome and binds to the spliceosomal SKIP protein via a binding site distinct from its active site.
Principal Findings
Here, we have studied the PPIL1 protein and its interaction with SKIP biochemically and by X-ray crystallography. A minimal linear binding epitope derived from the SKIP protein could be determined using a peptide array. A 36-residue region of SKIP centred on an eight-residue epitope suffices to bind PPIL1 in pull-down experiments. The crystal structure of PPIL1 in complex with the inhibitor cyclosporine A (CsA) was obtained at a resolution of 1.15 Å and exhibited two bound Cd2+ ions that enabled SAD phasing. PPIL1 residues that have previously been implicated in binding of SKIP are involved in the coordination of Cd2+ ions in the present crystal structure. Employing the present crystal structure, the determined minimal binding epitope and previously published NMR data [1], a molecular docking study was performed. In the docked model of the PPIL1·SKIP interaction, a proline residue of SKIP is buried in a hydrophobic pocket of PPIL1. This hydrophobic contact is encircled by several hydrogen bonds between the SKIP peptide and PPIL1.
We characterized a short, linear epitope of SKIP that is sufficient to bind the PPIL1 protein. Our data indicate that this SKIP peptide could function in recruiting PPIL1 into the core of the spliceosome. We present a molecular model for the binding mode of SKIP to PPIL1 which emphasizes the versatility of cyclophilin-type PPIases to engage in additional interactions with other proteins apart from active site contacts despite their limited surface area.
PMCID: PMC2848857  PMID: 20368803
17.  Serine/arginine-rich splicing factors belong to a class of intrinsically disordered proteins 
Nucleic Acids Research  2006;34(1):305-312.
Serine/arginine-rich (SR) splicing factors play an important role in constitutive and alternative splicing as well as during several steps of RNA metabolism. Despite the wealth of functional information about SR proteins accumulated to-date, structural knowledge about the members of this family is very limited. To gain a better insight into structure-function relationships of SR proteins, we performed extensive sequence analysis of SR protein family members and combined it with ordered/disordered structure predictions. We found that SR proteins have properties characteristic of intrinsically disordered (ID) proteins. The amino acid composition and sequence complexity of SR proteins were very similar to those of the disordered protein regions. More detailed analysis showed that the SR proteins, and their RS domains in particular, are enriched in the disorder-promoting residues and are depleted in the order-promoting residues as compared to the entire human proteome. Moreover, disorder predictions indicated that RS domains of SR proteins were completely unstructured. Two different classification methods, the charge-hydropathy measure and the cumulative distribution function (CDF) of the disorder scores, were in agreement with each other, and they both strongly predicted members of the SR protein family to be disordered. This study emphasizes the importance of the disordered structure for several functions of SR proteins, such as for spliceosome assembly and for interaction with multiple partners. In addition, it demonstrates the usefulness of order/disorder predictions for inferring protein structure from sequence.
PMCID: PMC1326245  PMID: 16407336
18.  Conservation of the Protein Composition and Electron Microscopy Structure of Drosophila melanogaster and Human Spliceosomal Complexes▿ † 
Molecular and Cellular Biology  2008;29(1):281-301.
Comprehensive proteomics analyses of spliceosomal complexes are currently limited to those in humans, and thus, it is unclear to what extent the spliceosome's highly complex composition and compositional dynamics are conserved among metazoans. Here we affinity purified Drosophila melanogaster spliceosomal B and C complexes formed in Kc cell nuclear extract. Mass spectrometry revealed that their composition is highly similar to that of human B and C complexes. Nonetheless, a number of Drosophila-specific proteins were identified, suggesting that there may be novel factors contributing specifically to splicing in flies. Protein recruitment and release events during the B-to-C transition were also very similar in both organisms. Electron microscopy of Drosophila B complexes revealed a high degree of structural similarity with human B complexes, indicating that higher-order interactions are also largely conserved. A comparison of Drosophila spliceosomes formed on a short versus long intron revealed only small differences in protein composition but, nonetheless, clear structural differences under the electron microscope. Finally, the characterization of affinity-purified Drosophila mRNPs indicated that exon junction complex proteins are recruited in a splicing-dependent manner during C complex formation. These studies provide insights into the evolutionarily conserved composition and structure of the metazoan spliceosome, as well as its compositional dynamics during catalytic activation.
PMCID: PMC2612486  PMID: 18981222
19.  Insights into the activation of the helicase Prp43 by biochemical studies and structural mass spectrometry 
Nucleic Acids Research  2013;42(2):1162-1179.
