Prespliceosome Assembly Research Articles

Inefficient recruitment of DDX39B impedes pre-spliceosome assembly on FOXP3 introns.

Forkhead box P3 (FOXP3) is the master fate-determining transcription factor in regulatory T (Treg) cells and is essential for their development, function, and homeostasis. Mutations in FOXP3 cause immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, and aberrant expression of FOXP3 has been implicated in other diseases such as multiple sclerosis and cancer. We previously demonstrated that pre-mRNA splicing of FOXP3 RNAs is highly sensitive to levels of DExD-box polypeptide 39B (DDX39B), and here we investigate the mechanism of this sensitivity. FOXP3 introns have cytidine (C)-rich/uridine (U)-poor polypyrimidine (py) tracts that are responsible for their inefficient splicing and confer sensitivity to DDX39B. We show that there is a deficiency in the assembly of commitment complexes (CCs) on FOXP3 introns, which is consistent with the lower affinity of U2AF2 for C-rich/U-poor py tracts. Our data indicate an even stronger effect on the conversion of CCs to pre-spliceosomes. We propose that this is due to an altered conformation that U2AF2 adopts when it binds to C-rich/U-poor py tracts and that this conformation has a lower affinity for DDX39B. As a consequence, CCs assembled on FOXP3 introns are defective in recruiting DDX39B, and this leads to the inefficient assembly of pre-spliceosome complexes.

Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

Structural insights into branch site proofreading by human spliceosome.

Selection of the pre-mRNA branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is crucial to prespliceosome (A complex) assembly. The RNA helicase PRP5 proofreads BS selection but the underlying mechanism remains unclear. Here we report the atomic structures of two sequential complexes leading to prespliceosome assembly: human 17S U2 snRNP and a cross-exon pre-A complex. PRP5 is anchored on 17S U2 snRNP mainly through occupation of the RNA path of SF3B1 by an acidic loop of PRP5; the helicase domain of PRP5 associates with U2 snRNA; the BS-interacting stem-loop (BSL) of U2 snRNA is shielded by TAT-SF1, unable to engage the BS. In the pre-A complex, an initial U2-BS duplex is formed; the translocated helicase domain of PRP5 stays with U2 snRNA and the acidic loop still occupies the RNA path. The pre-A conformation is specifically stabilized by the splicing factors SF1, DNAJC8 and SF3A2. Cancer-derived mutations in SF3B1 damage its association with PRP5, compromising BS proofreading. Together, these findings reveal key insights into prespliceosome assembly and BS selection or proofreading by PRP5.

Impaired Early Spliceosome Complex Assembly Underlies Gene Body Elongation Transcription Defect in SF3B1K700E

