A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly

Pre-mRNA splicing complex assembly is mediated by two specific pre-mRNA-snRNP interactions: U1 snRNP binds to the 5' splice site and U2 snRNP binds to the branch point. Here we show that unlike a purified U1 snRNP, which can bind to a 5' splice site, a partially purified U2 snRNP cannot interact with its target pre-mRNA sequence. We identify a previously uncharacterized activity, U2AF, that is required for the U2 snRNP-branch point interaction and splicing complex formation. Using RNA substrate exclusion and competition assays, we demonstrate that U2AF binds to the 3' splice site region prior to the U2 snRNP-branch point interaction. This provides an explanation for the necessity of the 3' splice site region in U2 snRNP binding and, hence, the first step of splicing.     

Structure and assembly of the SF3a splicing factor complex of U2 snRNP

SF3a is an evolutionarily conserved heterotrimeric complex essential for pre-mRNA splicing. It functions in spliceosome assembly within the mature U2 snRNP (small nuclear ribonucleoprotein particle), and its displacement from the spliceosome initiates the first step of the splicing reaction. We have identified a core domain of the yeast SF3a complex required for complex assembly and determined its crystal structure. The structure shows a bifurcated assembly of three subunits, Prp9, Prp11 and Prp21, with Prp9 interacting with Prp21 via a bidentate-binding mode, and Prp21 wrapping around Prp11. Structure-guided biochemical analysis also shows that Prp9 harbours a major binding site for stem-loop IIa of U2 snRNA. These findings provide mechanistic insights into the assembly of U2 snRNP.     

Domains of U4 and U6 snRNAs required for snRNP assembly and splicing complementation in Xenopus oocytes.

Structure-function relationships in the vertebrate U4-U6 snRNP have been analysed by assaying the ability of mutant RNAs to form U4-U6 snRNPs and to function in splicing complementation in Xenopus oocytes. The mutants define three categories of domain within the RNAs. First, domains which are not essential for splicing. These include regions of U6 which have previously been implicated in the capping and transport to the nucleus of U6 RNA as well as, less surprisingly, regions of U4 and U6 which have been poorly conserved in evolution. Second, domains whose mutation reduces U4-U6 snRNP assembly or stability. This group includes mutations in both the proposed U4-U6 interaction domain, and also, in the case of U6, in a highly conserve sequence flanking stem I of the interaction domain. These mutants are all defective in splicing. Third, regions not required for U4-U6 assembly, but required for splicing complementation. This category defines domains which are likely to be required for specific contacts with other components of the splicing machinery. Combinations of mutants in the U4 and U6 interaction domain are used to show that there are not only requirements for base complementarity but also for specific sequences in these regions.     

Modifications of U2 snRNA are required for snRNP assembly and pre-mRNA splicing.

Among the spliceosomal snRNAs, U2 has the most extensive modifications, including a 5' trimethyl guanosine (TMG) cap, ten 2'-O-methylated residues and 13 pseudouridines. At short times after injection, cellularly derived (modified) U2 but not synthetic (unmodified) U2 rescues splicing in Xenopus oocytes depleted of endogenous U2 by RNase H targeting. After prolonged reconstitution, synthetic U2 regenerates splicing activity; a correlation between the extent of U2 modification and U2 function in splicing is observed. Moreover, 5-fluorouridine-containing U2 RNA, a potent inhibitor of U2 pseudouridylation, specifically abolishes rescue by synthetic U2, while rescue by cellularly derived U2 is not affected. By creating chimeric U2 molecules in which some sequences are from cellularly derived U2 and others are from in vitro transcribed U2, we demonstrate that the functionally important modifications reside within the 27 nucleotides at the 5' end of U2. We further show that 2'-O-methylation and pseudouridylation activities reside in the nucleus and that the 5' TMG cap is not necessary for internal modification but is crucial for splicing activity. Native gel analysis reveals that unmodified U2 is not incorporated into the spliceosome. Examination of the U2 protein profile and glycerol-gradient analysis argue that U2 modifications directly contribute to conversion of the 12S to the 17S U2 snRNP particle, which is essential for spliceosome assembly.     