Splicing of precursor messenger RNA is a hallmark of eukaryotic cells, which is carried out by the spliceosome, a multi-megadalton ribonucleoprotein machinery. The splicing reaction removes non-coding regions (introns) and ligates coding regions (exons). The spliceosome is a highly dynamic ribonucleoprotein complex that undergoes dramatic structural changes during its assembly, the catalysis and its disassembly. The transitions between the different steps during the splicing cycle are promoted by eight conserved DExD/H box ATPases. The DEAH-box protein Prp43 is responsible for the disassembly of the intron-lariat spliceosome and its helicase activity is activated by the G-patch protein Ntr1. Here, we investigate the activation of Prp43 by Ntr1 in the presence and absence of RNA substrate by functional assays and structural proteomics. Residues 51–110 of Ntr1 were identified to be the minimal fragment that induces full activation. We found protein–protein cross-links that indicate that Prp43 interacts with the G-patch motif of Ntr1 through its C-terminal domains. Additionally, we report on functionally important RNA binding residues in both proteins and propose a model for the activation of the helicase.
PMCID: PMC3902948  PMID: 24165877
20.  U12-type Spliceosomal Introns of Insecta 
Most of eukaryotic genes are interrupted by introns that need to be removed from pre-mRNAs before they can perform their function. This is done by complex machinery called spliceosome. Many eukaryotes possess two separate spliceosomal systems that process separate sets of introns. The major (U2) spliceosome removes majority of introns, while minute fraction of intron repertoire is processed by the minor (U12) spliceosome. These two populations of introns are called U2-type and U12-type, respectively. The latter fall into two subtypes based on the terminal dinucleotides. The minor spliceosomal system has been lost independently in some lineages, while in some others few U12-type introns persist. We investigated twenty insect genomes in order to better understand the evolutionary dynamics of U12-type introns. Our work confirms dramatic drop of U12-type introns in Diptera, leaving these genomes just with a handful cases. This is mostly the result of intron deletion, but in a number of dipteral cases, minor type introns were switched to a major type, as well. Insect genes that harbor U12-type introns belong to several functional categories among which proteins binding ions and nucleic acids are enriched and these few categories are also overrepresented among these genes that preserved minor type introns in Diptera.
PMCID: PMC3291851  PMID: 22393306
U12-type introns; minor spliceosome; insect evolution.
21.  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
22.  Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing 
Nature structural & molecular biology  2013;20(12):10.1038/nsmb.2704.
The spliceosome is a dynamic ribonucleoprotein (RNP) machine that catalyzes the removal of introns in the two transesterification steps of eukaryotic pre-mRNA splicing. Here we used single molecule fluorescence resonance energy transfer to monitor the distance of the 5′ splice site (5′SS) and branchpoint (BP) of pre-mRNA in affinity-purified spliceosomes stalled by a mutation in the DExD/H-box helicase Prp2 immediately prior to the first splicing step. Addition of recombinant Prp2 together with NTP and protein cofactor Spp2 rearranges the spliceosome-substrate complex to reversibly explore conformations with proximal 5′SS and BP that accommodate chemistry. Addition of Cwc25 then strongly biases this equilibrium towards the proximal conformation, promoting efficient first-step splicing. The spliceosome thus functions as a biased Brownian ratchet machine where a helicase unlocks thermal fluctuations subsequently rectified by a cofactor “pawl”, a principle possibly widespread among the many helicase-driven RNPs.
PMCID: PMC3867266  PMID: 24240612
23.  The EF-G-like GTPase Snu114p Regulates Spliceosome Dynamics Mediated by Brr2p, a DExD/H-box ATPase 
Molecular cell  2006;23(3):389-399.
Binding of a pre-mRNA substrate triggers spliceosome activation while the release of the mRNA product triggers spliceosome disassembly. The mechanisms that underlie the regulation of these rearrangements remain unclear. We find evidence that the GTPase Snu114p mediates the regulation of spliceosome activation and disassembly. Specifically, both unwinding of U4/U6, required for spliceosome activation, and disassembly of the post-splicing U2/U6•U5•intron complex are repressed by Snu114p bound to GDP and derepressed by Snu114 bound to GTP or nonhydrolyzable GTP analogs. Further, similar to U4/U6 unwinding, spliceosome disassembly requires the DExD/H-box ATPase Brr2p. Together, our data define a common mechanism for regulating and executing spliceosome activation and disassembly. Although sequence similarity with EF-G suggests Snu114p functions as a molecular motor, our findings indicate that Snu114p functions as a classic regulatory G protein. We propose that Snu114p serves as a signal-dependent switch that transduces signals to Brr2p to control spliceosome dynamics.
PMCID: PMC3777414  PMID: 16885028
24.  Identification of the Major Spliceosomal RNAs in Dictyostelium discoideum Reveals Developmentally Regulated U2 Variants and Polyadenylated snRNAs†  
Eukaryotic Cell  2006;5(6):924-934.
