Human Spliceosome Research Articles

Controlled Trafficking of Multiple and Diverse Cations Prompts Nucleic Acid Hydrolysis.

Recent in crystallo reaction intermediates have detailed how nucleic acid hydrolysis occurs in the RNA ribonuclease H1 (RNase H1), a fundamental metalloenzyme involved in maintaining the human genome. At odds with the previous characterization, these in crystallo structures unexpectedly captured multiple metal ions (K+ and Mg2+) transiently bound in the vicinity of the two-metal-ion active site. Using multi-microsecond all-atom molecular dynamics and free-energy simulations, we investigated the functional implications of the dynamic exchange of multiple K+ and Mg2+ ions at the RNase H1 reaction center. We found that such ions are timely positioned at non-overlapping locations near the active site, at different stages of catalysis, being crucial for both reactants' alignment and leaving group departure. We also found that this cation trafficking is tightly regulated by variations of the solution's ionic strength and is aided by two conserved second-shell residues, E188 and K196, suggesting a mechanism for the cations' recruitment during catalysis. These results indicate that controlled trafficking of multi-cation dynamics, opportunely prompted by second-shell residues, is functionally essential to the complex enzymatic machinery of the RNase H1. These findings revise the current knowledge on the RNase H1 catalysis and open new catalytic possibilities for other similar metalloenzymes including, but not limited to, CRISPR-Cas9, group II intron ribozyme and the human spliceosome.

Synergistic roles for human U1 snRNA stem-loops in pre-mRNA splicing

ABSTRACT During spliceosome assembly, interactions that bring the 5′ and 3′ ends of an intron in proximity are critical for the production of mature mRNA. Here, we report synergistic roles for the stem-loops 3 (SL3) and 4 (SL4) of the human U1 small nuclear RNA (snRNA) in maintaining the optimal U1 snRNP function, and formation of cross-intron contact with the U2 snRNP. We find that SL3 and SL4 bind distinct spliceosomal proteins and combining a U1 snRNA activity assay with siRNA-mediated knockdown, we demonstrate that SL3 and SL4 act through the RNA helicase UAP56 and the U2 protein SF3A1, respectively. In vitro analysis using UV crosslinking and splicing assays indicated that SL3 likely promotes the SL4-SF3A1 interaction leading to enhancement of A complex formation and pre-mRNA splicing. Overall, these results highlight the vital role of the distinct contacts of SL3 and SL4 in bridging the pre-mRNA bound U1 and U2 snRNPs during the early steps of human spliceosome assembly.

Activation of Prp28 ATPase by phosphorylated Npl3 at a critical step of spliceosome remodeling

Splicing, a key step in the eukaryotic gene-expression pathway, converts precursor messenger RNA (pre-mRNA) into mRNA by excising introns and ligating exons. This task is accomplished by the spliceosome, a macromolecular machine that must undergo sequential conformational changes to establish its active site. Each of these major changes requires a dedicated DExD/H-box ATPase, but how these enzymes are activated remain obscure. Here we show that Prp28, a yeast DEAD-box ATPase, transiently interacts with the conserved 5′ splice-site (5′SS) GU dinucleotide and makes splicing-dependent contacts with the U1 snRNP protein U1C, and U4/U6.U5 tri-snRNP proteins, Prp8, Brr2, and Snu114. We further show that Prp28’s ATPase activity is potentiated by the phosphorylated Npl3, but not the unphosphorylated Npl3, thus suggesting a strategy for regulating DExD/H-box ATPases. We propose that Npl3 is a functional counterpart of the metazoan-specific Prp28 N-terminal region, which can be phosphorylated and serves as an anchor to human spliceosome.

Atomic structure of the minor spliceosome

Spliceosome About 1% of the human genome contains the so-called U12-type introns, which are spliced by the minor spliceosome. Compared with the major spliceosome, the composition, assembly, functional states, activation, regulation, and structure of the minor spliceosome have been enigmatic. Bai et al. assembled the activated human minor spliceosome in an in vitro U12-type intron-splicing assay and determined the structure by cryo–electron microscopy at 2.9-angstrom resolution. They found a number of exciting and unanticipated features, including five new proteins that play an important role in the activated minor spliceosome. Science , this issue p. [eabg0879][1] [1]: /lookup/doi/10.1126/science.abg0879

Structure of the activated human minor spliceosome.

