Characterization of Cwc2, U6 snRNA, and Prp8 interactions destabilized by Prp16 ATPase at the transition between the first and second steps of splicing.

The spliceosome performs two consecutive transesterification reactions using one catalytic center, thus requiring its rearrangement between the two catalytic steps of splicing. The Prp16 ATPase facilitates exit from the first-step conformation of the catalytic center by destabilizing some interactions important for catalysis. To better understand rearrangements within the Saccharomyces cerevisiae catalytic center, we characterize factors that modulate the function of Prp16: Cwc2, N-terminal domain of Prp8, and U6-41AACAAU46 region. Alleles of these factors were identified through genetic screens for mutants that correct cs defects of prp16-302 alleles. Several of the identified U6, cwc2, and prp8 alleles are located in close proximity of each other in cryo-EM structures of the spliceosomal catalytic conformations. Cwc2 and U6 interact with the intron sequences in the first step, but they do not seem to contribute to the stability of the second-step catalytic center. On the other hand, the N-terminal segment of Prp8 not only affects intron positioning for the first step, but it also makes important contacts in the proximity of the active site for both the first and second steps of splicing. By identifying interactions important for the stability of catalytic conformations, our genetic analyses indirectly inform us about features of the transition-state conformation of the spliceosome.

Tracing Allostery in the Spliceosome Ski2-like RNA Helicase Brr2.

RNA ATPases/helicases remodel substrate RNA-protein complexes in distinct ways. The different RNA ATPases/helicases, taking part in the spliceosome complex, reshape the RNA/RNA-protein contacts to enable premature-mRNA splicing. Among them, the bad response to refrigeration 2 (Brr2) helicase promotes U4/U6 small nuclear (sn)RNA unwinding via ATP-driven translocation of the U4 snRNA strand, thus playing a pivotal role during the activation, catalytic, and disassembly phases of splicing. The plastic Brr2 architecture consists of an enzymatically active N-terminal cassette (N-cassette) and a structurally similar but inactive C-terminal cassette (C-cassette). The C-cassette, along with other allosteric effectors and regulators, tightly and timely controls Brr2's function via an elusive mechanism. Here, microsecond-long molecular dynamics simulations, dynamical network theory, and community network analysis are combined to elucidate how allosteric effectors/regulators modulate the Brr2 function. We unexpectedly reveal that U4 snRNA itself acts as an allosteric regulator, amplifying the cross-talk of distal Brr2 domains and triggering a conformational reorganization of the protein. Our findings offer fundamental understanding into Brr2's mechanism of action and broaden our knowledge on the sophisticated regulatory mechanisms by which spliceosome ATPases/helicases control gene expression. This includes their allosteric regulation exerted by client RNA strands, a mechanism that may be broadly applicable to other RNA-dependent ATPases/helicases.

The spliceosome component Usp39 controls B cell development by regulating immunoglobulin gene rearrangement.

The spliceosome is a large ribonucleoprotein complex responsible for pre-mRNA splicing and genome stability maintenance. Disruption of the spliceosome activity may lead to developmental disorders and tumorigenesis. However, the physiological role that the spliceosome plays in B cell development and function is still poorly defined. Here, we demonstrate that ubiquitin-specific peptidase 39 (Usp39), a spliceosome component of the U4/U6.U5 tri-snRNP complex, is essential for B cell development. Ablation of Usp39 in B cell lineage blocks pre-pro-B to pro-B cell transition in the bone marrow, leading to a profound reduction of mature B cells in the periphery. We show that Usp39 specifically regulates immunoglobulin gene rearrangement in a spliceosome-dependent manner, which involves modulating chromatin interactions at the Igh locus. Moreover, our results indicate that Usp39 deletion reduces the pre-malignant B cells in Eµ-Myc transgenic mice and significantly improves their survival.

The role of splicing factor PRPF8 in breast cancer.

BACKGROUND: Alternative splicing is a mechanism to produce different proteins with diverse functions from one gene. Many splicing factors play an important role in cancer progression. PRPF8 is a core protein component of the spliceosome complex, U4/U6-U5 tri-snRNP. OBJECTIVE: However, PRPF8 involved in mRNA alternative splicing are rarely included in the prognosis. METHODS: We found that PRPF8 was expressed in all examined cancer types. Further analyses found that PRPF8 expression was significantly different between the breast cancer and paracancerous tissues. RESULTS: Survival analyses showed that PRPF8-high patients had a poor prognosis, and the expression of PRPF8 is associated with distant metastasis-free survival (DMFS) and post progression survival (PPS). Gene Set Enrichment Analysis (GSEA) has revealed that PRPF8 expression is correlated with TGF-ß, JAK-STAT, and cell cycle control pathways. Consistent with these results, upon PRPF8 silencing, the growth of MCF-7 cells was reduced, the ability of cell clone formation was weakened, and p⁢21 expression was increased. CONCLUSIONS: These results have revealed that PRPF8 is a significant factor for splicing in breast cancer progression.

