Spliceosome Activation Research Articles

The SF3b Complex is an Integral Component of the Spliceosome and Targeted by Natural Product-Based Inhibitors.

In this chapter, the essential role of the SF3b multi-protein complex will be discussed in the context of the overall spliceosome. SF3b is critical during spliceosome assembly for recognition of the branch point (BP)adenosine and, by de facto, selection of the 3' splice site. This complex is highly dynamic, undergoing significant conformational changes upon loading of the branch duplex RNA and in its relative positioning during spliceosomal remodeling from the A, pre-B, B, Bact and B* complexes. Ultimately, during the spliceosome activation phase, SF3b must be displaced to unmask the branch point adenosine for the first splicing reaction to occur. In certain cancers, such as the hematological malignancies CML, CLL and MDS, the SF3b subunit SF3B1 is frequently mutated. Recent studies suggest these mutations lead to inappropriate branch point selection and mis-splicing events that appear to be drivers of disease. Finally, the SF3b complex is the target for at least three different classes of natural product-based inhibitors. These inhibitors bind in the BP adenosine-binding pocket and demonstrate a pre-mRNA competitive mechanism of action resulting in either intron retention or exon skipping. These compounds are extremely useful as chemical probes to isolate and characterize early stages of spliceosome assembly. They are also being explored preclinically and clinically as possible agents for hematological cancers.

Mechanism of protein-guided folding of the active site U2/U6 RNA during spliceosome activation.

Spliceosome activation involves extensive protein and RNA rearrangements that lead to formation of a catalytically active U2/U6 RNA structure. At present, little is known about the assembly pathway of the latter and the mechanism whereby proteins aid its proper folding. Here, we report the cryo-electron microscopy structures of two human, activated spliceosome precursors (that is, pre-Bact complexes) at core resolutions of 3.9 and 4.2 angstroms. These structures elucidate the order of the numerous protein exchanges that occur during activation, the mutually exclusive interactions that ensure the correct order of ribonucleoprotein rearrangements needed to form the U2/U6 catalytic RNA, and the stepwise folding pathway of the latter. Structural comparisons with mature Bact complexes reveal the molecular mechanism whereby a conformational change in the scaffold protein PRP8 facilitates final three-dimensional folding of the U2/U6 catalytic RNA.

The integral spliceosomal component CWC15 is required for development in Arabidopsis

Efficient mRNA splicing is a prerequisite for protein biosynthesis and the eukaryotic splicing machinery is evolutionarily conserved among species of various phyla. At its catalytic core resides the activated splicing complex Bact consisting of the three small nuclear ribonucleoprotein complexes (snRNPs) U2, U5 and U6 and the so-called NineTeen complex (NTC) which is important for spliceosomal activation. CWC15 is an integral part of the NTC in humans and it is associated with the NTC in other species. Here we show the ubiquitous expression and developmental importance of the Arabidopsis ortholog of yeast CWC15. CWC15 associates with core components of the Arabidopsis NTC and its loss leads to inefficient splicing. Consistent with the central role of CWC15 in RNA splicing, cwc15 mutants are embryo lethal and additionally display strong defects in the female haploid phase. Interestingly, the haploid male gametophyte or pollen in Arabidopsis, on the other hand, can cope without functional CWC15, suggesting that developing pollen might be more tolerant to CWC15-mediated defects in splicing than either embryo or female gametophyte.

Minor Splicing Factors Zrsr1 and Zrsr2 Are Essential for Early Embryo Development and 2-Cell-Like Conversion.

Minor splicing plays an important role in vertebrate development. Zrsr1 and Zrsr2 paralog genes have essential roles in alternative splicing, mainly participating in the recognition of minor (U12) introns. To further explore their roles during early embryo development, we produced Zrsr1mu and Zrsr2mu mutant mice, containing truncating mutations within the second zinc finger domain. Both homozygous mutant mice were viable with a normal lifespan. When we crossed a homozygous Zrsr2mu/mu female with Zrsr1mu/mu male, the double heterozygotes were non-viable, giving rise to embryos that stopped developing mainly between the 2- and 4-cell stages, just after zygotic gene activation. RNA-seq analysis of Zrsr1/2mu 2-cell embryos showed altered gene and isoform expression of thousands of genes enriched in gene ontology terms and biological pathways related to ribosome, RNA transport, spliceosome, and essential zygotic gene activation steps. Alternative splicing was analyzed, showing a significant increase in intron retention in both U2 and U12 intron-containing genes related to cell cycle and mitotic nuclear division. Remarkably, both Zrsr1 and Zrsr2 were required for the conversion of mouse-induced pluripotent stem cells into 2C-like cells. According to our results, Zrsr1 or Zrsr2 are necessary for ZGA and both are indispensable for the conversion of induced pluripotent stem cells into 2C-like cells.

