Second Step Of Splicing Research Articles

Biochemical and genetic evidence supports Fyv6 as a second-step splicing factor in Saccharomyces cerevisiae.

Precursor mRNA (pre-mRNA) splicing is an essential process for gene expression in eukaryotes catalyzed by the spliceosome in two transesterification steps. The spliceosome is a large, highly dynamic complex composed of five small nuclear RNAs and dozens of proteins, some of which are needed throughout the splicing reaction while others only act during specific stages. The human protein FAM192A was recently proposed to be a splicing factor that functions during the second transesterification step, exon ligation, based on analysis of cryo-electron microscopy (cryo-EM) density. It was also proposed that Fyv6 might be the Saccharomyces cerevisiae functional and structural homolog of FAM192A; however, no biochemical or genetic data has been reported to support this hypothesis. Herein, we show that Fyv6 is a splicing factor and acts during exon ligation. Deletion of FYV6 results in genetic interactions with the essential splicing factors Prp8, Prp16, and Prp22 and decreases splicing in vivo of reporter genes harboring intron substitutions that limit the rate of exon ligation. When splicing is assayed in vitro, whole-cell extracts lacking Fyv6 accumulate first-step products and exhibit a defect in exon ligation. Moreover, loss of Fyv6 causes a change in 3' splice site (SS) selection in both a reporter gene and the endogenous SUS1 transcript in vivo. Together, these data suggest that Fyv6 is a component of the yeast spliceosome that influences 3' SS usage and the potential homolog of human FAM192A.

SWELLMAP 2, a phyB-Interacting Splicing Factor, Negatively Regulates Seedling Photomorphogenesis in Arabidopsis.

Light-triggered transcriptome reprogramming is critical for promoting photomorphogenesis in Arabidopsis seedlings. Nonetheless, recent studies have shed light on the importance of alternative pre-mRNA splicing (AS) in photomorphogenesis. The splicing factors splicing factor for phytochrome signaling (SFPS) and reduced red-light responses in cry1cry2 background1 (RRC1) are involved in the phytochrome B (phyB) signaling pathway and promote photomorphogenesis by controlling pre-mRNA splicing of light- and clock-related genes. However, splicing factors that serve as repressors in phyB signaling pathway remain unreported. Here, we report that the splicing factor SWELLMAP 2 (SMP2) suppresses photomorphogenesis in the light. SMP2 physically interacts with phyB and colocalizes with phyB in photobodies after light exposure. Genetic analyses show that SMP2 antagonizes phyB signaling to promote hypocotyl elongation in the light. The homologs of SMP2 in yeast and human belong to second-step splicing factors required for proper selection of the 3' splice site (3'SS) of an intron. Notably, SMP2 reduces the abundance of the functional REVEILLE 8 a (RVE8a) form, probably by determining the 3'SS, and thereby inhibits RVE8-mediated transcriptional activation of clock genes containing evening elements (EE). Finally, SMP2-mediated reduction of functional RVE8 isoform promotes phytochrome interacting factor 4 (PIF4) expression to fine-tune hypocotyl elongation in the light. Taken together, our data unveil a phyB-interacting splicing factor that negatively regulates photomorphogenesis, providing additional information for further mechanistic investigations regarding phyB-controlled AS of light- and clock-related genes.

Promoter-Bound Full-Length Intronic Circular RNAs-RNA Polymerase II Complexes Regulate Gene Expression in the Human Parasite Entamoeba histolytica.

Ubiquitous eukaryotic non-coding circular RNAs are involved in numerous co- and post-transcriptional regulatory mechanisms. Recently, we reported full-length intronic circular RNAs (flicRNAs) in Entamoeba histolytica, with 3′ss–5′ss ligation points and 5′ss GU-rich elements essential for their biogenesis and their suggested role in transcription regulation. Here, we explored how flicRNAs impact gene expression regulation. Using CLIP assays, followed by qRT-PCR, we identified that the RabX13 control flicRNA and virulence-associated flicRNAs were bound to the HA-tagged RNA Pol II C-terminus domain in E. histolytica transformants. The U2 snRNA was also present in such complexes, indicating that they belonged to transcription initiation/elongation complexes. Correspondingly, inhibition of the second step of splicing using boric acid reduced flicRNA formation and modified the expression of their parental genes and non-related genes. flicRNAs were also recovered from chromatin immunoprecipitation eluates, indicating that the flicRNA-Pol II complex was formed in the promoter of their cognate genes. Finally, two flicRNAs were found to be cytosolic, whose functions remain to be uncovered. Here, we provide novel evidence of the role of flicRNAs in gene expression regulation in cis, apparently in a widespread fashion, as an element bound to the RNA polymerase II transcription initiation complex, in E. histolytica.