PRP4 Promotes Skin Cancer by Inhibiting Production of Melanin, Blocking Influx of Extracellular Calcium, and Remodeling Cell Actin Cytoskeleton.

Pre-mRNA processing factor 4B (PRP4) has previously been shown to induce epithelial-mesenchymal transition (EMT) and drug resistance in cancer cell lines. As melanin plays an important photoprotective role in the risk of sun-induced skin cancers, we have investigated whether PRP4 can induce drug resistance and regulate melanin biosynthesis in a murine melanoma (B16F10) cell line. Cells were incubated with a crucial melanogenesis stimulator, alpha-melanocyte-stimulating hormone, followed by transfection with PRP4. This resulted in the inhibition of the production of melanin via the downregulation of adenylyl cyclase-cyclic adenosine 3',5'-monophosphate (AC)-(cAMP)-tyrosinase synthesis signaling pathway. Inhibition of melanin production by PRP4 leads to the promotion of carcinogenesis and induced drug resistance in B16F10 cells. Additionally, PRP4 overexpression upregulated the expression of ß-arrestin 1 and desensitized the extracellular calcium-sensing receptor (CaSR), which in turn, inhibited the influx of extracellular Ca2+ ions. The decreased influx of Ca2+ was confirmed by a decreased expression level of calmodulin. We have demonstrated that transient receptor potential cation channel subfamily C member 1 was involved in the influx of CaSR-induced Ca2+ via a decreasing level of its expression. Furthermore, PRP4 overexpression downregulated the expression of AC, decreased the synthesis of cAMP, and modulated the actin cytoskeleton by inhibiting the expression of Ras homolog family member A (RhoA). Our investigation suggests that PRP4 inhibits the production of melanin in B16F10 cells, blocks the influx of Ca2+ through desensitization of CaSR, and modulates the actin cytoskeleton through downregulating the AC-cAMP pathway; taken together, these observations collectively lead to the promotion of skin carcinogenesis.

PRP4 Kinase Domain Loss Nullifies Drug Resistance and Epithelial-Mesenchymal Transition in Human Colorectal Carcinoma Cells.

We have investigated the involvement of the pre-mRNA processing factor 4B (PRP4) kinase domain in mediating drug resistance. HCT116 cells were treated with curcumin, and apoptosis was assessed based on flow cytometry and the generation of reactive oxygen species (ROS). Cells were then transfected with PRP4 or pre-mRNA-processing-splicing factor 8 (PRP8), and drug resistance was analyzed both in vitro and in vivo. Furthermore, we deleted the kinase domain in PRP4 using GatewayTM technology. Curcumin induced cell death through the production of ROS and decreased the activation of survival signals, but PRP4 overexpression reversed the curcumin-induced oxidative stress and apoptosis. PRP8 failed to reverse the curcumin-induced apoptosis in the HCT116 colon cancer cell line. In xenograft mouse model experiments, curcumin effectively reduced tumour size whereas PRP4 conferred resistance to curcumin, which was evident from increasing tumour size, while PRP8 failed to regulate the curcumin action. PRP4 overexpression altered the morphology, rearranged the actin cytoskeleton, triggered epithelial-mesenchymal transition (EMT), and decreased the invasiveness of HCT116 cells. The loss of E-cadherin, a hallmark of EMT, was observed in HCT116 cells overexpressing PRP4. Moreover, we observed that the EMT-inducing potential of PRP4 was aborted after the deletion of its kinase domain. Collectively, our investigations suggest that the PRP4 kinase domain is responsible for promoting drug resistance to curcumin by inducing EMT. Further evaluation of PRP4-induced inhibition of cell death and PRP4 kinase domain interactions with various other proteins might lead to the development of novel approaches for overcoming drug resistance in patients with colon cancer.

Pre-mRNA processing factor 3 enhances the progression of keratinocyte-derived cutaneous squamous cell carcinoma by regulating the JAK2/STAT3 pathway.