Most eukaryotic mRNAs depend upon precise removal of introns by the spliceosome, a complex of RNAs and proteins. Splicing of pre-mRNA is known to take place in Dictyostelium discoideum, and we previously isolated the U2 spliceosomal RNA experimentally. In this study, we identified the remaining major spliceosomal RNAs in Dictyostelium by a bioinformatical approach. Expression was verified from 17 small nuclear RNA (snRNA) genes. All these genes are preceded by a putative noncoding RNA gene promoter. Immunoprecipitation showed that snRNAs U1, U2, U4, and U5, but not U6, carry the conserved trimethylated 5′ cap structure. A number of divergent U2 species are expressed in Dictyostelium. These RNAs carry the U2 RNA hallmark sequence and structure motifs but have an additional predicted stem-loop structure at the 5′ end. Surprisingly, and in contrast to the other spliceosomal RNAs in this study, the new U2 variants were enriched in the cytoplasm and were developmentally regulated. Furthermore, all of the snRNAs could also be detected as polyadenylated species, and polyadenylated U1 RNA was demonstrated to be located in the cytoplasm.
PMCID: PMC1489274  PMID: 16757740
25.  The unfoldomics decade: an update on intrinsically disordered proteins 
BMC Genomics  2008;9(Suppl 2):S1.
Our first predictor of protein disorder was published just over a decade ago in the Proceedings of the IEEE International Conference on Neural Networks (Romero P, Obradovic Z, Kissinger C, Villafranca JE, Dunker AK (1997) Identifying disordered regions in proteins from amino acid sequence. Proceedings of the IEEE International Conference on Neural Networks, 1: 90–95). By now more than twenty other laboratory groups have joined the efforts to improve the prediction of protein disorder. While the various prediction methodologies used for protein intrinsic disorder resemble those methodologies used for secondary structure prediction, the two types of structures are entirely different. For example, the two structural classes have very different dynamic properties, with the irregular secondary structure class being much less mobile than the disorder class. The prediction of secondary structure has been useful. On the other hand, the prediction of intrinsic disorder has been revolutionary, leading to major modifications of the more than 100 year-old views relating protein structure and function. Experimentalists have been providing evidence over many decades that some proteins lack fixed structure or are disordered (or unfolded) under physiological conditions. In addition, experimentalists are also showing that, for many proteins, their functions depend on the unstructured rather than structured state; such results are in marked contrast to the greater than hundred year old views such as the lock and key hypothesis. Despite extensive data on many important examples, including disease-associated proteins, the importance of disorder for protein function has been largely ignored. Indeed, to our knowledge, current biochemistry books don't present even one acknowledged example of a disorder-dependent function, even though some reports of disorder-dependent functions are more than 50 years old. The results from genome-wide predictions of intrinsic disorder and the results from other bioinformatics studies of intrinsic disorder are demanding attention for these proteins.
Disorder prediction has been important for showing that the relatively few experimentally characterized examples are members of a very large collection of related disordered proteins that are wide-spread over all three domains of life. Many significant biological functions are now known to depend directly on, or are importantly associated with, the unfolded or partially folded state. Here our goal is to review the key discoveries and to weave these discoveries together to support novel approaches for understanding sequence-function relationships.
Intrinsically disordered protein is common across the three domains of life, but especially common among the eukaryotic proteomes. Signaling sequences and sites of posttranslational modifications are frequently, or very likely most often, located within regions of intrinsic disorder. Disorder-to-order transitions are coupled with the adoption of different structures with different partners. Also, the flexibility of intrinsic disorder helps different disordered regions to bind to a common binding site on a common partner. Such capacity for binding diversity plays important roles in both protein-protein interaction networks and likely also in gene regulation networks. Such disorder-based signaling is further modulated in multicellular eukaryotes by alternative splicing, for which such splicing events map to regions of disorder much more often than to regions of structure. Associating alternative splicing with disorder rather than structure alleviates theoretical and experimentally observed problems associated with the folding of different length, isomeric amino acid sequences. The combination of disorder and alternative splicing is proposed to provide a mechanism for easily "trying out" different signaling pathways, thereby providing the mechanism for generating signaling diversity and enabling the evolution of cell differentiation and multicellularity. Finally, several recent small molecules of interest as potential drugs have been shown to act by blocking protein-protein interactions based on intrinsic disorder of one of the partners. Study of these examples has led to a new approach for drug discovery, and bioinformatics analysis of the human proteome suggests that various disease-associated proteins are very rich in such disorder-based drug discovery targets.
PMCID: PMC2559873  PMID: 18831774

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