A Snu114-GTP-Prp8 module forms a relay station for efficient splicing in yeast.

The single G protein of the spliceosome, Snu114, has been proposed to facilitate splicing as a molecular motor or as a regulatory G protein. However, available structures of spliceosomal complexes show Snu114 in the same GTP-bound state, and presently no Snu114 GTPase-regulatory protein is known. We determined a crystal structure of Snu114 with a Snu114-binding region of the Prp8 protein, in which Snu114 again adopts the same GTP-bound conformation seen in spliceosomes. Snu114 and the Snu114–Prp8 complex co-purified with endogenous GTP. Snu114 exhibited weak, intrinsic GTPase activity that was abolished by the Prp8 Snu114-binding region. Exchange of GTP-contacting residues in Snu114, or of Prp8 residues lining the Snu114 GTP-binding pocket, led to temperature-sensitive yeast growth and affected the same set of splicing events in vivo. Consistent with dynamic Snu114-mediated protein interactions during splicing, our results suggest that the Snu114–GTP–Prp8 module serves as a relay station during spliceosome activation and disassembly, but that GTPase activity may be dispensable for splicing.

Thermodynamic examination of pH and magnesium effect on U6 RNA internal loop.

U6 RNA contains a 1 × 2-nt internal loop that folds and unfold during spliceosomal assembly and activation. The 1 × 2 loop consists of a C67•A79 base pair that forms an additional hydrogen bond upon protonation, C67•A+79, and uracil (U80) that coordinates the catalytically essential magnesium ions. We designed a series of RNA and DNA constructs with a 1 × 2 loop sequence contained in the ISL, and its modifications, to measure the thermodynamic effects of protonation and magnesium binding using UV-visible thermal denaturation experiments. We show that the wild-type RNA construct gains 0.43 kcal/mol in 1 M KCl upon lowering the pH from 7.5 to 5.5; the presence of magnesium ions increases its stability by 2.17 kcal/mol at pH 7.5 over 1 M KCl. Modifications of the helix closing base pairs from C–G to U•G causes a loss in protonation-dependent stability and a decrease in stability in the presence of magnesium ions, especially in the C68U construct. A79G single-nucleotide bulge loop construct showed the largest gain in stability in the presence of magnesium ions. The DNA wild-type construct shows a smaller effect on stability upon lowering the pH and in the presence of magnesium ions, highlighting differences in RNA and DNA structures. A U6 RNA 1 × 2 loop sequence is rare in the databases examined.

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.

Nucleolin Interacts and Co-Localizes with Components of Pre-Catalytic Spliceosome Complexes

Nucleolin is an RNA binding protein that is involved in many post-transcriptional regulation steps of messenger RNAs in addition to its nucleolar role in ribosomal RNA transcription and assembly in pre-ribosomes. Acetylated nucleolin was found to be associated with nuclear speckles and to co-localize with the splicing factor SC35. Previous nuclear pull down of nucleolin identified several splicing components and factors involved in RNA polymerase II transcription associated with nucleolin. In this report, we show that these splicing components are specifics of the pre-catalytic A and B spliceosomes, while proteins recruited in the Bact, C and P complexes are absent from the nucleolin interacting proteins. Furthermore, we show that acetylated nucleolin co-localized with P-SF3B1, a marker of co-transcriptional active spliceosomes. P-SF3B1 complexes can be pulled down with nucleolin specific antibodies. Interestingly, the alternative splicing of Fibronectin at the IIICS and EDB sites was affected by nucleolin depletion. These data are consistent with a model where nucleolin could be a factor bridging RNA polymerase II transcription and assembly of pre-catalytic spliceosome similarly to its function in the co-transcriptional maturation of pre-rRNA.

Prp8 in a Reduced Spliceosome Lacks a Conserved Toggle that Correlates with Splicing Complexity across Diverse Taxa

Conformational rearrangements are critical to regulating the assembly and activity of the spliceosome. The spliceosomal protein Prp8 undergoes multiple conformational changes during the course of spliceosome assembly, activation, and catalytic activity. Most of these rearrangements of Prp8 involve the disposition of the C-terminal Jab-MPN and RH domains with respect to the core of Prp8. Here we use x-ray structural analysis to show that a previously characterized and highly conserved β-hairpin structure in the RH domain that acts as a toggle in the spliceosome is absent in Prp8 from the reduced spliceosome of the red alga Cyanidioschyzon merolae. Using comparative sequence analysis, we show that the presence or absence of this hairpin corresponds to the presence or absence of protein partners that interact with this hairpin as observed by x-ray and cryo-EM studies. The presence of the toggle correlates with increasing intron number suggesting a role in the regulation of splicing.

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.