The precise role of pre-mRNA processing factors (PRPs) in human tumorigenesis has not been yet explored. The object of the present study was to explore the effects of PRP3 in a common metastatic skin cancer, keratinocyte-derived cutaneous squamous cell carcinoma (cSCCs). RT-qPCR and western blotting were conducted to measure the expression levels of PRP3 in various cSCC cell lines and cSCC tissues. A benign epidermal keratinocyte cell line was transfected with a eukaryotic expression plasmid to overexpress PRP3. In addition, the endogenous expression level of PRP3 in cSCC cells was silenced using a short hairpin RNA method, and the role of PRP3 on cell proliferation and migration was examined by Cell Counting Kit-8, colony formation, wound healing assay and Transwell assays following knockdown in cSCC cells, and overexpression in keratinovcyte cells. Elevated levels of PRP3 mRNA and protein were noted in cSCC cell lines or cSCC tissues compared with actinic keratosis (AK) or benign epidermal keratinocyte cell line, respectively. Upregulation of PRP3 expression was found to be associated with poor clinical outcomes in patients with cSCCs. The upregulation of PRP3 promoted cell viability, metastasis and the activity of the JAK2/STAT3 pathway in epidermal keratinocyte cells. Interestingly, loss of PRP3 had no obvious impact on cell viability and migration in benign epidermal keratinocyte cells. Functionally, the inhibition of the JAK2/STAT3 pathway reversed the increased cell viability and migration of cSCC cells induced by PRP3. Taken together, the present observations indicated that PRP3 served as a tumor active factor in cSCCs by targeting the JAK2/STAT3 pathway. Moreover, it is implied that impeding the PRP3 activity may selectively constrain cancer cell growth and migration with limited effect on normal skin cells.

Prognostic potential of PRPF3 in hepatocellular carcinoma.

pre-mRNA processing factor 3 (PRPF3) is an RNA binding protein in a core component of the exon junction complex. Abnormal PRPF3 expression is potentially associated with carcinogenesis. However, the biological role of PRPF3 in hepatocellular carcinoma (HCC) remains to be determined. We analyzed PRPF3 expression via multiple gene expression databases and identified its genetic alterations and functional networks using cBioPortal. Co-expressed genes with PRPF3 and its regulators were identified using LinkedOmics. The correlations between PRPF3 and cancer immune infiltrates were investigated via Tumor Immune Estimation Resource (TIMER). PRPF3 was found up-regulated with amplification in tumor tissues in multiple HCC cohorts. High PRPF3 expression was associated with poorer overall survival (OS) and disease-free survival (DFS). Functional network analysis suggested that PRPF3 regulates spliceosome, DNA replication, and cell cycle signaling via pathways involving several cancer-related kinases and E2F family. Notably, PRPF3 expression was positively correlated with infiltrating levels of CD4+ T and CD8+ T cells, macrophages, neutrophils, and dendritic cells. PRPF3 expression showed strong correlations with diverse immune marker sets in HCC. These findings suggest that PRPF3 is correlated with prognosis and immune infiltrating in HCC, laying a foundation for further study of the immune regulatory role of PRPF3 in HCC.

Suppression of PRPF4 regulates pluripotency, proliferation, and differentiation in mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs) are characterized by their self-renewal and pluripotency and are capable of differentiating into all three germ layers. For this reason, mESCs are considered a very important model for stem cell research and clinical applications in regenerative medicine. The pre-mRNA processing factor 4 (PRPF4) gene is known to have a major effect on pre-mRNA splicing and is also known to affect tissue differentiation during development. In this study, we investigated the effects of PRPF4 knockdown on mESCs. First, we allowed mESCs to differentiate naturally and observed a significant decrease in PRPF4 expression during the differentiation process. We then artificially induced the knockdown of PRPF4 in mESCs and observed the changes in the phenotype. When PRPF4 was knocked down, various genes involved in mESC pluripotency showed significantly decreased expression. In addition, mESC proliferation increased abnormally, accompanied by a significant increase in mESC colony size. The formation of mESC embryoid bodies and teratomas was delayed following PRPF4 knockdown. Based on these results, the reduced expression of PRPF4 affects mESC phenotypes and is a key factor in mESC. SIGNIFICANCE OF THE STUDY: Our results indicate that PRPF4 affects the properties of mESCs. Suppression of PRPF4 resulted in a decrease in pluripotency of mESC and promoted proliferation. In addition, suppression of PRPF4 also resulted in decreased apoptosis. Moreover, the inhibition of PRPF4 reduced the ability to differentiate and formation of teratoma in mESC. Our results demonstrated that PRPF4 is a key factor of controlling mESC abilities.

