U1 snRNPs Research Articles

Insights into splicing: structure of the yeast U1 snRNP

Precursor‐mRNA splicing is catalyzed by a large and dynamic macromolecular assemblage termed the spliceosome. Integral to spliceosome function are five RNA‐protein complexes termed uridine‐rich small nuclear ribonucleoprotein particles, the U1, U2, U4, U5 and U6 snRNPs. An early and significant regulatory step in assembly of the spliceosome and splice‐site selection is U1 snRNP recognition of the junction between the 5′ exon and intron, a 5′ splice‐site. This pivotal event will act to: (1) trigger spliceosome assembly; (2) demarcate a 5′ splice‐site; and (3) contribute to spliceosome fidelity. In metazoans, U1 snRNP 5′ splice‐site selection is aided by transiently associated proteins known as alternative splicing factors that function to guide U1 snRNP to specific 5′ splice‐site(s). In contrast, Saccharomyces cerevisae has very few protein coding genes with more than a single intron. S. cerevisae U1 snRNP is an 18‐subunit, ~800 kDa assembly that has as integral subunits proteins that are alternative splicing factors in higher eukaryotes. We report the structure of a native S. cerevisae U1 snRNP determined by electron microscopy and present a working model that provides insight into the function of its protein subunits.Funding provided by the National Science Foundation; Howard Hughes Medical Institute

Discovery of a Splicing Regulator Required for Cell Cycle Progression

In the G1 phase of the cell division cycle, eukaryotic cells prepare many of the resources necessary for a new round of growth including renewal of the transcriptional and protein synthetic capacities and building the machinery for chromosome replication. The function of G1 has an early evolutionary origin and is preserved in single and multicellular organisms, although the regulatory mechanisms conducting G1 specific functions are only understood in a few model eukaryotes. Here we describe a new G1 mutant from an ancient family of apicomplexan protozoans. Toxoplasma gondii temperature-sensitive mutant 12-109C6 conditionally arrests in the G1 phase due to a single point mutation in a novel protein containing a single RNA-recognition-motif (TgRRM1). The resulting tyrosine to asparagine amino acid change in TgRRM1 causes severe temperature instability that generates an effective null phenotype for this protein when the mutant is shifted to the restrictive temperature. Orthologs of TgRRM1 are widely conserved in diverse eukaryote lineages, and the human counterpart (RBM42) can functionally replace the missing Toxoplasma factor. Transcriptome studies demonstrate that gene expression is downregulated in the mutant at the restrictive temperature due to a severe defect in splicing that affects both cell cycle and constitutively expressed mRNAs. The interaction of TgRRM1 with factors of the tri-SNP complex (U4/U6 & U5 snRNPs) indicate this factor may be required to assemble an active spliceosome. Thus, the TgRRM1 family of proteins is an unrecognized and evolutionarily conserved class of splicing regulators. This study demonstrates investigations into diverse unicellular eukaryotes, like the Apicomplexa, have the potential to yield new insights into important mechanisms conserved across modern eukaryotic kingdoms.

A Gene Therapeutic Approach to Correct Splice Defects with Modified U1 and U6 snRNPs

Splicing is an essential cellular process to generate mature transcripts from pre-mRNA. It requires the splice factor U1 small nuclear ribonucleoprotein (U1), which promotes exon recognition by base-pairing interaction with the splice donor site (SD). After U1 dissociation, exon recognition is maintained by U6 small nuclear ribonucleoproteins (U6). It has been shown that SD mutations lower the binding affinity of U1 and cause splice defects in about 10% of patients with monogenetic diseases. U1 isoforms specifically designed to bind the mutated SD with increased affinity can correct these splice defects. We investigated the applicability of this gene therapeutic approach for different mutated SD positions. A minigene-based splicing assay was established to study a typical SD derived from the gene BBS1. We found that mutations at seven SD positions caused splice defects. In four cases, mutation-adapted U1 isoforms completely corrected these splice defects. Partial correction was found for splice defects induced by the mutation at SD position +5. The limited therapeutic efficacy at this position was alleviated by applying a combined treatment with mutation-adapted U1 and U6. The sequence complementarity between U6 and three SD positions (+4, +5,and +6) was relevant for the outcome of the therapy. Between 30 and 100% of the normal transcripts can be restored. The treatment significantly decreased both exon skipping and intron retention. Massive missplicing of off-target transcripts was not detected. Our study helps to assess the therapeutic efficacy of mutation-adapted U snRNAs in gene therapy and illustrates their strong potential to correct splice defects, which cause many different inherited conditions.

