Commitment of yeast pre-mRNA to the splicing pathway requires a novel U1 small nuclear ribonucleoprotein polypeptide, Prp39p.

The binding of a U1 small nuclear ribonucleoprotein (snRNP) particle to the 5' splice site region of a pre-mRNA is a primary step of intron recognition. In this report, we identify a novel 75-kDa polypeptide of Saccharomyces cerevisiae, Prp39p, necessary for the stable interaction of mRNA precursors with the snRNP components of the pre-mRNA splicing machinery. In vivo, temperature inactivation or metabolic depletion of Prp39p blocks pre-mRNA splicing and causes growth arrest. Analyses of cell extracts reveal a specific and dramatic increase in the electrophoretic mobility of the U1 snRNP particle upon Prp39p depletion and demonstrate that extracts deficient in Prp39p activity are unable to form either the CC1 or CC2 commitment complex band characteristic of productive U1 snRNP/pre-mRNA association. Immunological studies establish that Prp39p is uniquely associated with the U1 snRNP and is recruited with the U1 snRNP into splicing complexes. On the basis of these and related observations, we propose that Prp39p functions, at least in part, prior to stable branch point recognition by the U1 snRNP particle to facilitate or stabilize the U1 snRNP/5' splice site interaction.     

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.     

Identification by mass spectrometry and functional analysis of novel proteins of the yeast [U4/U6.U5] tri-snRNP.

The 25S [U4/U6.U5] tri-snRNP (small nuclear ribonucleoprotein) is a central unit of the nuclear pre-mRNA splicing machinery. The U4, U5 and U6 snRNAs undergo numerous rearrangements in the spliceosome, and knowledge of all of the tri-snRNP proteins is crucial to the detailed investigation of the RNA dynamics during the spliceosomal cycle. Here we characterize by mass spectrometric methods the proteins of the purified [U4/U6.U5] tri-snRNP from the yeast Saccharomyces cerevisiae. In addition to the known tri-snRNP proteins (only one, Lsm3p, eluded detection), we identified eight previously uncharacterized proteins. These include four Sm-like proteins (Lsm2p, Lsm5p, Lsm6p and Lsm7p) and four specific proteins named Snu13p, Dib1p, Snu23p and Snu66p. Snu13p comprises a putative RNA-binding domain. Interestingly, the Schizosaccharomyces pombe orthologue of Dib1p, Dim1p, was previously assigned a role in cell cycle progression. The role of Snu23p, Snu66p and, additionally, Spp381p in pre-mRNA splicing was investigated in vitro and/or in vivo. Finally, we show that both tri-snRNPs and the U2 snRNP are co-precipitated with protein A-tagged versions of Snu23p, Snu66p and Spp381p from extracts fractionated by glycerol gradient centrifugation. This suggests that these proteins, at least in part, are also present in a [U2.U4/U6.U5] tetra-snRNP complex.     

Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly

HeLa cell nuclear splicing extracts have been prepared that are specifically and efficiently depleted of U1, U2, or U4/U6 snRNPs by antisense affinity chromatography using biotinylated 2'-OMe RNA oligonucleotides. Removal of each snRNP particle prevents pre-mRNA splicing but arrests spliceosome formation at different stages of assembly. Mixing extracts depleted for different snRNP particles restores formation of functional splicing complexes. Specific binding of factors to the 3' splice site region is still detected in snRNP-depleted extracts. Depletion of U1 snRNP impairs stable binding of U2 snRNP to the pre-mRNA branch site. This role of U1 snRNP in promoting stable preslicing complex formation is independent of the U1 snRNA-5' splice site interaction.     

Direct interactions between pre-mRNA and six U2 small nuclear ribonucleoproteins during spliceosome assembly.