Introduction: Recurrent mutations in splicing factors (SFs) such as SF3B1, U2AF1 and SRSF2 are common in clonal myeloid disorders. These mutations are typically hemizygous, exclusive to each other, and non-synonymous, pointing to neomorphic functions. Molecular mechanisms connecting the mutations to disease pathogenesis remain unclear. Previous studies predominantly focused on the well-established role of splicing factors in pre-mRNA splicing, seeking to explain how mutations in these SFs leading to changes in the abundance of key regulatory genes through their effects on alternative splicing. These studies are limited by inconsistent and relatively low isoform changes across mutations and independent datasets. They also don't explain the mutual exclusivity of these mutations. In this study, we explored a novel mechanism that link SF mutations to disease biology: dysregulation of RNA Polymerase II (Pol2) transcriptional kinetics arising from impaired spliceosome assembly. Methods: We generated inducible isogenic K562 cell lines that express single mutant alleles (SF3B1 K700E and U2AF1 S34F,and corresponding wild-type controls). A novel combinatory approach of AAV-intron trap with CRISPR/Cas9 (Boddu et al, Comms Biol, 2021) was utilized for gene editing. RNAPII transcription kinetics changes were assessed, at an early post-induction time point of 4 days (Fig1A), using ChIP-seq, and nascent-transcriptome assays (including GRO-seq and transient transcriptome-time lapse sequencing or TT-TL seq). Spliceosome assembly kinetics were assayed with non-denaturing gels. Co-transcriptional splicing efficiency (CoSE) was assessed using nascent long-read sequencing (LRS) (Reimer, Mol Cell 2021). Key findings in K562 cell lines were validated in CD34+ progenitor populations from MDS-derived patient samples (and healthy volunteer controls). Results: ChIP-seq for Pol 2 (both total (Fig1B) and elongation-specific Ser2P (Fig1C) showed a redistribution of Pol2 into gene-bodies, suggesting a failure of Pol2 elongation in SF3B1 K700E. This Pol2 redistribution was dependent on gene length and intron number (long, multi-intron genes most affected). GRO-seq confirmed the ChIP-seq results (Fig1D). Additionally, spike-in normalized TT-seq showed significantly reduced nascent transcription in gene bodies in SF3B1 K700E (Fig 1E), consistent with a gene-body elongation rate defect. A similar redistribution of Pol2 into gene bodies was noted by ChIP-seq and TT-seq in U2AF1S34F, pointing to a convergent transcriptional response among distinct SF mutations. Genome-wide CoSE was noted to be lower in SF3B1 K700Eindicating an associated splicing assembly defect (Fig1F). Changes to transcription due to SF3B1 mutations were similarly observed, using low-input Pol2 CUT&RUN, in CD34+ progenitor populations isolated from human MDS patient samples (Fig1G). Of pathologic relevance, this transcriptional dysregulation led to generation of R-loops, DNA damage, and S-phase arrest. Given close coordination of splicing and transcription, and role of SF3B1 and U2AF1 in early spliceosome formation, we speculated if mutant SF3B1 leads to defective assembly of early spliceosomes. Compared to wild-type protein, SF3B1 K700E was noted to have significantly impaired interactions with U2AF1, U2AF2, HTATSF1 (Fig1H). We then determined how spliceosome assembly kinetics of SF3B1 K700E differed from those of SF3B1 WTusing an in vitro splicing assay. The transition from E- to A- complex intermediates was reduced in SF3B1 K700E(Fig1I). Furthermore, disrupting the early pre-spliceosome assembly (through loss of DDX46 or HTATSF1 using both siRNA and inducible shRNA (Fig1J) knockdown systems) resulted in a transcriptional response similar to SF3B1 K700E. Based on recent cryo-EM structures of Pol II-U1 snRNP interactions (Zhang et al, Science, 2021), we speculate that the impaired transition to A/B complex in SF3B1 K700E prevents the release of U1 snRNP and release of Pol2, leading to intron-predominant defective Pol II elongation (Fig1K). Conclusion: Here, we found that SF3B1 and U2AF1 mutations impair Pol2 transcription elongation along gene bodies, leading to pathologic R-loops and replication stress. This elongation results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1.

Structure of a transcribing RNA polymerase II-U1 snRNP complex.

To initiate cotranscriptional splicing, RNA polymerase II (Pol II) recruits the U1 small nuclear ribonucleoprotein particle (U1 snRNP) to nascent precursor messenger RNA (pre-mRNA). Here, we report the cryo-electron microscopy structure of a mammalian transcribing Pol II-U1 snRNP complex. The structure reveals that Pol II and U1 snRNP interact directly. This interaction positions the pre-mRNA 5' splice site near the RNA exit site of Pol II. Extension of pre-mRNA retains the 5' splice site, leading to the formation of a "growing intron loop." Loop formation may facilitate scanning of nascent pre-mRNA for the 3' splice site, functional pairing of distant intron ends, and prespliceosome assembly. Our results provide a starting point for a mechanistic analysis of cotranscriptional spliceosome assembly and the biogenesis of mRNA isoforms by alternative splicing.

Prp5-Spt8/Spt3 interaction mediates a reciprocal coupling between splicing and transcription.