The transition in spliceosome assembly from complex E to complex A purges surplus U1 snRNPs from alternative splice sites

Spliceosomes are assembled in stages. The first stage forms complex E, which is characterized by the presence of U1 snRNPs base-paired to the 5′ splice site, components recognizing the 3′ splice site and proteins thought to connect them. The splice sites are held in close proximity and the pre-mRNA is committed to splicing. Despite this, the sites for splicing appear not to be fixed until the next complex (A) forms. We have investigated the reasons why 5′ splice sites are not fixed in complex E, using single molecule methods to determine the stoichiometry of U1 snRNPs bound to pre-mRNA with one or two strong 5′ splice sites. In complex E most transcripts with two alternative 5′ splice sites were bound by two U1 snRNPs. However, the surplus U1 snRNPs were displaced during complex A formation in an ATP-dependent process requiring an intact 3′ splice site. This process leaves only one U1 snRNP per complex A, regardless of the number of potential sites. We propose a mechanism for selection of the 5′ splice site. Our results show that constitutive splicing components need not be present in a fixed stoichiometry in a splicing complex.

A U1-U2 snRNP Interaction Network during Intron Definition

The assembly of prespliceosomes is responsible for selection of intron sites for splicing. U1 and U2 snRNPs recognize 5' splice sites and branch sites, respectively; although there is information regarding the composition of these complexes, little is known about interaction among the components or between the two snRNPs. Here we describe the protein network of interactions linking U1 and U2 snRNPs with the ATPase Prp5, important for branch site recognition and fidelity during the first steps of the reaction, using fission yeast Schizosaccharomyces pombe. The U1 snRNP core protein U1A binds to a novel SR-like protein, Rsd1, which has homologs implicated in transcription. Rsd1 also contacts S. pombe Prp5 (SpPrp5), mediated by SR-like domains in both proteins. SpPrp5 then contacts U2 snRNP through SF3b, mediated by a conserved DPLD motif in Prp5. We show that mutations in this motif have consequences not only in vitro (defects in prespliceosome formation) but also in vivo, yielding intron retention and exon skipping defects in fission yeast and altered intron recognition in budding yeast Saccharomyces cerevisiae, indicating that the U1-U2 network provides critical, evolutionarily conserved contacts during intron definition.

Turning on the Spliceosome

The spliceosome is the complex macromolecular machine responsible for removing introns from pre-mRNAs. The processes of spliceosome assembly and activation rely on the coordinated interactions of many dozens of spliceosome components to identify splice sites in a pre-mRNA, build a spliceosome, and activate the spliceosome for catalysis by formation of an active site prior to transesterification. The activation step itself likely involves many intermediates. It results in loss of the U1 and U4 snRNPs from the spliceosome, removal of SF3 from the branchsite, and several conformational rearrangements of the snRNAs and pre-mRNA prior to lariat formation. These assembly and activation events are best studied using endogenous spliceosome components found in whole or nuclear cell extracts. We recently demonstrated that a single molecule technique (CoSMoS: Co-localization Single Molecule Spectroscopy) in combination with yeast genetic engineering and chemical biology provides a powerful method for studying spliceosome assembly in S. cerevisiae whole cell lysate (Hoskins et al., Science, v331, pg. 1289-95 (2011)). That study provided significant novel insight into the kinetics of the assembly reaction. We are now extending these results to spliceosome activation. By monitoring the relative association and dissociation kinetics of the U1, U4, U5, NTC, and SF3b spliceosome components on single pre-mRNAs, we are able to definitively order the snRNP association and dissociation events involved in spliceosome activation.