Solution structures of the SURP domains and the subunit-assembly mechanism within the splicing factor SF3a complex in 17S U2 snRNP

The SF3a complex, consisting of SF3a60, SF3a66, and SF3a120, in 17S U2 snRNP is crucial to spliceosomal assembly. SF3a120 contains two tandem SURP domains (SURP1 and SURP2), and SURP2 is responsible for binding to SF3a60. We found that the SURP2 fragment forms a stable complex with an SF3a60 fragment (residues 71-107) and solved its structure by NMR spectroscopy. SURP2 exhibits a fold of the alpha1-alpha2-3(10)-alpha3 topology, and the SF3a60 fragment forms an amphipathic alpha helix intimately contacting alpha1 of SURP2. We also solved the SURP1 structure, which has the same fold as SURP2. The protein-binding interface of SURP2 is quite similar to the corresponding surface of SURP1, except for two amino acid residues. One of them, Leu169, is characteristic of SF3a120 SURP2 among SURP domains. Mutagenesis showed that this single Leu residue is the critical determinant for complex formation, which reveals the protein recognition mechanism in the subunit assembly.     

Rds3p is required for stable U2 snRNP recruitment to the splicing apparatus

Rds3p is a well-conserved 12-kDa protein with five CxxC zinc fingers that has been implicated in the activation of certain drug transport genes and in the pre-mRNA splicing pathway. Here we show that Rds3p resides in the yeast spliceosome and is essential for splicing in vitro. Rds3p purified from yeast stably associates with at least five U2 snRNP proteins, Cus1p, Hsh49p, Hsh155p, Rse1p, and Ist3p/Snu17p, and with the Yra1p RNA export factor. A mutation upstream of the first Rds3p zinc finger causes the conditional release of the putative branchpoint nucleotide binding protein, Ist3p/Snu17p, and weakens Rse1p interaction with the Rds3p complex. The resultant U2 snRNP particle migrates exceptionally slowly in polyacrylamide gels, suggestive of a disorganized structure. U2 snRNPs depleted of Rds3p fail to form stable prespliceosomes, although U2 snRNA stability is not affected. Metabolic depletion of Yra1p blocks cell growth but not splicing, suggesting that Yra1p association with Rds3p relates to Yra1p's role in RNA trafficking. Together these data establish Rds3p as an essential component of the U2 snRNP SF3b complex and suggest a new link between the nuclear processes of pre-mRNA splicing and RNA export.     

Characterization of novel SF3b and 17S U2 snRNP proteins,including a human Prp5p homologue and an SF3b DEAD-box protein

Mass spectrometry was used to identify novel proteins associated with the human 17S U2 snRNP and one of its stable subunits, SF3b. Several additional proteins were identified, demonstrating that 17S U2 snRNPs are significantly more complex than previously thought. Two of the newly identified proteins, namely the DEAD-box proteins SF3b125 and hPrp5 (a homologue of Saccharomyces cerevisiae Prp5p) were characterized further. Immunodepletion experiments with HeLa nuclear extract indicated that hPrp5p plays an important role in pre-mRNA splicing, acting during or prior to prespliceosome assembly. The SF3b-associated protein SF3b125 dissociates at the time of 17S U2 formation, raising the interesting possibility that it might facilitate the assembly of the 17S U2 snRNP. Finally, immunofluorescence/FISH studies revealed a differential subnuclear distribution of U2 snRNA, hPrp5p and SF3b125, which were enriched in Cajal bodies, versus SF3b155 and SF3a120, which were not; a model for 17S U2 snRNP assembly based on these findings is presented. Taken together, these studies provide new insight into the composition of the 17S U2 snRNP and the potential function of several of its proteins.     

Discrete domains of human U6 snRNA required for the assembly of U4/U6 snRNP and splicing complexes.

U6 snRNA sequences required for assembly of U4/U6 snRNP and splicing complexes were determined by in vitro reconstitution of snRNPs. Both mutagenesis and chemical modification/interference assays identify a U6 snRNA domain required for U4/U6 snRNP formation. The results support the existence of a U4/U6 snRNA interaction domain previously proposed on the basis of phylogenetic evidence. In addition, two short U6 snRNA regions flanking the U4/U6 interaction domain are essential to assemble the U4/U6 snRNP into splicing complexes. These two regions may represent binding sites for splicing factors or may facilitate the formation of an alternative U6 snRNA secondary structure during spliceosome assembly.     