The minor spliceosome mediates splicing of the rare but essential U12-type precursor messenger RNA. Here, we report the atomic features of the activated human minor spliceosome determined by cryo-electron microscopy at 2.9-angstrom resolution. The 5' splice site and branch point sequence of the U12-type intron are recognized by the U6atac and U12 small nuclear RNAs (snRNAs), respectively. Five newly identified proteins stabilize the conformation of the catalytic center: The zinc finger protein SCNM1 functionally mimics the SF3a complex of the major spliceosome, the RBM48-ARMC7 complex binds the γ-monomethyl phosphate cap at the 5' end of U6atac snRNA, the U-box protein PPIL2 coordinates loop I of U5 snRNA and stabilizes U5 small nuclear ribonucleoprotein (snRNP), and CRIPT stabilizes U12 snRNP. Our study provides a framework for the mechanistic understanding of the function of the human minor spliceosome.

Rewards of divergence in sequences, 3-D structures and dynamics of yeast and human spliceosome SF3b complexes.

The evolution of homologous and functionally equivalent multiprotein assemblies is intriguing considering sequence divergence of constituent proteins. Here, we studied the implications of protein sequence divergence on the structure, dynamics and function of homologous yeast and human SF3b spliceosomal subcomplexes. Human and yeast SF3b comprise of 7 and 6 proteins respectively, with all yeast proteins homologous to their human counterparts at moderate sequence identity. SF3b6, an additional component in the human SF3b, interacts with the N-terminal extension of SF3b1 while the yeast homologue Hsh155 lacks the equivalent region. Through detailed homology studies, we show that SF3b6 is absent not only in yeast but in multiple lineages of eukaryotes implying that it is critical in specific organisms. We probed for the potential role of SF3b6 in the spliceosome assembled form through structural and flexibility analyses. By analysing normal modes derived from anisotropic network models of SF3b1, we demonstrate that when SF3b1 is bound to SF3b6, similarities in the magnitude of residue motions (0.86) and inter-residue correlated motions (0.94) with Hsh155 are significantly higher than when SF3b1 is considered in isolation (0.21 and 0.89 respectively). We observed that SF3b6 promotes functionally relevant ‘open-to-close’ transition in SF3b1 by enhancing concerted residue motions. Such motions are found to occur in the Hsh155 without SF3b6. The presence of SF3b6 influences motions of 16 residues that interact with U2 snRNA/branchpoint duplex and supports the participation of its interface residues in long-range communication in the SF3b1. These results advocate that SF3b6 potentially acts as an allosteric regulator of SF3b1 for BPS selection and might play a role in alternative splicing. Furthermore, we observe variability in the relative orientation of SF3b4 and in the local structure of three β-propeller domains of SF3b3 with reference to their yeast counterparts. Such differences influence the inter-protein interactions of SF3b between these two organisms. Together, our findings highlight features of SF3b evolution and suggests that the human SF3b may have evolved sophisticated mechanisms to fine tune its molecular function.

Structural Insights into the Roles of Metazoan-Specific Splicing Factors in the Human Step 1 Spliceosome

Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4Å core resolution and 4.5-5.7Å at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome's architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome's catalytically active RNP (ribonucleoprotein) conformation. It reveals the spatial organization of the metazoan-specific proteins PPWD1, WDR70, FRG1, and CIR1 in human C complexes, indicating they stabilize functionally important protein domains and RNA structures rearranged/repositioned during the Bact to C transition. Structural comparisons with human Bact, C∗, and P complexes reveal an intricate cascade of RNP rearrangements during splicing catalysis, with intermediate RNP conformations not found in yeast, and additionally elucidate the structural basis for the sequential recruitment of metazoan-specific spliceosomal proteins.

Unbiased Identification of Key Residues in Splicing Protein Dib1

Pre‐messenger RNA (pre‐mRNA) is transcribed from a DNA segment, and then post‐transcriptionally modified before being translated into protein. To prepare the pre‐mRNA for proper protein production, the spliceosome facilitates the removal of non‐coding introns from the pre‐mRNA and the ligation of exons together, forming the mature messenger RNA (mRNA) in a process known as pre‐messenger RNA splicing. The spliceosome is a large ribonucleoprotein complex composed of 5‐snRNA and over 90 proteins that work in concert to facilitate pre‐mRNA splicing and ensure its accuracy. Due to its complexity and evolutionary importance to survival, mutations of the spliceosome have been linked to diseases. For instance, Burn‐McKeown syndrome is a craniofacial disorder caused by abnormalities in the gene for the U5‐15k protein, a small protein centered within human spliceosomes and an ortholog of Dib1 in yeast.Dib1 is a 17‐kDa thioredoxin‐like protein that is associated with the U5 small nuclear ribonucleoprotein (snRNP) in the U5/U4.U6 complex. Located in the catalytic groove of the pre‐B complex of the spliceosome, Dib1 must vacate the complex so the spliceosome can complete its assembly. However, this “gate‐keeping” mechanism controlling this departure is still unknown. This project intends to discover residues that are crucial to the function of Dib1 by an unbiased method. To identify key residues in Dib1, a randomly mutagenized dib1 plasmid library was constructed. A temperature sensitive (ts) yeast screen was then performed with this library in order to identify Dib1 residues important for cell growth. From this screen, we have identified a new dib1 mutant and will discuss the significance of its location in Dib1 and its relevance to splicing.Support or Funding InformationTrinity University, ASBMB Distinguished Undergraduate Scholarship, NIH‐NIGMS R15GM120720, and The Welch Foundation