Transcription and pre-mRNA splicing are coupled to promote gene expression and regulation. However, mechanisms by which transcription and splicing influence each other are still under investigation. The ATPase Prp5p is required for pre-spliceosome assembly and splicing proofreading at the branch-point region. From an open UV mutagenesis screen for genetic suppressors of prp5 defects and subsequent targeted testing, we identify components of the TBP-binding module of the Spt–Ada–Gcn5 Acetyltransferase (SAGA) complex, Spt8p and Spt3p. Spt8Δ and spt3Δ rescue the cold-sensitivity of prp5-GAR allele, and prp5 mutants restore growth of spt8Δ and spt3Δ strains on 6-azauracil. By chromatin immunoprecipitation (ChIP), we find that prp5 alleles decrease recruitment of RNA polymerase II (Pol II) to an intron-containing gene, which is rescued by spt8Δ. Further ChIP-seq reveals that global effects on Pol II-binding are mutually rescued by prp5-GAR and spt8Δ. Inhibited splicing caused by prp5-GAR is also restored by spt8Δ. In vitro assays indicate that Prp5p directly interacts with Spt8p, but not Spt3p. We demonstrate that Prp5p's splicing proofreading is modulated by Spt8p and Spt3p. Therefore, this study reveals that interactions between the TBP-binding module of SAGA and the spliceosomal ATPase Prp5p mediate a balance between transcription initiation/elongation and pre-spliceosome assembly.

Blocking late stages of splicing quickly limits pre-spliceosome assembly in vivo

ABSTRACTPre-messenger RNA splicing involves multi-step assembly of the large spliceosome complexes that catalyse the two consecutive trans-esterification reactions, resulting in intron removal. There is evidence that proof-reading mechanisms monitor the fidelity of this complex process. Transcripts that fail these fidelity tests are thought to be directed to degradation pathways, permitting the splicing factors to be recycled. While studying the roles of splicing factors in vivo, in budding yeast, we performed targeted depletion of individual proteins, and analysed the effect on co-transcriptional spliceosome assembly and splicing efficiency. Unexpectedly, depleting factors such as Prp16 or Prp22, that are known to function at the second catalytic step or later in the splicing pathway, resulted in a defect in the first step of splicing, and accumulation of arrested spliceosomes. Through a kinetic analysis of newly synthesized RNA, we observed that a second step splicing defect (the primary defect) was rapidly followed by the first step of splicing defect. Our results show that knocking down a splicing factor can quickly lead to a recycling defect with splicing factors sequestered in stalled complexes, thereby limiting new rounds of splicing. We demonstrate that this ‘feed-back’ effect can be minimized by depleting the target protein more gradually or only partially, allowing a better separation between primary and secondary effects. Our findings indicate that splicing surveillance mechanisms may not always cope with spliceosome assembly defects, and suggest that work involving knock-down of splicing factors or components of other large complexes should be carefully monitored to avoid potentially misleading conclusions.

Defining the NSD2 interactome: PARP1 PARylation reduces NSD2 histone methyltransferase activity and impedes chromatin binding

NSD2 is a histone methyltransferase that specifically dimethylates histone H3 lysine 36 (H3K36me2), a modification associated with gene activation. Dramatic overexpression of NSD2 in t(4;14) multiple myeloma (MM) and an activating mutation of NSD2 discovered in acute lymphoblastic leukemia are significantly associated with altered gene activation, transcription, and DNA damage repair. The partner proteins through which NSD2 may influence critical cellular processes remain poorly defined. In this study, we utilized proximity-based labeling (BioID) combined with label-free quantitative MS to identify high confidence NSD2 interacting partners in MM cells. The top 24 proteins identified were involved in maintaining chromatin structure, transcriptional regulation, RNA pre-spliceosome assembly, and DNA damage. Among these, an important DNA damage regulator, poly(ADP-ribose) polymerase 1 (PARP1), was discovered. PARP1 and NSD2 have been found to be recruited to DNA double strand breaks upon damage and H3K36me2 marks are enriched at damage sites. We demonstrate that PARP1 regulates NSD2 via PARylation upon oxidative stress. In vitro assays suggest the PARylation significantly reduces NSD2 histone methyltransferase activity. Furthermore, PARylation of NSD2 inhibits its ability to bind to nucleosomes and further get recruited at NSD2-regulated genes, suggesting PARP1 regulates NSD2 localization and H3K36me2 balance. This work provides clear evidence of cross-talk between PARylation and histone methylation and offers new directions to characterize NSD2 function in DNA damage response, transcriptional regulation, and other pathways.