Highly purified mammalian spliceosomal complex B contains more than 30 specific protein components. We have carried out UV cross-linking studies to determine which of these components directly contacts pre-mRNA in purified prespliceosomal and spliceosomal complexes. We show that heterogeneous nuclear ribonucleoproteins cross-link in the nonspecific complex H but not in the B complex. U2AF65, which binds to the 3' splice site, is the only splicing factor that cross-links in purified prespliceosomal complex E. U2AF65 and the U1 small nuclear ribonucleoprotein particle (snRNP) are subsequently destabilized, and a set of six spliceosome-associated proteins (SAPs) cross-links to the pre-mRNA in the prespliceosomal complex A. These proteins require the 3' splice site for binding and cross-link to an RNA containing only the branch site and 3' splice site. Significantly, all six of these SAPs are specifically associated with U2 snRNP. These proteins and a U5 snRNP component cross-link in the fully assembled B complex. Previous work detected an ATP-dependent, U2 snRNP-associated factor that protects a 30- to 40-nucleotide region surrounding the branchpoint sequence from RNase digestion. Our data indicate that the six U2 snRNP-associated SAPs correspond to this branchpoint protection factor. Four of the snRNP proteins that are in intimate contact with the pre-mRNA are conserved between Saccharomyces cerevisiae and humans, consistent with the possibility that these factors play key roles in mediating snRNA-pre-mRNA interactions during the splicing reaction.     

The 5'' end domain of U2 snRNA is required to establish the interaction of U2 snRNP with U2 auxiliary factor(s) during mammalian spliceosome assembly.

Stable association of U2 snRNP with the branchpoint sequence of mammalian pre-mRNAs requires binding of a non-snRNP protein to the polypyrimidine tract. In order to determine how U2 snRNP contacts this protein, we have used an RNA containing the consensus 5' and the (Py)n-AG 3' splice sites but lacking the branchpoint sequence so as to prevent direct U2 snRNA base pairing to the branchpoint. Different approaches including electrophoretic separation of RNP complexes formed in nuclear extracts, RNase T1 protection immunoprecipitation assays with antibodies against snRNPs and UV cross-linking experiments coupled to immunoprecipitations allowed us to demonstrate that at least three splicing factors contact this RNA at 0 degree C without ATP. As expected, U1 snRNP interacts with the region comprising the 5' splice site. A protein of approximately 65,000 molecular weight recognizes the RNA specifically at the 5' boundary of the polypyrimidine tract. It could be either the U2 auxiliary factor (U2AF) (Zamore and Green (1989) PNAS 86, 9243-9247), the polypyrimidine tract binding protein (pPTB) (Garcia-Blanco et al. (1989) Genes and Dev. 3, 1874-1886) or a mixture of both. U2 snRNP also contacts the RNA in a way depending on p65 binding, thereby further arguing that the latter may correspond to the previously characterized U2AF and pPTB. Cleavage of U2 snRNA sequence by a complementary oligonucleotide and RNase H led us to conclude that the 5' terminus of U2 snRNA is required to ensure the contact between U2 snRNP and p65 bound to the RNA. More importantly, this conclusion can be extended to authentic pre-mRNAs. When we have used a human beta-globin pre-mRNA instead of the above artificial substrate, RNA bound p65 became precipitable by anti-(U2) RNP and anti-Sm antibodies except when the 5' end of U2 snRNA was selectively cleaved.     

Mer1p is a modular splicing factor whose function depends on the conserved U2 snRNP protein Snu17p

Mer1p activates the splicing of at least three pre-mRNAs (AMA1, MER2, MER3) during meiosis in the yeast Saccharomyces cerevisiae. We demonstrate that enhancer recognition by Mer1p is separable from Mer1p splicing activation. The C-terminal KH-type RNA-binding domain of Mer1p recognizes introns that contain the Mer1p splicing enhancer, while the N-terminal domain interacts with the spliceosome and activates splicing. Prior studies have implicated the U1 snRNP and recognition of the 5′ splice site as key elements in Mer1p-activated splicing. We provide new evidence that Mer1p may also function at later steps of spliceosome assembly. First, Mer1p can activate splicing of introns that have mutated branch point sequences. Secondly, Mer1p fails to activate splicing in the absence of the non-essential U2 snRNP protein Snu17p. Thirdly, Mer1p interacts with the branch point binding proteins Mud2p and Bbp1p and the U2 snRNP protein Prp11p by two-hybrid assays. We conclude that Mer1p is a modular splicing regulator that can activate splicing at several early steps of spliceosome assembly and depends on the activities of both U1 and U2 snRNP proteins to activate splicing.     

The yeast U5 snRNP coisolated with the U1 snRNP has an unexpected protein composition and includes the splicing factor Aar2p.