Visualizing group II intron dynamics between the first and second steps of splicing

Group II introns are ubiquitous self-splicing ribozymes and retrotransposable elements evolutionarily and chemically related to the eukaryotic spliceosome, with potential applications as gene-editing tools. Recent biochemical and structural data have captured the intron in multiple conformations at different stages of catalysis. Here, we employ enzymatic assays, X-ray crystallography, and molecular simulations to resolve the spatiotemporal location and function of conformational changes occurring between the first and the second step of splicing. We show that the first residue of the highly-conserved catalytic triad is protonated upon 5’-splice-site scission, promoting a reversible structural rearrangement of the active site (toggling). Protonation and active site dynamics induced by the first step of splicing facilitate the progression to the second step. Our insights into the mechanism of group II intron splicing parallels functional data on the spliceosome, thus reinforcing the notion that these evolutionarily-related molecular machines share the same enzymatic strategy.

Insights into the Involvement of Spliceosomal Mutations in Myelodysplastic Disorders from Analysis of SACY-1/DDX41 in Caenorhabditis elegans.

Mutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia (AML). DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase that is recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of AML. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here, we conduct a comprehensive analysis of the Caenorhabditis elegans DDX41 ortholog, SACY-1 Biochemical analyses defined SACY-1 as a component of the C. elegans spliceosome, and genetic analyses revealed synthetic lethal interactions with spliceosomal components. We used the auxin-inducible degradation system to analyze the consequence of SACY-1 depletion on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-dependent and splicing-independent mechanisms. Altered 3' splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 in the second step of splicing. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing. The transcriptome changes observed after SACY-1 depletion suggest that disruption of the spliceosome induces a stress response, which could contribute to the cellular phenotypes conferred by sacy-1 mutant alleles. Multiple sacy-1/ddx41 missense mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that their incorporation into the spliceosome is detrimental. Antagonistic variants that perturb the function of the spliceosome may be relevant to the disease-causing mutations, including DDX41, affecting highly conserved components of the spliceosome in humans.

Structural basis for the second step of group II intron splicing

The group II intron and the spliceosome share a common active site architecture and are thought to be evolutionarily related. Here we report the 3.7 Å crystal structure of a eukaryotic group II intron in the lariat-3′ exon form, immediately preceding the second step of splicing, analogous to the spliceosomal P complex. This structure reveals the location of the intact 3′ splice site within the catalytic core of the group II intron. The 3′-OH of the 5′ exon is positioned in close proximity to the 3′ splice site for nucleophilic attack and exon ligation. The active site undergoes conformational rearrangements with the catalytic triplex having different configurations before and after the second step of splicing. We describe a complete model for the second step of group II intron splicing that incorporates a dynamic catalytic triplex being responsible for creating the binding pocket for 3′ splice site capture.

Activating the branch-forming splicing pathway by reengineering the ribozyme component of a natural group II intron

When assayed in vitro, group IIC self-splicing introns, which target bacterial Rho-independent transcription terminators, generally fail to yield branched products during splicing despite their possessing a seemingly normal branchpoint. Starting with intron O.i.I1 from Oceanobacillus iheyensis, whose crystallographically determined structure lacks branchpoint-containing domain VI, we attempted to determine what makes this intron unfit for in vitro branch formation. A major factor was found to be the length of the helix at the base of domain VI: 4 base pairs (bp) are required for efficient branching, even though a majority of group IIC introns have a 3-bp helix. Equally important for lariat formation is the removal of interactions between ribozyme domains II and VI, which are specific to the second step of splicing. Conversely, mismatching of domain VI and its proposed first-step receptor in subdomain IC1 was found to be detrimental; these data suggest that the intron-encoded protein may promote branch formation partly by modulating the equilibrium between conformations specific to the first and second steps of splicing. As a practical application, we show that by making just two changes to the O.i.I1 ribozyme, it is possible to generate sufficient amounts of lariat intron for the latter to be purified and used in kinetic assays in which folding and reaction are uncoupled.

Prevalent and distinct spliceosomal 3'-end processing mechanisms for fungal telomerase RNA.