U2AF65 enhances milk synthesis and growth of bovine mammary epithelial cells by positively regulating the mTOR-SREBP-1c signalling pathway.

U2 snRNP auxiliary factor 65kDa (U2AF65) is a splicing factor that promotes prespliceosome assembly. The function of U2AF65 in alternative splicing has been identified; however, the essential physiological role of U2AF65 remains poorly understood. In this study, we investigated the regulatory role of U2AF65 in milk synthesis and growth of bovine mammary epithelial cells (BMECs). Our results showed that U2AF65 localizes in the nucleus. Treatment with amino acids (Met and Leu) and hormones (prolactin and β-estradiol) upregulated the expression of U2AF65 in these cells. U2AF65 overexpression increased the synthesis of β-casein, triglycerides, and lactose; increased cell viability; and promoted proliferation of BMECs. Furthermore, our results showed that U2AF65 positively regulated mTOR phosphorylation and expression of mature mRNA of mTOR and SREBP-1c. Collectively, our findings demonstrate that U2AF65 regulates the mRNA expression of signalling molecules (mTOR and SREBP-1c) involved in milk synthesis and growth of BMECs, possibly via controlling the splicing and maturation of these mRNAs. U2 snRNP auxiliary factor 65kDa (U2AF65) is a splicing factor that promotes prespliceosome assembly. The essential physiological role of U2AF65 remains poorly understood. In the present study, we confirmed that U2AF65 functions as a positive regulator of milk synthesis in and proliferation of bovine mammary epithelial cells via the mTOR-SREBP-1c signalling pathway. Therefore, our study uncovers the regulatory role of U2AF65 in milk synthesis and cell proliferation.

Synergistic assembly of human pre-spliceosomes across introns and exons.

Most human genes contain multiple introns, necessitating mechanisms to effectively define exons and ensure their proper connection by spliceosomes. Human spliceosome assembly involves both cross-intron and cross-exon interactions, but how these work together is unclear. We examined in human nuclear extracts dynamic interactions of single pre-mRNA molecules with individual fluorescently tagged spliceosomal subcomplexes to investigate how cross-intron and cross-exon processes jointly promote pre-spliceosome assembly. U1 subcomplex bound to the 5' splice site of an intron acts jointly with U1 bound to the 5' splice site of the next intron to dramatically increase the rate and efficiency by which U2 subcomplex is recruited to the branch site/3' splice site of the upstream intron. The flanking 5' splice sites have greater than additive effects implying distinct mechanisms facilitating U2 recruitment. This synergy of 5' splice sites across introns and exons is likely important in promoting correct and efficient splicing of multi-intron pre-mRNAs.

Splicing inhibition of U2AF65 leads to alternative exon skipping

U2 snRNP auxiliary factor 65 kDa (U2AF(65)) is a general splicing factor that contacts polypyrimidine (Py) tract and promotes prespliceosome assembly. In this report, we show that U2AF(65) stimulates alternative exon skipping in spinal muscular atrophy (SMA)-related survival motor neuron (SMN) pre-mRNA. A stronger 5' splice-site mutation of alternative exon abolishes the stimulatory effects of U2AF(65). U2AF(65) overexpression promotes its own binding only on the weaker, not the stronger, Py tract. We further demonstrate that U2AF(65) inhibits splicing of flanking introns of alternative exon in both three-exon and two-exon contexts. Similar U2AF(65) effects were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we demonstrate that U2AF(65) even inhibits general splicing of adenovirus major late (Ad ML) or β-globin pre-mRNA. Thus, we conclude that U2AF(65) possesses a splicing Inhibitory function that leads to alternative exon skipping.