Smu1 and RED are required for activation of spliceosomal B complexes assembled on short introns

Human pre-catalytic spliceosomes contain several proteins that associate transiently just prior to spliceosome activation and are absent in yeast, suggesting that this critical step is more complex in higher eukaryotes. We demonstrate via RNAi coupled with RNA-Seq that two of these human-specific proteins, Smu1 and RED, function both as alternative splicing regulators and as general splicing factors and are required predominantly for efficient splicing of short introns. In vitro splicing assays reveal that Smu1 and RED promote spliceosome activation, and are essential for this step when the distance between the pre-mRNA’s 5′ splice site (SS) and branch site (BS) is sufficiently short. This Smu1-RED requirement can be bypassed when the 5′ and 3′ regions of short introns are physically separated. Our observations suggest that Smu1 and RED relieve physical constraints arising from a short 5′SS-BS distance, thereby enabling spliceosomes to overcome structural challenges associated with the splicing of short introns.

Mechanism of 5' splice site transfer for human spliceosome activation.

The prespliceosome, comprising U1 and U2 small nuclear ribonucleoproteins (snRNPs) bound to the precursor messenger RNA 5' splice site (5'SS) and branch point sequence, associates with the U4/U6.U5 tri-snRNP to form the fully assembled precatalytic pre-B spliceosome. Here, we report cryo-electron microscopy structures of the human pre-B complex captured before U1 snRNP dissociation at 3.3-angstrom core resolution and the human tri-snRNP at 2.9-angstrom resolution. U1 snRNP inserts the 5'SS-U1 snRNA helix between the two RecA domains of the Prp28 DEAD-box helicase. Adenosine 5'-triphosphate-dependent closure of the Prp28 RecA domains releases the 5'SS to pair with the nearby U6 ACAGAGA-box sequence presented as a mobile loop. The structures suggest that formation of the 5'SS-ACAGAGA helix triggers remodeling of an intricate protein-RNA network to induce Brr2 helicase relocation to its loading sequence in U4 snRNA, enabling Brr2 to unwind the U4/U6 snRNA duplex to allow U6 snRNA to form the catalytic center of the spliceosome.

A Combined in silico, in vitro and Clinical Approach to Characterize Novel Pathogenic Missense Variants in PRPF31 in Retinitis Pigmentosa.

At least six different proteins of the spliceosome, including PRPF3, PRPF4, PRPF6, PRPF8, PRPF31, and SNRNP200, are mutated in autosomal dominant retinitis pigmentosa (adRP). These proteins have recently been shown to localize to the base of the connecting cilium of the retinal photoreceptor cells, elucidating this form of RP as a retinal ciliopathy. In the case of loss-of-function variants in these genes, pathogenicity can easily be ascribed. In the case of missense variants, this is more challenging. Furthermore, the exact molecular mechanism of disease in this form of RP remains poorly understood. In this paper we take advantage of the recently published cryo EM-resolved structure of the entire human spliceosome, to predict the effect of a novel missense variant in one component of the spliceosome; PRPF31, found in a patient attending the genetics eye clinic at Bristol Eye Hospital. Monoallelic variants in PRPF31 are a common cause of autosomal dominant retinitis pigmentosa (adRP) with incomplete penetrance. We use in vitro studies to confirm pathogenicity of this novel variant PRPF31 c.341T > A, p.Ile114Asn. This work demonstrates how in silico modeling of structural effects of missense variants on cryo-EM resolved protein complexes can contribute to predicting pathogenicity of novel variants, in combination with in vitro and clinical studies. It is currently a considerable challenge to assign pathogenic status to missense variants in these proteins.

Structural Basis of Nuclear pre-mRNA Splicing: Lessons from Yeast.