A novel yeast U2 snRNP protein, Snu17p, is required for the first catalytic step of splicing and for progression of spliceosome assembly

We have isolated and microsequenced Snu17p, a novel yeast protein with a predicted molecular mass of 17 kDa that contains an RNA recognition motif. We demonstrate that Snu17p binds specifically to the U2 small nuclear ribonucleoprotein (snRNP) and that it is part of the spliceosome, since the pre-mRNA and the lariat-exon 2 are specifically coprecipitated with Snu17p. Although the SNU17 gene is not essential, its knockout leads to a slow-growth phenotype and to a pre-mRNA splicing defect in vivo. In addition, the first step of splicing is dramatically decreased in extracts prepared from the snu17 deletion (snu17Delta) mutant. This defect is efficiently reversed by the addition of recombinant Snu17p. To investigate the step of spliceosome assembly at which Snu17p acts, we have used nondenaturing gel electrophoresis. In Snu17p-deficient extracts, the spliceosome runs as a single slowly migrating complex. In wild-type extracts, usually at least two distinct complexes are observed: the prespliceosome, or B complex, containing the U2 but not the U1 snRNP, and the catalytically active spliceosome, or A complex, containing the U2, U6, and U5 snRNPs. Northern blot analysis and affinity purification of the snu17Delta spliceosome showed that it contains the U1, U2, U6, U5, and U4 snRNPs. The unexpected stabilization of the U1 snRNP and the lack of dissociation of the U4 snRNP suggest that loss of Snu17p inhibits the progression of spliceosome assembly prior to U1 snRNP release and after [U4/U6.U5] tri-snRNP addition.     

Partial purification of the yeast U2 snRNP reveals a novel yeast pre-mRNA splicing factor required for pre-spliceosome assembly.

We have partially purified the U2 snRNP of Saccharomyces cerevisiae. Identification of some proteins consistently found in the purified fractions by nanoelectrospray mass spectrometry indicated the presence of a novel splicing factor named Rse1p. The RSE1 gene is essential and codes for a 148.2 kDa protein. We demonstrated that Rse1p associates specifically with U2 snRNA at low salt concentrations. In addition, we showed that Rse1p is a component of the pre-spliceosome. Depletion of Rse1p and analysis of a conditional mutant indicated that Rse1p was required for efficient splicing in vivo. In vitro Rse1p is required for the formation of pre-spliceosomes. Database searches revealed that Rse1p is conserved in humans and that it belongs to a large protein family that includes polyadenylation factors and DNA repair proteins. The characteristics of Rse1p suggest that its human homologue could be a subunit of the SF3 splicing factor.     

Conserved domains of human U4 snRNA required for snRNP and spliceosome assembly.

U4 snRNA is phylogenetically highly conserved and organized in several domains. To determine the function of each of the domains of human U4 snRNA in the multi-step process of snRNP and spliceosome assembly, we used reconstitution procedures in combination with snRNA mutagenesis. The highly conserved 5' terminal domain of U4 snRNA consists of the stem I and stem II regions that have been proposed to base pair with U6 snRNA, and the 5' stem-loop structure. We found that each of these structural elements is essential for spliceosome assembly. However, only the stem II region is required for U4-U6 interaction, and none of these elements for Sm protein binding. In contrast, the 3' terminal domain of U4 snRNA containing the Sm binding site is dispensable for both U4-U6 interaction and spliceosome assembly. Our results support an organization of the U4 snRNP into multiple functional domains, each of which acts at distinct stages of snRNP and spliceosome assembly.     

CUGBP2 directly interacts with U2 17S snRNP components and promotes U2 snRNA binding to cardiac troponin T pre-mRNA

CUGBP2 (ETR-3/NAPOR/BRUNOL3) promotes inclusion of cardiac troponin T (cTNT) exon 5 via binding between positions 21 and 74 of the downstream intron. The molecular mechanism by which CUGBP2 activates cTNT exon 5 inclusion is unknown. Our results suggest that CUGBP2 promotes exon inclusion by a novel mechanism in which CUGBP2 directly interacts with components of the activated U2 snRNP and enhances binding of U2 snRNP to the branch site located upstream of the exon. Using an in vitro splicing assay, we show that recombinant CUGBP2 enhances complex A formation of a cTNT pre-mRNA. Enhanced complex A assembly requires both the upstream and downstream introns consistent with dual requirements for the downstream CUGBP2-binding site and an upstream branch site for U2 snRNP binding. We also show that CUGBP2 enhances binding of U2 snRNA to the cTNT pre-mRNA consistent with enhanced complex A assembly. Purification of CUGBP2-interacting proteins using tandem affinity purification leads to the demonstration that the core 17S U2 snRNP components, SF3b145 and SF3b49 bind directly to CUGBP2. We conclude that CUGBP2 activates exon inclusion by forming direct interactions with components of the 17S snRNP complex and recruits and/or stabilizes binding of U2 snRNP.     