Telomerase RNA (TER) is an essential component of the telomerase ribonucleoprotein complex. The mechanism for TER 3′-end processing is highly divergent among different organisms. Here we report a unique spliceosome-mediated TER 3′-end cleavage mechanism in Neurospora crassa which is distinct from that found specifically in the fission yeast Schizosaccharomyces pombe. While the S. pombe TER intron contains the canonical 5′-splice site GUAUGU, the N. crassa TER intron contains a non-canonical 5′-splice site AUAAGU that alone prevents the second step of splicing and promotes spliceosomal cleavage. The unique N. crassa TER 5′-splice site sequence is evolutionarily conserved in TERs from Pezizomycotina and early branching Taphrinomycotina species. This suggests that the widespread and basal N. crassa-type spliceosomal cleavage mechanism is more ancestral than the S. pombe-type. The discovery of a prevalent, yet distinct, spliceosomal cleavage mechanism throughout diverse fungal clades furthers our understanding of TER evolution and non-coding RNA processing.

PRPF8 Defects Cause Missplicing In Myeloid Malignancies

Recent application of whole exome next generation sequencing has led to the discovery of a new class of somatic mutations affecting various components of the spliceosomal machinery in myeloid neoplasms. All affected spliceosomal genes, except for LUC7L2 (which might function in the U1snRNP-related pathway) encode U2snRNP associated proteins, but the molecular consequences of individual mutations remain unclear.Based on 3 index cases where somatic PRPF8 mutations (M1307I, A687P and G1750E) were observed, we have screened a much larger cohort of patients with MDS and related conditions for the presence of acquired lesions affecting this gene. Somatic PRPF8 mutations and heterozygous deletions affecting the corresponding locus were found in 9/352 and 24/450 cases, respectively. Del17p13 was associated with haploinsufficient PRPF8 expression, but down-modulation of PRPF8 was also found in about 10% of additional cases with diploid 17p. PRPF8 mutations were mutually exclusive with other spliceosomal mutations, in particular with SF3B1. Serial studies of clonal architecture revealed that somatic PRPF8 mutation is an early event.PRPF8 encodes the largest and evolutionarily most conserved spliceosomal protein. It is essential for pre-mRNA splicing and required in all tissues. Germline mutations in human PRPF8 are associated with an autosomal dominant form of retinitis pigmentosa type 13.In total, around 1/2 of the PRPF8 mutant and del17p deletion cases were found in pAML and sAML. Accordingly, MDS with PRPF8 lesions conveyed poor prognosis as shown by KM statistics. Phenotypically, PRPF8 defects correlated with the presence of ring sideroblasts (83% of mutants and 65% of deletion cases), likely overseen in the context of a high blast count. Additionally, most of the cases also displayed pseudo Pelger-Huet anomaly.In in vitro experiments, decreased expression levels of PRPF8 due to experimental knockdown by lentiviral shRNA were associated with increased cellular proliferation and clonogenicity, suggesting that PRPF8 has a tumor suppressor role in myeloid malignancies.When we studied the consequences of PRPF8 defects in yeast models, mutations in PRPF8 abrogated a block in the second step of splicing introduced by an experimental intron mutation. RNA-sequencing of primary mutant and deletion cases as well as engineered shRNA knockdown cell lines showed that loss of PRPF8 function or mutation resulted in a global missplicing defect. Some of the most misspliced genes included those involved in mitochondrial function such as NDUFAF6, SFXN2, RPS24, and SLC25A19 and, perhaps most significantly, GATA1 which was mispliced in samples with either low or mutant PRPF8 as well as in the knockdown K562 cells. Similarly, analysis in patients with RS and defective PRPF8 showed common expression profiles of genes involved in mitochondrial electron transport chain complexes. These changes may correspond to ring sideroblasts associated with PRPF8 defects while missplicing of RNA helicases (frequently mutated in MDS, see other abstract from this group), may explain the mechanisms of lost tumor suppressor function.In sum, we describe here another important somatic spliceosomal mutation associated with myeloid neoplastic transformation and unique phenotypic features. Disclosures:Maciejewski:NIH: Research Funding; Aplastic anemia&MDS International Foundation: Research Funding.