Noncoding introns are removed from nuclear precursor messenger RNA (pre-mRNA) in a two-step phosphoryl transfer reaction by the spliceosome, a dynamic multimegadalton enzyme. Cryo-electron microscopy (cryo-EM) structures of the Saccharomyces cerevisiae spliceosome were recently determined in eight key states. Combined with the wealth of available genetic and biochemical data, these structures have revealed new insights into the mechanisms of spliceosome assembly, activation, catalysis, and disassembly. The structures show how a single RNA catalytic center forms during activation and accomplishes both steps of the splicing reaction. The structures reveal how spliceosomal helicases remodel the spliceosome for active site formation, substrate docking, reaction product undocking, and spliceosome disassembly and how they facilitate splice site proofreading. Although human spliceosomes contain additional proteins, their cryo-EM structures suggest that the underlying mechanism is conserved across all eukaryotes. In this review, we summarize the current structural understanding of pre-mRNA splicing.

Structural Insights into Nuclear pre-mRNA Splicing in Higher Eukaryotes.

The spliceosome is a highly complex, dynamic ribonucleoprotein molecular machine that undergoes numerous structural and compositional rearrangements that lead to the formation of its active site. Recent advances in cyroelectron microscopy (cryo-EM) have provided a plethora of near-atomic structural information about the inner workings of the spliceosome. Aided by previous biochemical, structural, and functional studies, cryo-EM has confirmed or provided a structural basis for most of the prevailing models of spliceosome function, but at the same time allowed novel insights into splicing catalysis and the intriguing dynamics of the spliceosome. The mechanism of pre-mRNA splicing is highly conserved between humans and yeast, but the compositional dynamics and ribonucleoprotein (RNP) remodeling of the human spliceosome are more complex. Here, we summarize recent advances in our understanding of the molecular architecture of the human spliceosome, highlighting differences between the human and yeast splicing machineries.

Structures of the human spliceosomes before and after release of the ligated exon

Pre-mRNA splicing is executed by the spliceosome, which has eight major functional states each with distinct composition. Five of these eight human spliceosomal complexes, all preceding exon ligation, have been structurally characterized. In this study, we report the cryo-electron microscopy structures of the human post-catalytic spliceosome (P complex) and intron lariat spliceosome (ILS) at average resolutions of 3.0 and 2.9 Å, respectively. In the P complex, the ligated exon remains anchored to loop I of U5 small nuclear RNA, and the 3′-splice site is recognized by the junction between the 5′-splice site and the branch point sequence. The ATPase/helicase Prp22, along with the ligated exon and eight other proteins, are dissociated in the P-to-ILS transition. Intriguingly, the ILS complex exists in two distinct conformations, one with the ATPase/helicase Prp43 and one without. Comparison of these three late-stage human spliceosomes reveals mechanistic insights into exon release and spliceosome disassembly.

A human postcatalytic spliceosome structure reveals essential roles of metazoan factors for exon ligation.

During exon ligation, the Saccharomyces cerevisiae spliceosome recognizes the 3'-splice site (3'SS) of precursor messenger RNA (pre-mRNA) through non-Watson-Crick pairing with the 5'SS and the branch adenosine, in a conformation stabilized by Prp18 and Prp8. Here we present the 3.3-angstrom cryo-electron microscopy structure of a human postcatalytic spliceosome just after exon ligation. The 3'SS docks at the active site through conserved RNA interactions in the absence of Prp18. Unexpectedly, the metazoan-specific FAM32A directly bridges the 5'-exon and intron 3'SS of pre-mRNA and promotes exon ligation, as shown by functional assays. CACTIN, SDE2, and NKAP-factors implicated in alternative splicing-further stabilize the catalytic conformation of the spliceosome during exon ligation. Together these four proteins act as exon ligation factors. Our study reveals how the human spliceosome has co-opted additional proteins to modulate a conserved RNA-based mechanism for 3'SS selection and to potentially fine-tune alternative splicing at the exon ligation stage.

High-throughput RNAi screening reveals cancer-selective lethal targets in the RNA spliceosome.

Novel therapeutic strategies for non-small-cell lung cancer (NSCLC) are urgently needed. RNA splicing, orchestrated by the spliceosome, is deregulated in many forms of cancer, including NSCLC. Here, we performed high-throughput screening with a small interfering RNA library targeting all annotated human spliceosome proteins to identify cancer-selective lethal targets in the RNA splicing machinery. Silencing of several spliceosome proteins reduced cell viability in a panel of NSCLC cell lines, but not in non-malignant lung fibroblasts and epithelial cells. Interestingly, the cancer-selective lethal target set comprised all seven Sm proteins that, together with small nuclear RNA, form the core structure of most spliceosome subunits. Interfering with Sm protein expression induced apoptosis in NSCLC cells, but not in non-malignant cells. In silico analysis revealed that Sm proteins are frequently upregulated in NSCLC. For several Sm proteins, increased expression showed a positive correlation with disease severity. Together, our results suggest that the Sm proteins represent particularly useful novel targets for selective treatment of NSCLC.