PRPF4 is a novel therapeutic target for the treatment of breast cancer by influencing growth, migration, invasion, and apoptosis of breast cancer cells via p38 MAPK signaling pathway.

Pre-mRNA processing factor 4 (PRPF4), a core protein in U4/U6 snRNP, maintains snRNP structures by interacting with PRPF3 and cyclophilin H. Expression of the PRPF4 gene affects cell survival as well as apoptosis and is responsible for retinitis pigmentosa (RP). Proteomics analysis shows that PRPF4 may be a therapeutic target in human cancers. Nevertheless, the exact function and role of the PRPF4 gene are unclear. In this study, we assessed the expression of PRPF4 gene in human breast cancer cells. First, we confirmed that the PRPF4 gene was overexpressed in various breast cancer cell lines. Next, using breast cancer cell lines MCF7 and MDA-MB-468, we established stable cell lines with PRPF4 gene knockdown. We also performed microarray analysis to investigate molecular mechanisms underlying PRPF4 activity. All cell lines with PRPF4 gene knockdown exhibited reduced cell proliferation, remarkable reduction in anchorage-independent colony formation capacity, and reduction of PCNA protein, which is a marker cell of proliferation. Reduced expression of the PRPF4 gene induced apoptosis and changes in the expression of associated apoptotic markers in breast cancer cell lines. Knockdown of the PRPF4 gene reduced cellular capacity for migration and invasion (the key hallmarks of human cancers) and decreased the expression of genes involved in epithelial-mesenchymal transition (EMT). Microarray results showed that the expression of PPIP5K1, PPIPK2, and YWHAE genes was reduced at the transcriptional level, leading to reduced phosphorylation of p38 MAPK. These findings suggest that knockdown of PRPF4 gene slows down breast cancer progression via suppression of p38 MAPK phosphorylation. In conclusion, the PRPF4 gene plays an important role in the growth of breast cancer cells and is therefore a potential therapeutic target.

Precursor RNA processing 3 is required for male fertility, and germline stem cell self-renewal and differentiation via regulating spliceosome function in Drosophila testes.

The nuclear pre-mRNA spliceosome is a large complex containing five small nuclear ribonucleoprotein particles (snRNPs) and many splicing factors. Messenger RNAs (mRNAs) are generated from pre-mRNAs by the process of RNA splicing, which is conserved in eukaryotes. Precursor RNA processing 3 (Prp3) is a U4/U6-associated snRNP whose function remains largely unknown. In the present study, using genetic manipulation of a Drosophila melanogaster testis model, we demonstrated that Prp3 is essential for male fertility in Drosophila. Prp3 deficiency in germline stem cells (GSCs) and early cyst cells resulted in abnormal structure of testes and maintenance defects of GSCs and cyst stem cells. Knockdown of Prp3 in spermatogonia and early cyst cells mediated tumor formation caused by differentiation defects. Using an in vitro assay, knockdown of Prp3 decreased proliferation and increased cell death, and controlled the spliceosome function via regulating spliceosome subunits expression in Drosophila S2 cells. We also identified two other splicing factors in the Prp complex (Prp19 and Prp8), which mimicked the phenotype of Prp3 in the Drosophila stem cell niche. Our results revealed a significant role of precursor RNA processing factors in male testes, indicating that Prp3, a key spliceosome component in the Prp complex, is essential for male fertility, and germline stem cell self-renewal and differentiation, via regulating the spliceosome function in Drosophila testes.

Uncovering the signaling landscape controlling breast cancer cell migration identifies novel metastasis driver genes.

Ttriple-negative breast cancer (TNBC) is an aggressive and highly metastatic breast cancer subtype. Enhanced TNBC cell motility is a prerequisite of TNBC cell dissemination. Here, we apply an imaging-based RNAi phenotypic cell migration screen using two highly motile TNBC cell lines (Hs578T and MDA-MB-231) to provide a repository of signaling determinants that functionally drive TNBC cell motility. We have screened ~4,200 target genes individually and discovered 133 and 113 migratory modulators of Hs578T and MDA-MB-231, respectively, which are linked to signaling networks predictive for breast cancer progression. The splicing factors PRPF4B and BUD31 and the transcription factor BPTF are essential for cancer cell migration, amplified in human primary breast tumors and associated with metastasis-free survival. Depletion of PRPF4B, BUD31 and BPTF causes primarily down regulation of genes involved in focal adhesion and ECM-interaction pathways. PRPF4B is essential for TNBC metastasis formation in vivo, making PRPF4B a candidate for further drug development.