Visualizing the ai5γ group IIB intron

It has become apparent that much of cellular metabolism is controlled by large well-folded noncoding RNA molecules. In addition to crystallographic approaches, computational methods are needed for visualizing the 3D structure of large RNAs. Here, we modeled the molecular structure of the ai5γ group IIB intron from yeast using the crystal structure of a bacterial group IIC homolog. This was accomplished by adapting strategies for homology and de novo modeling, and creating a new computational tool for RNA refinement. The resulting model was validated experimentally using a combination of structure-guided mutagenesis and RNA structure probing. The model provides major insights into the mechanism and regulation of splicing, such as the position of the branch-site before and after the second step of splicing, and the location of subdomains that control target specificity, underscoring the feasibility of modeling large functional RNA molecules.

Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression

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

A Weak Spliceosome-Binding Domain of Yju2 Functions in the First Step and Bypasses Prp16 in the Second Step of Splicing

Yju2 is an essential splicing factor required for the first catalytic step after the action of Prp2. We dissected the structure of Yju2 and found that the amino (Yju2-N) and carboxyl (Yju2-C) halves of the protein can be separated and reconstituted for Yju2 function both in vivo and in vitro. Yju2-N has a weak affinity for the spliceosome but functions in promoting the first reaction, with the second reaction being severely impeded. The association of Yju2-N with the spliceosome is stabilized by the presence of Yju2-C at both the precatalytic and postcatalytic stages. Strikingly, Yju2-N supported a low level of the second reaction even in the absence of Prp16. Prp16 is known to mediate destabilization of Yju2 and Cwc25 after the first reaction to allow progression of the second reaction. We propose that in the absence of the C domain, Yju2-N is not stably associated with the spliceosome after lariat formation, and thus bypasses the need for Prp16. We also showed, by UV cross-linking, that Yju2 directly contacts U2 snRNA primarily in the helix II region both pre- and postcatalytically and in the branch-binding region only at the precatalytic stage, suggesting a possible role for Yju2 in positioning the branch point during the first reaction.

Intronic sequence elements impede exon ligation and trigger a discard pathway that yields functional telomerase RNA in fission yeast

The fission yeast telomerase RNA (TER1) precursor harbors an intron immediately downstream from its mature 3' end. Unlike most introns, which are removed from precursor RNAs by the spliceosome in two sequential but tightly coupled transesterification reactions, TER1 only undergoes the first cleavage reaction during telomerase RNA maturation. The mechanism underlying spliceosome-mediated 3' end processing has remained unclear. We now demonstrate that a strong branch site (BS), a long distance to the 3' splice site (3' SS), and a weak polypyrimidine (Py) tract act synergistically to attenuate the transition from the first to the second step of splicing. The observation that a strong BS antagonizes the second step of splicing in the context of TER1 suggests that the BS-U2 snRNA interaction is disrupted after the first step and thus much earlier than previously thought. The slow transition from first to second step triggers the Prp22 DExD/H-box helicase-dependent rejection of the cleaved products and Prp43-dependent "discard" of the splicing intermediates. Our findings explain how the spliceosome can function in 3' end processing and provide new insights into the mechanism of splicing.

Brr2p-mediated conformational rearrangements in the spliceosome during activation and substrate repositioning

Brr2p is one of eight RNA helicases involved in pre-mRNA splicing. Detailed understanding of the functions of Brr2p and other spliceosomal helicases has been limited by lack of knowledge of their in vivo substrates. To address this, sites of direct Brr2p-RNA interaction were identified by in vivo UV cross-linking in budding yeast. Cross-links identified in the U4 and U6 small nuclear RNAs (snRNAs) suggest U4/U6 stem I as a Brr2p substrate during spliceosome activation. Further Brr2p cross-links were identified in loop 1 of the U5 snRNA and near splice sites and 3' ends of introns, suggesting the possibility of a previously uncharacterized function for Brr2p in the catalytic center of the spliceosome. Consistent with this, mutant brr2-G858R reduced second-step splicing efficiency and enhanced cross-linking to 3' ends of introns. Furthermore, RNA sequencing indicated preferential inhibition of splicing of introns with structured 3' ends. The Brr2-G858Rp cross-linking pattern in U6 was consistent with an open conformation for the catalytic center of the spliceosome during first-to-second-step transition. We propose a previously unsuspected function for Brr2p in driving conformational rearrangements that lead to competence for the second step of splicing.