Structural basis for the interaction between the first SURP domain of the SF3A1 subunit in U2 snRNP and the human splicing factor SF1

SURP domains are exclusively found in splicing‐related proteins in all eukaryotes. SF3A1, a component of the U2 snRNP, has two tandem SURP domains, SURP1, and SURP2. SURP2 is permanently associated with a specific short region of SF3A3 within the SF3A protein complex whereas, SURP1 binds to the splicing factor SF1 for recruitment of U2 snRNP to the early spliceosomal complex, from which SF1 is dissociated during complex conversion. Here, we determined the solution structure of the complex of SURP1 and the human SF1 fragment using nuclear magnetic resonance (NMR) methods. SURP1 adopts the canonical topology of α1–α2–3₁₀–α3, in which α1 and α2 are connected by a single glycine residue in a particular backbone conformation, allowing the two α‐helices to be fixed at an acute angle. A hydrophobic patch, which is part of the characteristic surface formed by α1 and α2, specifically contacts a hydrophobic cluster on a 16‐residue α‐helix of the SF1 fragment. Furthermore, whereas only hydrophobic interactions occurred between SURP2 and the SF3A3 fragment, several salt bridges and hydrogen bonds were found between the residues of SURP1 and the SF1 fragment. This finding was confirmed through mutational studies using bio‐layer interferometry. The study also revealed that the dissociation constant between SURP1 and the SF1 fragment peptide was approximately 20 μM, indicating a weak or transient interaction. Collectively, these results indicate that the interplay between U2 snRNP and SF1 involves a transient interaction of SURP1, and this transient interaction appears to be common to most SURP domains, except for SURP2.     

A novel genetic screen for snRNP assembly factors in yeast identifies a conserved protein, Sad1p, also required for pre-mRNA splicing

The assembly pathway of spliceosomal snRNPs in yeast is poorly understood. We devised a screen to identify mutations blocking the assembly of newly synthesized U4 snRNA into a functional snRNP. Fifteen mutant strains failing either to accumulate the newly synthesized U4 snRNA or to assemble a U4/U6 particle were identified and categorized into 13 complementation groups. Thirteen previously identified splicing-defective prp mutants were also assayed for U4 snRNP assembly defects. Mutations in the U4/U6 snRNP components Prp3p, Prp4p, and Prp24p led to disassembly of the U4/U6 snRNP particle and degradation of the U6 snRNA, while prp17-1 and prp19-1 strains accumulated free U4 and U6 snRNA. A detailed analysis of a newly identified mutant, the sad1-1 mutant, is presented. In addition to having the snRNP assembly defect, the sad1-1 mutant is severely impaired in splicing at the restrictive temperature: the RP29 pre-mRNA strongly accumulates and splicing-dependent production of beta-galactosidase from reporter constructs is abolished, while extracts prepared from sad1-1 strains fail to splice pre-mRNA substrates in vitro. The sad1-1 mutant is the only splicing-defective mutant analyzed whose mutation preferentially affects assembly of newly synthesized U4 snRNA into the U4/U6 particle. SAD1 encodes a novel protein of 52 kDa which is essential for cell viability. Sad1p localizes to the nucleus and is not stably associated with any of the U snRNAs. Sad1p contains a putative zinc finger and is phylogenetically highly conserved, with homologues identified in human, Caenorhabditis elegans, Arabidospis, and Drosophila.     

The human U4/U6 snRNP contains 60 and 90kD proteins that are structurally homologous to the yeast splicing factors Prp4p and Prp3p.

Immunoaffinity-purified human 25S [U4/U6.U5] tri-snRNPs harbor a set of polypeptides, termed the tri-snRNP proteins, that are not present in Mono Q-purified 20S U5 snRNPs or 10S U4/U6 snRNPs and that are important for tri-snRNP complex formation (Behrens SE, Lührmann R, 1991, Genes & Dev 5:1439-1452). Biochemical and immunological characterization of HeLa [U4/U6.U5] tri-snRNPs led to the identification of two novel proteins with molecular weights of 61 and 63kD that are distinct from the previously described 15.5, 20, 27, 60, and 90kD tri-snRNP proteins. For the initial characterization of tri-snRNP proteins that interact directly with U4/U6 snRNPs, immunoaffinity chromatography with an antibody directed against the 60kD protein was performed. We demonstrate that the 60 and 90kD tri-snRNP proteins specifically associate with the U4/U6 snRNP at salt concentrations where the tri-snRNP complex has dissociated. The primary structures of the 60kD and 90kD proteins were determined by cloning and sequencing their respective cDNAs. The U4/U6-60kD protein possesses a C-terminal WD domain that contains seven WD repeats and thus belongs to the WD-protein family, whose best-characterized members include the Gbeta subunits of heterotrimeric G proteins. A database homology search revealed a significant degree of overall homology (57.8% similarity, 33.9% identity) between the human 60kD protein and the Saccharomyces cerevisiae U4/U6 snRNP protein Prp4p. Two additional, previously undetected WD repeats (with seven in total) were also identified in Prp4p, consistent with the possibility that 60kD/Prp4p, like beta-transducin, may adopt a propeller-like structure. The U4/U6-90kD protein was shown to exhibit significant homology, particularly in its C-terminal half, with the S. cerevisiae splicing factor Prp3p, which also associates with the yeast U4/U6 snRNP. Interestingly, U4/U6-90kD shares short regions of homology with E. coli RNase III, including a region encompassing its double-stranded RNA binding domain. Based on their structural similarity with essential splicing factors in yeast, the human U4/U6-60kD and 90kD proteins are likely also to play important roles in the mammalian splicing process.     