Rearrangements within the U6 snRNA Core during the Transition between the Two Catalytic Steps of Splicing.

The RNA catalytic core of spliceosomes as visualized by cryoelectron microscopy (cryo-EM) remains unchanged at different stages of splicing. However, we demonstrate that mutations within the core of yeast U6 snRNA modulate conformational changes between the two catalytic steps. We propose that the intramolecular stem-loop (ISL) of U6 exists in two competing states, changing between a default, non-catalytic conformation and a transient, catalytic conformation. Whereas stable interactions in the catalytic triplex promote catalysis and their disruptions favor exit from the catalytic conformation, destabilization of the lower ISL stem promotes catalysis and its stabilization supports exit from the catalytic conformation. Thus, in addition to the catalytic triplex, U6-ISL acts as an important dynamic component of the catalytic center. The relative flexibility of the lower U6-ISL stem is conserved across eukaryotes. Similar features are found in U6atac and domain V of group II introns, arguing for the generality of the proposed mechanism.

A Genome-wide Haploid Genetic Screen Identifies Regulators of Glutathione Abundance and Ferroptosis Sensitivity.

The tripeptide glutathione suppresses the iron-dependent, non-apoptotic cell death process of ferroptosis. How glutathione abundance is regulated in the cell and how this regulation alters ferroptosis sensitivity is poorly understood. Using genome-wide human haploid genetic screening technology coupled to fluorescence-activated cell sorting (FACS), we directly identify genes that regulate intracellular glutathione abundance and characterize their role in ferroptosis regulation. Disruption of the ATP binding cassette (ABC)-family transporter multidrug resistance protein 1 (MRP1) prevents glutathione efflux from the cell and strongly inhibits ferroptosis. High levels of MRP1 expression decrease sensitivity to certain pro-apoptotic chemotherapeutic drugs, while collaterally sensitizing to all tested pro-ferroptotic agents. By contrast, disruption of KEAP1 and NAA38, leading to the stabilization of the transcription factor NRF2, increases glutathione levels but only weakly protects from ferroptosis. This is due in part to concomitant NRF2-mediated upregulation of MRP1. These results pinpoint glutathione efflux as an unanticipated regulator of ferroptosis sensitivity.

Regulation of Yki/Yap subcellular localization and Hpo signaling by a nuclear kinase PRP4K.

Hippo (Hpo) signaling pathway controls tissue growth by regulating the subcellular localization of Yorkie (Yki)/Yap via a cytoplasmic kinase cassette containing an upstream kinase Hpo/MST1/2 and a downstream kinase Warts (Wts)/Lats1/2. Here we show that PRP4K, a kinase involved in mRNA splicing, phosphorylates Yki/Yap in the nucleus to prevent its nuclear accumulation and restrict Hpo pathway target gene expression. PRP4K inactivation accelerates whereas excessive PRP4K inhibits Yki-driven tissue overgrowth. PRP4K phosphorylates a subset of Wts/Lats1/2 sites on Yki/Yap to inhibit the binding of Yki/Yap to the Scalloped (Sd)/TEAD transcription factor and exclude Yki/Yap nuclear localization depending on nuclear export. Furthermore, PRP4K inhibits proliferation and invasiveness of cultured breast cancer cells and its high expression correlates with good prognosis in breast cancer patients. Our study unravels an unanticipated layer of Hpo pathway regulation and suggests that PRP4K-mediated Yki/Yap phosphorylation in the nucleus provides a fail-safe mechanism to restrict aberrant pathway activation.

Loss of PRP4K drives anoikis resistance in part by dysregulation of epidermal growth factor receptor endosomal trafficking.