The Cellular EJC Interactome Reveals Higher-Order mRNP Structure and an EJC-SR Protein Nexus

In addition to sculpting eukaryotic transcripts by removing introns, pre-mRNA splicing greatly impacts protein composition of the emerging mRNP. The exon junction complex (EJC), deposited upstream of exon-exon junctions after splicing, is a major constituent of spliced mRNPs. Here, we report comprehensive analysis of the endogenous human EJC protein and RNA interactomes. We confirm that the major "canonical" EJC occupancy site in vivo lies 24 nucleotides upstream of exon junctions and that the majority of exon junctions carry an EJC. Unexpectedly, we find that endogenous EJCs multimerize with one another and with numerous SR proteins to form megadalton sized complexes in which SR proteins are super-stoichiometric to EJC core factors. This tight physical association may explain known functional parallels between EJCs and SR proteins. Further, their protection of long mRNA stretches from nuclease digestion suggests that endogenous EJCs and SR proteins cooperate to promote mRNA packaging and compaction.

CEF1/CDC5 alleles modulate transitions between catalytic conformations of the spliceosome

Conformational change within the spliceosome is required between the first and second catalytic steps of pre-mRNA splicing. A prior genetic screen for suppressors of an intron mutant that stalls between the two steps yielded both prp8 and non-prp8 alleles that suppressed second-step splicing defects. We have now identified the strongest non-prp8 suppressors as alleles of the NTC (Prp19 complex) component, CEF1. These cef1 alleles generally suppress second-step defects caused by a variety of intron mutations, mutations in U6 snRNA, or deletion of the second-step protein factor Prp17, and they can activate alternative 3' splice sites. Genetic and functional interactions between cef1 and prp8 alleles suggest that they modulate the same event(s) in the first-to-second-step transition, most likely by stabilization of the second-step spliceosome; in contrast, alleles of U6 snRNA that also alter this transition modulate a distinct event, most likely by stabilization of the first-step spliceosome. These results implicate a myb-like domain of Cef1/CDC5 in interactions that modulate conformational states of the spliceosome and suggest that alteration of these events affects splice site use, resulting in alternative splicing-like patterns in yeast.

Modelling Reveals Kinetic Advantages of Co-Transcriptional Splicing

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.

The splice is right: Guarantors of fidelity in pre-mRNA splicing: FIGURE 1.

Two recent papers, one from the Staley laboratory (Koodathingal and colleagues) and the other from the Cheng laboratory (Tseng and colleagues), show that the RNA-dependent ATPase Prp16, which is required for the second step of splicing, acts to reject slowly splicing pre-mRNAs immediately before the first catalytic reaction in pre-mRNA splicing. The results answer long-investigated questions about the actions of Prp16 and provide a wealth of molecular details on the proofreading process in pre-mRNA splicing. The discussion here reviews and integrates the results of the two papers and describes the implications for proofreading in splicing.

The U2AF35-related protein Urp contacts the 3′ splice site to promote U12-type intron splicing and the second step of U2-type intron splicing

The U2AF35-related protein Urp has been implicated previously in splicing of the major class of U2-type introns. Here we show that Urp is also required for splicing of the minor class of U12-type introns. Urp is recruited in an ATP-dependent fashion to the U12-type intron 3' splice site, where it promotes formation of spliceosomal complexes. Remarkably, Urp also contacts the 3' splice site of a U2-type intron, but in this case is specifically required for the second step of splicing. Thus, through recognition of a common splicing element, Urp facilitates distinct steps of U2- and U12-type intron splicing.

Localization of the Fission Yeast U5.U2/U6 Spliceosome Subunits

The spliceosome is a dynamic macromolecular machine that catalyzes the excision of introns from pre-mRNA to generate protein-coding transcripts. The megadalton-sized spliceosome is composed of four small nuclear RNPs (U1, U2, U5, and U4/U6) and numerous pre-mRNA splicing factors. The formation of an active spliceosome is hypothesized to occur in a stepwise manner requiring the assembly and disassembly of large multiprotein/RNA complexes. A promising structural approach to obtain information about spliceosome complexes is single-particle cryo-electron microscopy (cryo-EM), a powerful technique that is ideal for determining the structures of large dynamic complexes at protein concentrations too low for crystallization. Formerly, our group determined structure of the fission yeast U5.U2/U6 spliceosome complex by cryo-EM. This U5.U2/U6 spliceosome complex contains the U2, U5, and U6 snRNAs, pre-mRNA splicing intermediates, U2 and U5 snRNP proteins, the Nineteen Complex (NTC), and second-step splicing factors. However, the location of these subunits in the complex was not determined. Using antibody labeling and single particle EM we are now localizing these individual subunits within the density map of the U5.U2/U6 spliceosome complex. This work now enables us to propose a structural model for U5.U2/U6 organization.