An Allosteric Network for Spliceosome Activation Revealed by High-Throughput Suppressor Analysis inSaccharomyces cerevisiae

Selection of suppressor mutations that correct growth defects caused by substitutions in an RNA or protein can reveal functionally important molecular structures and interactions in living cells. This approach is particularly useful for the study of complex biological pathways involving many macromolecules, such as premessenger RNA (pre-mRNA) splicing. When a sufficiently large number of suppressor mutations is obtained and structural information is available, it is possible to generate detailed models of molecular function. However, the laborious and expensive task of identifying suppressor mutations in whole-genome selections limits the utility of this approach. Here I show that a custom targeted sequencing panel can greatly accelerate the identification of suppressor mutations in the Saccharomyces cerevisiae genome. Using a panel that targets 112 genes encoding pre-mRNA splicing factors, I identified 27 unique mutations in six protein-coding genes that each overcome the cold-sensitive block to spliceosome activation caused by a substitution in U4 small nuclear RNA. When mapped to existing structures of spliceosomal complexes, the identified suppressors implicate specific molecular contacts between the proteins Brr2, Prp6, Prp8, Prp31, Sad1, and Snu114 as functionally important in an early step of catalytic activation of the spliceosome. This approach shows great promise for elucidating the allosteric cascade of molecular interactions that direct accurate and efficient pre-mRNA splicing and should be broadly useful for understanding the dynamics of other complex biological assemblies or pathways.

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.

Function and Dynamics of the LSm2-8 Protein Ring during Spliceosome Activation

LSm2-8, a heteroheptameric protein ring, decorates the 3′ end of the U6 snRNA and is a stable component of U6 snRNP, U4/U6 di-snRNP, U4/U6.U5 tri-snRNP, and the spliceosome B complex. However, it is released during spliceosome activation, during which the active site for pre-mRNA splicing is created. While Lsm2-8 is known to stabilize the U6 snRNA in the cell, any potential function during spliceosome assembly or activation is unclear. Here, we characterize Lsm2-8 ejection during spliceosome activation using Colocalization Single Molecule Spectroscopy (CoSMoS). Our CoSMoS data reveals that LSm ring leaves after both release of U4 snRNP and arrival of Nine Teen Complex (NTC) on pre-mRNA. This indicates that a conformational change must occur within the NTC after spliceosome binding since available cryo-EM structures indicate that Lsm2-8 and NTC binding are mutually exclusive with one another. Thus, our data provides evidence for an intermediate spliceosome complex between B and Bact that has not been yet observed by cryo-EM. This unexpected late release of the Lsm2-8 ring also suggests an extended role during spliceosome activation. Based on sequence homology to bacterial Hfq proteins, it is possible that Lsm2-8 may be promoting formation of a RNA duplex between the U2 and U6 snRNAs. Using in vitro annealing assay, we observed that LSm ring forms a ternary complex in the presence U6 and U2 RNAs, which is dependent on base pairing. These results indicate that LSm ring may help in stabilizing U2/U6 helix II during activation and is retained on spliceosome until U4 unwinding is complete. After U4 is unwound from U6, LSm is replaced by NTC components that takeover stabilization of helix II during later stages of activation and splicing.

Single-Molecule Analysis of Spliceosome Activation Kinetics Reveals Multiple Intermediate States

During pre-mRNA splicing, the spliceosome needs to be activated in an ATP-dependent manner in order to carry out the catalytic steps of the splicing reaction. During activation, the protein composition of the spliceosome changes dramatically and includes loss of the B complex proteins (Spp381, Snu23 and Prp38) and Snu66. Using colocalization single molecule spectroscopy (CoSMoS), we show that Spp381, Snu23, and Snu66 release is dependent on ATP by carrying out reactions at both high and low ATP concentrations. By comparing the characteristic dwell times for these proteins at high ATP, we found that Spp381 and Snu23 behave similar to each other and with kinetics distinct from Snu66. Specifically, at both ATP concentrations, Snu66 shows much longer-lived populations than Spp381 and Snu23. This suggests that Snu66 is bound more stably to the spliceosome than the other two. These results are consistent with previous biochemical and structural studies suggesting that Spp381, Snu23, and Prp38 form a protein subcomplex of the spliceosome. Together, our results suggest that this subcomplex and Snu66 likely have two different roles in the activation step and give rise to multiple activation intermediates as they are sequentially released from the spliceosome.