Anoikis acts as a critical barrier to metastasis by inducing cell death upon cancer cell detachment from the extracellular matrix (ECM), thereby preventing tumor cell dissemination to secondary sites. The induction of anoikis requires the lysosomal-mediated downregulation of epidermal growth factor receptors (EGFRs) leading to termination of pro-survival signaling. In this study, we demonstrate that depletion of pre-mRNA splicing factor 4 kinase (PRP4K; also known as PRPF4B) causes dysregulation of EGFR trafficking and anoikis resistance. We also report a novel cytoplasmic localization of PRP4K at the late endosome, and demonstrate both nuclear and cytoplasmic localization in breast, lung and ovarian cancer tissue. Mechanistically, depletion of PRP4K leads to reduced EGFR degradation following cell detachment from the ECM and correlates with increased TrkB, vimentin and Zeb1 expression. As a result, PRP4K loss promotes sustained growth factor signaling and increased cellular resistance to anoikis in vitro and in a novel zebrafish xenotransplantation model of anoikis sensitivity, as well as increased metastasis in a mouse model of ovarian cancer. Thus, PRP4K may serve as a potential biomarker of anoikis sensitivity in ovarian and other epithelial cancers.

Dual RNA Processing Roles of Pat1b via Cytoplasmic Lsm1-7 and Nuclear Lsm2-8 Complexes.

Pat1 RNA-binding proteins, enriched in processing bodies (P bodies), are key players in cytoplasmic 5' to 3' mRNA decay, activating decapping of mRNA in complex with the Lsm1-7 heptamer. Using co-immunoprecipitation and immunofluorescence approaches coupled with RNAi, we provide evidence for a nuclear complex of Pat1b with the Lsm2-8 heptamer, which binds to the spliceosomal U6 small nuclear RNA (snRNA). Furthermore, we establish the set of interactions connecting Pat1b/Lsm2-8/U6 snRNA/SART3 and additional U4/U6.U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) components in Cajal bodies, the site of snRNP biogenesis. RNA sequencing following Pat1b depletion revealed the preferential upregulation of mRNAs normally found in P bodies and enriched in 3' UTR AU-rich elements. Changes in >180 alternative splicing events were also observed, characterized by skipping of regulated exons with weak donor sites. Our data demonstrate the dual role of a decapping enhancer in pre-mRNA processing as well as in mRNA decay via distinct nuclear and cytoplasmic Lsm complexes.

A close-up look at the spliceosome, at last.

Major developments in cryo-electron microscopy in the past three or four years have led to the solution of a number of spliceosome structures at high resolution, e.g., the fully assembled but not yet active spliceosome (Bact), the spliceosome just after the first step of splicing (C), and the spliceosome activated for the second step (C*). Therefore 30 years of genetics and biochemistry of the spliceosome can now be interpreted at the structural level. I have closely examined the RNase H domain of Prp8 in each of the structures. Interestingly, the RNase H domain has different and unexpected roles in each of the catalytic steps of splicing.

USP15 regulates dynamic protein-protein interactions of the spliceosome through deubiquitination of PRP31.

Post-translational modifications contribute to the spliceosome dynamics by facilitating the physical rearrangements of the spliceosome. Here, we report USP15, a deubiquitinating enzyme, as a regulator of protein-protein interactions for the spliceosome dynamics. We show that PRP31, a component of U4 snRNP, is modified with K63-linked ubiquitin chains by the PRP19 complex and deubiquitinated by USP15 and its substrate targeting factor SART3. USP15SART3 makes a complex with USP4 and this ternary complex serves as a platform to deubiquitinate PRP31 and PRP3. The ubiquitination and deubiquitination status of PRP31 regulates its interaction with the U5 snRNP component PRP8, which is required for the efficient splicing of chromosome segregation related genes, probably by stabilizing the U4/U6.U5 tri-snRNP complex. Collectively, our data suggest that USP15 plays a key role in the regulation of dynamic protein-protein interactions of the spliceosome.

Molecular architecture of the Saccharomyces cerevisiae activated spliceosome.

The activated spliceosome (Bact) is in a catalytically inactive state and is remodeled into a catalytically active machine by the RNA helicase Prp2, but the mechanism is unclear. Here, we describe a 3D electron cryomicroscopy structure of the Saccharomyces cerevisiae Bact complex at 5.8-angstrom resolution. Our model reveals that in Bact, the catalytic U2/U6 RNA-Prp8 ribonucleoprotein core is already established, and the 5' splice site (ss) is oriented for step 1 catalysis but occluded by protein. The first-step nucleophile-the branchsite adenosine-is sequestered within the Hsh155 HEAT domain and is held 50 angstroms away from the 5'ss. Our structure suggests that Prp2 adenosine triphosphatase-mediated remodeling leads to conformational changes in Hsh155's HEAT domain that liberate the first-step reactants for catalysis.