Chemical Inhibition of Pre-mRNA Splicing in Living Saccharomyces cerevisiae

The spliceosome mediates precursor mRNA splicing in eukaryotes, including the model organism Saccharomyces cerevisiae (yeast). Despite decades of study, no chemical inhibitors of yeast splicing invivo are available. We have developed a system to efficiently inhibit splicing and block proliferation in living yeast cells using compounds that target the human spliceosome protein SF3B1. Potent inhibition is observed in yeast expressing a chimeric protein containing portions of human SF3B1. However, only a single point mutation in the yeast homolog of SF3B1 is needed for selective inhibition of splicing by pladienolide B, herboxidiene, or meayamycin in liquid culture. Mutations that enable inhibition also improve splicing of branch sites containing mismatches between the intron and small nuclear RNA-suggesting a link between inhibitor sensitivity and usage of weak branch sites in humans. This approach provides powerful new tools for manipulating splicing in live yeast and studies of spliceosome inhibitors.

Molecular Mechanisms of pre-mRNA Splicing through Structural Biology of the Spliceosome.

Precursor messenger RNA (pre-mRNA) splicing is executed by the spliceosome. In the past 3 years, cryoelectron microscopy (cryo-EM) structures have been elucidated for a majority of the yeast spliceosomal complexes and for a few human spliceosomes. During the splicing reaction, the dynamic spliceosome has an immobile core of about 20 protein and RNA components, which are organized around a conserved splicing active site. The divalent metal ions, coordinated by U6 small nuclear RNA (snRNA), catalyze the branching reaction and exon ligation. The spliceosome also contains a mobile but compositionally stable group of about 13 proteins and a portion of U2 snRNA, which facilitate substrate delivery into the splicing active site. The spliceosomal transitions are driven by the RNA-dependent ATPase/helicases, resulting in the recruitment and dissociation of specific splicing factors that enable the reaction. In summary, the spliceosome is a protein-directed metalloribozyme.

Structural studies of the spliceosome: past, present and future perspectives.

The spliceosome is a multi-subunit RNA-protein complex involved in the removal of non-coding segments (introns) from between the coding regions (exons) in precursors of messenger RNAs (pre-mRNAs). Intron removal proceeds via two transesterification reactions, occurring between conserved sequences at intron-exon junctions. A tightly regulated, hierarchical assembly with a multitude of structural and compositional rearrangements posed a great challenge for structural studies of the spliceosome. Over the years, X-ray crystallography dominated the field, providing valuable high-resolution structural information that was mostly limited to individual proteins and smaller sub-complexes. Recent developments in the field of cryo-electron microscopy allowed the visualisation of fully assembled yeast and human spliceosomes, providing unprecedented insights into substrate recognition, catalysis, and active site formation. This has advanced our mechanistic understanding of pre-mRNA splicing enormously.

Structures of the human pre-catalytic spliceosome and its precursor spliceosome

The pre-catalytic spliceosome (B complex) is preceded by its precursor spliceosome (pre-B complex) and followed by the activated spliceosome (Bact complex). The pre-B-to-B and B-to-Bact transitions are driven by the ATPase/helicases Prp28 and Brr2, respectively. In this study, we report the cryo-electron microscopy structures of the human pre-B complex and the human B complex at an average resolution of 5.7 and 3.8 Å, respectively. In the pre-B complex, U1 and U2 small nuclear ribonucleoproteins (snRNPs) associate with two edges of the tetrahedron-shaped U4/U6.U5 tri-snRNP. The pre-mRNA is yet to be recognized by U5 or U6 small nuclear RNA (snRNA), and loop I of U5 snRNA remains unengaged. In the B complex, U1 snRNP and Prp28 are dissociated, the 5’-exon is anchored to loop I of U5 snRNA, and the 5′-splice site is recognized by U6 snRNA through duplex formation. In sharp contrast to S. cerevisiae, most components of U2 snRNP and tri-snRNP, exemplified by Brr2, undergo pronounced rearrangements in the human pre-B-to-B transition. Structural analysis reveals mechanistic insights into the assembly and activation of the human spliceosome.