EIF4A3 Phosphorylation by CDKs Affects NMD during the Cell Cycle

Exon junction complexes (EJCs) loaded onto spliced mRNAs during splicing serve as molecular markers for various post-transcriptional gene-regulatory processes, including nonsense-mediated mRNA decay (NMD). Although the composition and structure of EJCs are well characterized, the mechanism regulating EJC deposition remains unknown. Here we find that threonine 163 (T163) within the RNA-binding motif of eIF4A3 (a core EJC component) is phosphorylated by cyclin-dependent protein kinases 1 and 2 in a cell cycle-dependent manner. T163 phosphorylation hinders binding of eIF4A3 to spliced mRNAs and other EJC components. Instead, it promotes association of eIF4A3 with CWC22, which guides eIF4A3 to an active spliceosome. These molecular events ensure the fidelity of specific deposition of the EJC ∼20-24 nt upstream of an exon-exon junction. Accordingly, NMD is affected by T163 phosphorylation. Collectively, our data provide evidence that T163 phosphorylation affects EJC formation and, consequently, NMD efficiency in a cell cycle-dependent manner.

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.

RNA Polymerase II Phosphorylated on CTD Serine 5 Interacts with the Spliceosome during Co-transcriptional Splicing

SummaryThe highly intronic nature of protein coding genes in mammals necessitates a co-transcriptional splicing mechanism as revealed by mNET-seq analysis. Immunoprecipitation of MNase-digested chromatin with antibodies against RNA polymerase II (Pol II) shows that active spliceosomes (both snRNA and proteins) are complexed to Pol II S5P CTD during elongation and co-transcriptional splicing. Notably, elongating Pol II-spliceosome complexes form strong interactions with nascent transcripts, resulting in footprints of approximately 60 nucleotides. Also, splicing intermediates formed by cleavage at the 5′ splice site are associated with nearly all spliced exons. These spliceosome-bound intermediates are frequently ligated to upstream exons, implying a sequential, constitutive, and U12-dependent splicing process. Finally, lack of detectable spliced products connected to the Pol II active site in human HeLa or murine lymphoid cells suggests that splicing does not occur immediately following 3′ splice site synthesis. Our results imply that most mammalian splicing requires exon definition for completion.

The tri-snRNP specific protein FgSnu66 is functionally related to FgPrp4 kinase in Fusarium graminearum.

Deletion of Prp4, the only kinase among spliceosome components, is not lethal in Fusarium graminearum but Fgprp4 mutants have severe growth defects and produced spontaneous suppressors. To identify novel suppressor mutations of Fgprp4, we sequenced the genome of suppressor S37 that was normal in growth but only partially recovered for intron splicing and identified a tandem duplication of 9-aa in the tri-snRNP component FgSNU66. Among the 19 additional suppressor strains found to have mutations in FgSNU66 (out of 260 screened), five had the same 9-aa duplication event with S37 and another five had the R477H/C mutation. The rest had nonsense or G-to-D mutations in the C-terminal 27-aa (CT27) region of FgSnu66, which is absent in its yeast ortholog. Truncation of this C-terminal region reduced the interaction of FgSnu66 with FgHub1 but increased its interaction with FgPrp8 and FgPrp6. Five phosphorylation sites were identified in FgSnu66 by phosphoproteomic analysis and the T418A-S420A-S422A mutation was shown to reduce virulence. Overall, our results showed that mutations in FgSNU66 can suppress deletion of Fgprp4, which has not been reported in other organisms, and the C-terminal tail of FgSnu66 plays a role in its interaction with key tri-snRNP components during spliceosome activation.

Structural basis for spliceosome assembly

Molecular Biology The spliceosome removes noncoding sequences from precursor RNA and ligates coding sequences into useful mRNA. The pre-spliceosome (A complex) associates with a small nuclear ribonucleoprotein (snRNP) complex called U4/U6.U5 tri-snRNP to form the pre-B complex, which is converted into the precatalytic B complex. Bai et al. solved the cryo–electron microscopy structures of the pre-B and B complexes isolated from yeast. These structures show the U1 and U2 snRNPs and allow modeling of the A complex to reveal the early steps of spliceosome assembly and activation. Science , this issue p. [1423][1] [1]: /lookup/doi/10.1126/science.aau0325

Structures of the fully assembled Saccharomyces cerevisiae spliceosome before activation.

The precatalytic spliceosome (B complex) is preceded by the pre-B complex. Here we report the cryo-electron microscopy structures of the Saccharomyces cerevisiae pre-B and B complexes at average resolutions of 3.3 to 4.6 and 3.9 angstroms, respectively. In the pre-B complex, the duplex between the 5' splice site (5'SS) and U1 small nuclear RNA (snRNA) is recognized by Yhc1, Luc7, and the Sm ring. In the B complex, U1 small nuclear ribonucleoprotein is dissociated, the 5'-exon-5'SS sequences are translocated near U6 snRNA, and three B-specific proteins may orient the precursor messenger RNA. In both complexes, U6 snRNA is anchored to loop I of U5 snRNA, and the duplex between the branch point sequence and U2 snRNA is recognized by the SF3b complex. Structural analysis reveals the mechanism of assembly and activation for the yeast spliceosome.