Functional mammalian spliceosomal complex E contains SMN complex proteins in addition to U1 and U2 snRNPs

Spliceosomes remove introns from primary gene transcripts. They assemble de novo on each intron through a series of steps that involve the incorporation of five snRNP particles and multiple non-snRNP proteins. In mammals, all the intermediate complexes have been characterized on one transcript (MINX), with the exception of the very first, complex E. We have purified this complex by two independent procedures using antibodies to either U1-A or PRPF40A proteins, which are known to associate at an early stage of assembly. We demonstrate that the purified complexes are functional in splicing using commitment assays. These complexes contain components expected to be in the E complex and a number of previously unrecognized factors, including survival of motor neurons (SMN) and proteins of the SMN-associated complex. Depletion of the SMN complex proteins from nuclear extracts inhibits formation of the E complex and causes non-productive complexes to accumulate. This suggests that the SMN complex stabilizes the association of U1 and U2 snRNPs with pre-mRNA. In addition, the antibody to PRPF40A precipitated U2 snRNPs from nuclear extracts, indicating that PRPF40A associates with U2 snRNPs.

Composition of yeast snRNPs and snoRNPs in the absence of trimethylguanosine caps reveals nuclear cap binding protein as a gained U1 component implicated in the cold-sensitivity of tgs1Δ cells

Small nuclear and nucleolar RNAs that program pre-mRNA splicing and rRNA processing have a signature 5′-trimethylguanosine (TMG) cap. Whereas the mechanism of TMG synthesis by Tgs1 methyltransferase has been elucidated, we know little about whether or how RNP biogenesis, structure and function are perturbed when TMG caps are missing. Here, we analyzed RNPs isolated by tandem-affinity purification from TGS1 and tgs1Δ yeast strains. The protein and U-RNA contents of total SmB-containing RNPs were similar. Finer analysis revealed stoichiometric association of the nuclear cap-binding protein (CBP) subunits Sto1 and Cbc2 with otherwise intact Mud1- and Nam8-containing U1 snRNPs from tgs1Δ cells. CBP was not comparably enriched in Lea1-containing U2 snRNPs from tgs1Δ cells. Moreover, CBP was not associated with mature Nop58-containing C/D snoRNPs or mature Cbf5- and Gar1-containing H/ACA snoRNPs from tgs1Δ cells. The protein composition and association of C/D snoRNPs with the small subunit (SSU) processosome were not grossly affected by absence of TMG caps, nor was the composition of H/ACA snoRNPs. The cold-sensitive (cs) growth defect of tgs1Δ yeast cells could be suppressed by mutating the cap-binding pocket of Cbc2, suggesting that ectopic CBP binding to the exposed U1 m7G cap in tgs1Δ cells (not lack of TMG caps per se) underlies the cs phenotype.

A mutation in a rare type of intron in a sodium-channel gene results in aberrant splicing and causes myotonia

Many mutations in the skeletal-muscle sodium-channel gene SCN4A have been associated with myotonia and/or periodic paralysis, but so far all of these mutations are located in exons. We found a patient with myotonia caused by a deletion/insertion located in intron 21 of SCN4A, which is an AT-AC type II intron. This is a rare class of introns that, despite having AT-AC boundaries, are spliced by the major or U2-type spliceosome. The patient's skeletal muscle expressed aberrantly spliced SCN4A mRNA isoforms generated by activation of cryptic splice sites. In addition, genetic suppression experiments using an SCN4A minigene showed that the mutant 5' splice site has impaired binding to the U1 and U6 snRNPs, which are the cognate factors for recognition of U2-type 5' splice sites. One of the aberrantly spliced isoforms encodes a channel with a 35-amino acid insertion in the cytoplasmic loop between domains III and IV of Nav1.4. The mutant channel exhibited a marked disruption of fast inactivation, and a simulation in silico showed that the channel defect is consistent with the patient's myotonic symptoms. This is the first report of a disease-associated mutation in an AT-AC type II intron, and also the first intronic mutation in a voltage-gated ion channel gene showing a gain-of-function defect.