Structural basis for the molecular recognition between human splicing factors U2AF65 and SF1/mBBP

The essential splicing factors SF1 and U2AF play an important role in the recognition of the pre-mRNA 3' splice site during early spliceosome assembly. The structure of the C-terminal RRM (RRM3) of human U2AF(65) complexed to an N-terminal peptide of SF1 reveals an extended negatively charged helix A and an additional helix C. Helix C shields the potential RNA binding surface. SF1 binds to the opposite, helical face of RRM3. It inserts a conserved tryptophan into a hydrophobic pocket between helices A and B in a way that strikingly resembles part of the molecular interface in the U2AF heterodimer. This molecular recognition establishes a paradigm for protein binding by a subfamily of noncanonical RRMs.     

The yeast PRP6 gene encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein, and the PRP9 gene encodes a protein required for U2 snRNP binding.

PRP6 and PRP9 are two yeast genes involved in pre-mRNA splicing. Incubation at 37 degrees C of strains that carry temperature-sensitive mutations at these loci inhibits splicing, and in vivo experiments suggested that they might be involved in commitment complex formation (P. Legrain and M. Rosbash, Cell 57:573-583, 1989). To examine the specific role that the PRP6 and PRP9 products may play in splicing or pre-mRNA transport to the cytoplasm, we have characterized in vitro splicing and spliceosome assembly in extracts derived from prp6 and prp9 mutant strains. We have also characterized RNAs that are specifically immunoprecipitated with the PRP6 and PRP9 proteins. Both approaches indicate that PRP6 encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein and that the PRP9 protein is required for a stable U2 snRNP-substrate interaction. The results are discussed with reference to the previously observed in vivo phenotypes of these mutants.     

Esf2p, a U3-associated factor required for small-subunit processome assembly and compaction

Esf2p is the Saccharomyces cerevisiae homolog of mouse ABT1, a protein previously identified as a putative partner of the TATA-element binding protein. However, large-scale studies have indicated that Esf2p is primarily localized to the nucleolus and that it physically associates with pre-rRNA processing factors. Here, we show that Esf2p-depleted cells are defective for pre-rRNA processing at the early nucleolar cleavage sites A0 through A2 and consequently are inhibited for 18S rRNA synthesis. Esf2p was stably associated with the 5' external transcribed spacer (ETS) and the box C+D snoRNA U3, as well as additional box C+D snoRNAs and proteins enriched within the small-subunit (SSU) processome/90S preribosomes. Esf2p colocalized on glycerol gradients with 90S preribosomes and slower migrating particles containing 5' ETS fragments. Strikingly, upon Esf2p depletion, chromatin spreads revealed that SSU processome assembly and compaction are inhibited and glycerol gradient analysis showed that U3 remains associated within 90S preribosomes. This suggests that in the absence of proper SSU processome assembly, early pre-rRNA processing is inhibited and U3 is not properly released from the 35S pre-rRNAs. The identification of ABT1 in a large-scale analysis of the human nucleolar proteome indicates that its role may also be conserved in mammals.     

Three protein factors (SF1, SF3 and U2AF) function in pre-splicing complex formation in addition to snRNPs.

The splicing of nuclear messenger RNA precursors can be reproduced in vitro with fractions obtained after chromatography of HeLa cell nuclear extracts. Here we report the chromatographic separation of three protein factors: SF1, SF3 and U2AF. All factors function early in the splicing reaction, in the assembly of a pre-splicing complex. Likewise, all factors are essential for the production of spliced RNA. In addition to their distinct chromatographic properties, the splicing factors can be distinguished by their sensitivities to heat and N-ethylmaleimide. All activities can be detected in a cytoplasmic A-100 fraction from HeLa cells. The fact that SF1, SF3 and U2AF are essential factors in pre-splicing complex formation raises the possibility that SF1 and/or SF3 participate in the interaction of U2 snRNP with the branch point in addition to U2AF.