ZRSR2 Mutations Cause Dysregulated RNA Splicing in MDS

Recurrent somatic mutations have been uncovered in several components of the spliceosome in Myelodysplastic Syndrome (MDS). Recent high throughput sequencing of large cohorts of MDS has established RNA splicing as the pathway most frequently targeted by somatic mutations. These findings implicate dysregulated RNA splicing in the pathogenesis of MDS. However, the mechanism linking aberrant splicing to the development of MDS is unknown. ZRSR2, a frequently mutated spliceosome gene in MDS is located on the X chromosome. Somatic alterations of ZRSR2 are typically inactivating mutations (frameshift indels, nonsense point mutations or splice site mutations) which are observed predominantly in males. Mutations in ZRSR2are more prevalent in MDS subtypes without ring sideroblasts and chronic myelomonocytic leukemia (CMML) and are associated with elevated bone marrow blasts and higher rate of progression to AML. Although ZRSR2 has been suggested to interact with other splice proteins, U2AF2 and SRSF2, at the 3΄ splice sites during the pre-spliceosome assembly, its precise role in RNA splicing remains unexplored.In this study, we demonstrate that deficiency of ZRSR2 leads to impaired splicing of U12-type introns which are dependent upon the minor spliceosome. RNA-Sequencing of MDS bone marrow harboring inactivating mutations of ZRSR2 revealed aberrant retention of U12-type introns as compared to bone marrow with wild-type ZRSR2. In addition, several U12-type introns displayed mis-splicing associated with recognition of cryptic splice-site. In contrast, the splicing of U2-type introns (dependent upon the major spliceosome) was largely unaffected. The mis-spliced introns were found in several key genes including PTEN, MAPK1, MAPK3, BRAF and E2F2, and the impaired splicing of several introns in ZRSR2 mutant bone marrow was validated experimentally. Further, short hairpin RNA (shRNA) mediated knockdown of ZRSR2 in MDS/AML TF1 cells led to impaired splicing specifically of U12-type introns. We also observe that ectopic expression of ZRSR2 in stable knockdown 293T cells resulted in increase in splicing efficiency of U12-type introns.Further, the downregulation of ZRSR2 in leukemia cells results in reduced growth and clonogenic potential in leukemia cell lines. Moreover, knockdown of ZRSR2 in human CD34+ cells displayed altered differentiation potential towards erythroid and myeloid lineages in vitro.Overall, the dysregulated RNA splicing of U12-type introns in ZRSR2 mutant samples affects several crucial genes involved in cell cycle, signaling, RNA binding and transport. These genes are potential mediators of MDS phenotype. Ours is a first study which demonstrates the functional consequences of ZRSR2 mutations in MDS and identifies a specific role of ZRSR2 in regulating RNA splicing. It underlines aberrant splicing of U12-type introns as a distinctive feature of ZRSR2 mutant MDS. DisclosuresKohlmann:MLL Munich Leukemia Laboratory, Munich, Germany: Employment. Grossmann:MLL Munich Leukemia Laboratory, Munich, Germany: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

UAP56- a key player with surprisingly diverse roles in pre-mRNA splicing and nuclear export

Transcripts contain introns that are usually removed from premessenger RNA (MRNA) in the process of pre-mRNA splicing. After splicing, the mature RNA is exported from the nucleus to the cytoplasm. The splicing and export processes are coupled. UAP56 protein, which is ubiquitously present in organisms from yeasts to humans, is a DExD/H-box family RNA helicase that is an essential splicing factor with various functions in the prespliceosome assembly and mature spliceosome assembly. Collective evidence indicates that UAP56 has an essential role in mRNA nuclear export. This mini-review summarizes recent evidence for the role of UAP56 in pre-mRNA splicing and nuclear export.