Structure of the spliceosomal U4 snRNP core domain and its implication for snRNP biogenesis.

The spliceosome is a dynamic macromolecular machine that assembles on pre-mRNA substrates and catalyses the excision of non-coding intervening sequences (introns)1-3. Four of the five major components of the spliceosome, U1, U2, U4 and U5 snRNPs, contain seven Sm proteins (SmB/B’, SmD1, SmD2, SmD3, SmE, SmF and SmG) in common4,5. Following export of the U1, U2, U4 and U5 snRNAs to the cytoplasm6,7, the seven Sm proteins chaperoned by the survival of motor neurons (SMN) complex assemble around a single-stranded, U-rich sequence called the Sm site in each snRNA, to form the core domain of the respective snRNP particle8,9. Core domain formation is a prerequisite for re-import into the nucleus10, where these snRNPs mature via addition of their particle-specific proteins. Here we present a crystal structure of the U4 snRNP core domain at 3.6 Å resolution, detailing how the Sm site heptad (AUUUUUG) binds inside the central hole of the heptameric ring of Sm proteins, interacting one-to-one with SmE-SmG-SmD3-SmB-SmD1-SmD2-SmF. An irregular backbone conformation of the Sm site sequence combined with the asymmetric structure of the heteromeric protein ring allows each base to interact in a distinct manner with four key residues at equivalent positions in the L3 and L5 loops of the Sm fold. A comparison of this structure with the U1 snRNP at 5.5 Å resolution11,12 reveals snRNA-dependent structural changes outside the Sm fold, which may facilitate the binding of particle-specific proteins that is crucial to biogenesis of spliceosomal snRNPs.

RNA-Seq analysis in mutant zebrafish reveals role of U1C protein in alternative splicing regulation

Precise 5' splice-site recognition is essential for both constitutive and regulated pre-mRNA splicing. The U1 small nuclear ribonucleoprotein particle (snRNP)-specific protein U1C is involved in this first step of spliceosome assembly and important for stabilizing early splicing complexes. We used an embryonically lethal U1C mutant zebrafish, hi1371, to investigate the potential genomewide role of U1C for splicing regulation. U1C mutant embryos contain overall stable, but U1C-deficient U1 snRNPs. Surprisingly, genomewide RNA-Seq analysis of mutant versus wild-type embryos revealed a large set of specific target genes that changed their alternative splicing patterns in the absence of U1C. Injection of ZfU1C cRNA into mutant embryos and in vivo splicing experiments in HeLa cells after siRNA-mediated U1C knockdown confirmed the U1C dependency and specificity, as well as the functional conservation of the effects observed. In addition, sequence motif analysis of the U1C-dependent 5' splice sites uncovered an association with downstream intronic U-rich elements. In sum, our findings provide evidence for a new role of a general snRNP protein, U1C, as a mediator of alternative splicing regulation.

Crystal Structure of a Ten-Subunit Human Spliceosomal U1 snRNP at 5.5 Å Resolution

Most eukaryotic protein-coding genes contain non-coding regions (introns) that separate those coding for protein (exons). The introns must be excised and exons spliced together from the precursor-mRNA transcript of such genes. This process (RNA splicing) is catalyzed by the spliceosome, integral to which are five RNA-protein complexes (U1, U2, U4, U5 and U6 snRNPs). A first step in RNA splicing, functioning to initiate spliceosome assembly, involves recognition of the junction between the 5′-exon and intron (5′-splice site) by U1 snRNP.