RBM5/Luca-15/H37 Regulates Fas Alternative Splice Site Pairing after Exon Definition

RBM5/Luca-15/H37 is a gene frequently inactivated in lung cancers and overexpressed in breast tumors. Its protein product has been detected in prespliceosomal complexes and modulates cell proliferation and Fas-mediated apoptosis. We report that RBM5 is a component of complexes involved in 3' splice site recognition and regulates alternative splicing of apoptosis-related genes, including the Fas receptor, switching between isoforms with antagonistic functions in programmed cell death. In contrast with classical mechanisms of splicing regulation, RBM5 does not affect early events of splice site recognition that lead to Fas exon 6 definition. Instead, RBM5 inhibits the transition between prespliceosomal complexes assembled around exon 6 to mature spliceosomes assembled on the flanking introns and promotes sequence-specific pairing of the distal splice sites. An OCRE domain important for RBM5 function contacts components of the U4/5/6 tri-snRNP, consistent with the idea that RBM5 modulates splice site pairing after prespliceosome assembly and exon definition.

Distinct activities of the DExD/H-box splicing factor hUAP56 facilitate stepwise assembly of the spliceosome

The essential splicing factor human UAP56 (hUAP56) is a DExD/H-box protein known to promote prespliceosome assembly. Here, using a series of hUAP56 mutants that are defective for ATP-binding, ATP hydrolysis, or dsRNA unwindase/helicase activity, we assess the relative contributions of these biochemical functions to pre-mRNA splicing. We show that prespliceosome assembly requires hUAP56's ATP-binding and ATPase activities, which, unexpectedly, are required for hUAP56 to interact with U2AF(65) and be recruited into splicing complexes. Surprisingly, we find that hUAP56 is also required for mature spliceosome assembly, which requires, in addition to the ATP-binding and ATPase activities, hUAP56's dsRNA unwindase/helicase activity. We demonstrate that hUAP56 directly contacts U4 and U6 snRNAs and can promote unwinding of the U4/U6 duplex, and that both these activities are dependent on U2AF(65). Our results indicate that hUAP56 first interacts with U2AF(65) in an ATP-dependent manner, and subsequently with U4/U6 snRNAs to facilitate stepwise assembly of the spliceosome.

The U11-48K protein contacts the 5' splice site of U12-type introns and the U11-59K protein.

Little is currently known about proteins that make contact with the pre-mRNA in the U12-dependent spliceosome and thereby contribute to intron recognition. Using site-specific cross-linking, we detected an interaction between the U11-48K protein and U12-type 5' splice sites (5'ss). This interaction did not require branch point recognition and was sensitive to 5'ss mutations, suggesting that 48K interacts with the 5'ss during the first steps of prespliceosome assembly in a sequence-dependent manner. RNA interference-induced knockdown of 48K in HeLa cells led to reduced cell growth and the inhibition of U12-type splicing, as well as the activation of cryptic, U2-type splice sites, suggesting that 48K plays a critical role in U12-type intron recognition. 48K knockdown also led to reduced levels of U11/U12 di-snRNP, indicating that 48K contributes to the stability and/or formation of this complex. In addition to making contact with the 5'ss, 48K interacts with the U11-59K protein, a protein at the interface of the U11/U12 di-snRNP. These studies provide important insights into the protein-mediated recognition of the U12-type 5'ss, as well as functionally important interactions within the U11/U12 di-snRNP.

Splicing Factor SPF30 Bridges an Interaction between the Prespliceosome Protein U2AF35 and Tri-small Nuclear Ribonucleoprotein Protein hPrp3

Spliceosome assembly is a dynamic process involving the sequential recruitment and rearrangement of small nuclear ribonucleoproteins (snRNPs) on a pre-mRNA substrate. Here we identify several spliceosome protein interactions with different domains of human splicing factor SPF30 that have the potential to mediate the addition of the tri-snRNP to the prespliceosome. In particular, we show that the C-terminal tails of SmD1, SmD3, and the protein Lsm4 interact with the central Tudor domain of SPF30. We identify a novel interaction between the N-terminal domain of SPF30 and U2AF35, a prespliceosome protein that has a role in recognizing the 3' splice site and recruiting U2 snRNP. We also show that the C terminus of SPF30 interacts with a middle domain of hPrp3, a component of U4/U6 di-snRNP and the tri-snRNP. Importantly, we show that the U2AF35 and hPrp3 interactions with SPF30 can occur simultaneously, thereby potentially linking 3' splice site recognition with tri-snRNP addition. Finally, we note that SPF30 and its partner-interacting domains are not conserved in yeast, suggesting this interaction network may play an important role in the complex splicing observed in higher eukaryotes.

Interactions of the yeast SF3b splicing factor.

The U2 snRNP promotes prespliceosome assembly through interactions that minimally involve the branchpoint binding protein, Mud2p, and the pre-mRNA. We previously showed that seven proteins copurify with the yeast (Saccharomyces cerevisiae) SF3b U2 subcomplex that associates with the pre-mRNA branchpoint region: Rse1p, Hsh155p, Hsh49p, Cus1p, and Rds3p and unidentified subunits p10 and p17. Here proteomic and genetic studies identify Rcp10p as p10 and show that it contributes to SF3b stability and is necessary for normal cellular Cus1p accumulation and for U2 snRNP recruitment in splicing. Remarkably, only the final 53 amino acids of Rcp10p are essential. p17 is shown to be composed of two accessory splicing factors, Bud31p and Ist3p, the latter of which independently associates with the RES complex implicated in the nuclear pre-mRNA retention. A directed two-hybrid screen reveals a network of prospective interactions that includes previously unreported intra-SF3b contacts and SF3b interactions with the RES subunit Bud13p, the Prp5p DExD/H-box protein, Mud2p, and the late-acting nineteen complex. These data establish the concordance of yeast and mammalian SF3b complexes, implicate accessory splicing factors in U2 snRNP function, and support SF3b contribution from early pre-mRNP recognition to late steps in splicing.

Human Splicing Factor SF3a, but Not SF1, Is Essential for Pre-mRNA Splicing In Vivo

The three subunits of human splicing factor SF3a are essential for the formation of the functional 17S U2 snRNP and prespliceosome assembly in vitro. RNAi-mediated depletion indicates that each subunit is essential for viability of human cells. Knockdown of single subunits results in a general block in splicing strongly suggesting that SF3a is a constitutive splicing factor in vivo. In contrast, splicing of several endogenous and reporter pre-mRNAs is not affected after knockdown of SF1, which functions at the onset of spliceosome assembly in vitro and is essential for cell viability. Thus, SF1 may only be required for the splicing of a subset of pre-mRNAs. We also observe a reorganization of U2 snRNP components in SF3a-depleted cells, where U2 snRNA and U2-B'' are significantly reduced in nuclear speckles and the nucleoplasm, but still present in Cajal bodies. Together with the observation that the 17S U2 snRNP cannot be detected in extracts from SF3a-depleted cells, our results provide further evidence for a function of Cajal bodies in U2 snRNP biogenesis.

Arginine-Serine-Rich Domains Bound at Splicing Enhancers Contact the Branchpoint to Promote Prespliceosome Assembly

Exonic splicing enhancers (ESEs) are required for splicing of certain pre-mRNAs and function by providing binding sites for serine-arginine (SR) proteins, which contain an arginine-serine-rich (RS) domain. How an RS domain bound at the ESE promotes splicing is poorly understood. We have developed an RNA-protein crosslinking procedure to identify the target of the ESE-bound RS domain. Using this approach, we show that the ESE-bound RS domain specifically contacts the pre-mRNA branchpoint. The interaction between the ESE-bound RS domain and the branchpoint occurs in the prespliceosome and is dependent upon the same splicing signals, biochemical factors, and reaction conditions required to support prespliceosome assembly. Analysis of RS domain mutants demonstrates that the ability to interact with the branchpoint, to promote prespliceosome assembly, and to support splicing are related activities. We conclude that the ESE-bound RS domain functions by contacting the branchpoint to promote prespliceosome assembly.