Excessive RNA Splicing and Inhibition of HIV-1 Replication Induced by Modified U1 Small Nuclear RNAs

HIV-1 RNA undergoes a complex splicing process whereby over 40 different mRNA species are produced by alternative splicing. In addition, approximately half of the RNA transcripts remain unspliced and either are used to encode Gag and Gag-Pol proteins or are packaged into virions as genomic RNA. It has previously been shown that HIV-1 splicing is regulated by cis elements that bind to cellular factors. These factors either enhance or repress definition of exons that are flanked by the HIV-1 3' splice sites. Here we report that expression of modified U1 snRNPs with increased affinity to HIV-1 downstream 5' splice sites and to sequences within the first tat coding exon act to selectively increase splicing at the upstream 3' splice sites in cotransfected 293T cells. This results in a decrease of unspliced viral RNA levels and an approximately 10-fold decrease in virus production. In addition, excessive splicing of viral RNA is concomitant with a striking reduction in the relative amounts of Gag processing intermediates and products. We also show that T cell lines expressing modified U1 snRNAs exhibit reduced HIV-1 replication. Our results suggest that induction of excessive HIV-1 RNA splicing may be a novel strategy to inhibit virus replication in human patients.

The differential interaction of snRNPs with pre-mRNA reveals splicing kinetics in living cells

Precursor messenger RNA (pre-mRNA) splicing is catalyzed by the spliceosome, a large ribonucleoprotein (RNP) complex composed of five small nuclear RNP particles (snRNPs) and additional proteins. Using live cell imaging of GFP-tagged snRNP components expressed at endogenous levels, we examined how the spliceosome assembles in vivo. A comprehensive analysis of snRNP dynamics in the cell nucleus enabled us to determine snRNP diffusion throughout the nucleoplasm as well as the interaction rates of individual snRNPs with pre-mRNA. Core components of the spliceosome, U2 and U5 snRNPs, associated with pre-mRNA for 15-30 s, indicating that splicing is accomplished within this time period. Additionally, binding of U1 and U4/U6 snRNPs with pre-mRNA occurred within seconds, indicating that the interaction of individual snRNPs with pre-mRNA is distinct. These results are consistent with the predictions of the step-wise model of spliceosome assembly and provide an estimate on the rate of splicing in human cells.

U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation.

SummaryIn higher eukaryotes, U1 snRNP forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6, however its abundance far exceeds that of the other snRNPs. Here, we used antisense morpholino oligonucleotide (AMO) to U1 snRNA for functional U1 snRNP knockdown in HeLa cells and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. Remarkably, in addition to inhibiting splicing, U1 snRNP knockdown caused premature cleavage and polyadenylation (PCPA) in numerous pre-mRNAs at cryptic polyadenylation signals (PASs), frequently in introns near (< 5 kb) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA AMO or the U2 snRNP inactivating drug, spliceostatin A, unless U1 AMO was also included. We further show that U1 snRNA-pre-mRNA base pairing was required to suppress PCPA from nearby cryptic PASs located in introns. These findings reveal a critical splicing-independent function for U1 snRNP in protecting the transcriptome, which we propose explains its overabundance.

Coevolution of Drosophila snf Protein and Its snRNA Targets

SNF is a protein that is found in the U1 and U2 snRNPs (small nuclear ribonucleoproteins) of Drosophila. Its mammalian counterparts are two homologous proteins, U1A and U2B''. In vivo, these proteins segregate to the U1 and U2 snRNPs, respectively, where they bind distinct RNA hairpins. The RNA binding properties and mechanism of U1A have been studied extensively, but much less is known about SNF and U2B'' binding to their RNA targets. By comparing thermodynamic aspects of SNF-RNA interactions with those of U1A-RNA interactions, we find that SNF binds its RNA targets in a manner that is distinct from that of U1A. In vitro, SNF is able to bind both Drosophila U1 stem-loop II and U2 stem-loop IV with high affinity, although it binds stem-loop II more tightly than it binds stem-loop IV. Intriguingly, SNF is unable to bind human U2 stem-loop IV, which suggests that both the protein and RNAs have coevolved to interact with each other such that a single protein can bind RNAs that are more commonly bound by two distinct proteins.

Specific splicing defects in S. pombe carrying a degron allele of the Survival of Motor Neuron gene

Spinal muscular atrophy results from deletions or mutations in the survival of motor neuron (SMN1) gene. The SMN protein has an essential role in the biogenesis of spliceosomal snRNPs, but the link between a defect in this process and specific splicing inhibition of pre-mRNAs has not been established. In this study, we report the construction of a temperature-degron (td) allele of the Schizosaccharomyces pombe SMN protein and show that its depletion at 37 degrees C affects splicing and formation of U1, U2, U4 and U5 snRNPs, but not of U6 and U3 ribonucleoproteins. The function of the tdSMN allele in snRNP assembly is already perturbed at 25 degrees C, suggesting a deleterious effect of the tag at this temperature. Using a genome-wide approach, we report that introns react unequally to lower levels of snRNPs in tdSMN cells and that increasing the length of the polypyrimidine tract can improve the splicing efficiency of some, but not all, affected introns. Altogether, our results suggest that the defects observed in tdSMN fission yeast cells mimic splicing deficits observed in SMN-deficient metazoan cells.

Exon Definition Complexes Contain the Tri-snRNP and Can Be Directly Converted into B-like Precatalytic Splicing Complexes

The first step in splicing of pre-mRNAs with long introns is exon definition, where U1 and U2 snRNPs bind at opposite ends of an exon. After exon definition, these snRNPs must form a complex across the upstream intron to allow splicing catalysis. Exon definition and conversion of cross-exon to cross-intron spliceosomal complexes are poorly understood. Here we demonstrate that, in addition to U1 and U2 snRNPs, cross-exon complexes contain U4, U5, and U6 (which form the tri-snRNP). Tri-snRNP docking involves the formation of U2/U6 helix II. This interaction is stabilized by a 5' splice site (SS)-containing oligonucleotide, which can bind the tri-snRNP and convert the cross-exon complex into a cross-intron, B-like complex. Our data suggest that the switch from cross-exon to cross-intron complexes can occur directly when an exon-bound tri-snRNP interacts with an upstream 5'SS, without prior formation of a cross-intron A complex, revealing an alternative spliceosome assembly pathway.

Establishment of an in vitro trans-splicing system in Trypanosoma brucei that requires endogenous spliced leader RNA

In trypanosomes a 39 nucleotide exon, the spliced leader (SL) is donated to all mRNAs from a small RNA, the SL RNA, by trans-splicing. Since the discovery of trans-splicing in trypanosomes two decades ago, numerous attempts failed to reconstitute the reaction in vitro. In this study, a crude whole-cell extract utilizing the endogenous SL RNA and synthetic tubulin pre-mRNA were used to reconstitute the trans-splicing reaction. An RNase protection assay was used to detect the trans-spliced product. The reaction was optimized and shown to depend on ATP and intact U2 and U6 snRNPs. Mutations introduced at the polypyrimidine tract and the AG splice site reduced the reaction efficiency. To simplify the assay, RT–PCR and quantitative real-time PCR assays were established. The system was used to examine the structural requirements for SL RNA as a substrate in the reaction. Interestingly, synthetic SL RNA assembled poorly to its cognate particle and was not utilized in the reaction. However, SL RNA synthesized in cells lacking Sm proteins, which is defective in cap-4 modification, was active in the reaction. This study is the first step towards further elucidating the mechanism of trans-splicing, an essential reaction which determines the trypanosome transcriptome.

Interpreting a Low Resolution Map of Human U1 snRNP Using Anomalous Scatterers

SummaryWe recently determined the crystal structure of the functional core of human U1 snRNP, consisting of nine proteins and one RNA, based on a 5.5 Å resolution electron density map. At 5–7 Å resolution, α helices and β sheets appear as rods and slabs, respectively, hence it is not possible to determine protein fold de novo. Using inverse beam geometry, accurate anomalous signals were obtained from weakly diffracting and radiation sensitive P1 crystals. We were able to locate anomalous scatterers with positional errors below 2 Å. This enabled us not only to place protein domains of known structure accurately into the map but also to trace an extended polypeptide chain, of previously undetermined structure, using selenomethionine derivatives of single methionine mutants spaced along the sequence. This method of Se-Met scanning, in combination with structure prediction, is a powerful tool for building a protein of unknown fold into a low resolution electron density map.