We describe the purification and characterization of a 16S U5 snRNP from the yeast Saccharomyces cerevisiae and the identification of its proteins. In contrast to the human 20S U5 snRNP, it has a comparatively simple protein composition. In addition to the Sm core proteins, it contains only two of the U5 snRNP specific proteins, Prp8p and Snu114p. Interestingly, the 16S U5 snRNP contains also Aar2p, a protein that was previously implicated in splicing of the two introns of the MATa1 pre-mRNA. Here, we demonstrate that Aar2p is essential and required for in vivo splicing of U3 precursors. However, it is not required for splicing in vitro. Aar2p is associated exclusively with this simple form of the U5 snRNP (Aar2-U5), but not with the [U4/U6.U5] tri-snRNP or spliceosomal complexes. Consistent with this, we show that depletion of Aar2p interferes with later rounds of splicing, suggesting that it has an effect when splicing depends on snRNP recycling. Remarkably, the Aar2-U5 snRNP is invariably coisolated with the U1 snRNP regardless of the purification protocol used. This is consistent with the previously suggested cooperation between the U1 and U5 snRNPs prior to the catalytic steps of splicing. Electron microscopy of the Aar2-U5 snRNP revealed that, despite the comparatively simple protein composition, the yeast Aar2-U5 snRNP appears structurally similar to the human 20S U5 snRNP. Thus, the basic structural scaffold of the Aar2-U5 snRNP seems to be essentially determined by Prp8p, Snu114p, and the Sm proteins.     

Association of U2 snRNP with the spliceosomal complex E.

In metazoans, the E complex is operationally defined as an ATP-independent spliceosomal complex that elutes as a single peak on a gel filtration column and can be chased into spliced products in the presence of an excess of competitor pre-mRNA. The A complex is the first ATP-dependent functional spliceosomal complex. U1 snRNP first binds tightly to the 5'splice site in the E complex and U2 snRNP first binds tightly to the branch site in the A complex. In this study, we have generated and characterized a monoclonal antibody (mAb 4G8) directed against SAP 62, a component of U2 snRNP and a subunit of the essential mammalian splicing factor SF3a. We show that this antibody is highly specific for SAP 62, detecting only SAP 62 on Western blots and immunoprecipitating only SAP 62 from nuclear extracts. The anti-SAP 62 antibody also immunoprecipitates U2 snRNP and the A complex. Significantly, however, we find that the E complex is also efficiently immunoprecipitated by the anti-SAP 62 antibody. This antibody does not cross-react with any E complex-specific components, indicating that SAP 62 itself is associated with the E complex. To determine whether other U2 snRNP components are associated with the E complex, we used antibodies to the U2 snRNP proteins B"and SAP 155. These antibodies also specifically immunoprecipitate the E complex. These observations indicate that U2 snRNP is associated with the E complex. However, we find that U2 snRNP is not as tightly bound in the E complex as it is in the A complex. The possible significance of the weak association of U2 snRNP with the E complex is discussed.     

Functional interaction of a novel 15.5kD [U4/U6.U5] tri-snRNP protein with the 5' stem-loop of U4 snRNA.

Activation of the spliceosome for splicing catalysis requires the dissociation of U4 snRNA from the U4/U6 snRNA duplex prior to the first step of splicing. We characterize an evolutionarily conserved 15.5 kDa protein of the HeLa [U4/U6.U5] tri-snRNP that binds directly to the 5' stem-loop of U4 snRNA. This protein shares a novel RNA recognition motif with several RNP-associated proteins, which is essential, but not sufficient for RNA binding. The 15.5kD protein binding site on the U4 snRNA consists of an internal purine-rich loop flanked by the stem of the 5' stem-loop and a stem comprising two base pairs. Addition of an RNA oligonucleotide comprising the 5' stem-loop of U4 snRNA (U4SL) to an in vitro splicing reaction blocked the first step of pre-mRNA splicing. Interestingly, spliceosomal C complex formation was inhibited while B complexes accumulated. This indicates that the 15.5kD protein, and/or additional U4 snRNP proteins associated with it, play an important role in the late stage of spliceosome assembly, prior to step I of splicing catalysis. Our finding that the 15.5kD protein also efficiently binds to the 5' stem-loop of U4atac snRNA indicates that it may be shared by the [U4atac/U6atac.U5] tri-snRNP of the minor U12-type spliceosome.     

A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins.

The U1 snRNP is essential for recognition of the pre-mRNA 5'-splice site and the subsequent assembly of the spliceosome. Yeast U1 snRNP is considerably more complex than its metazoan counterpart, which suggests possible differences between yeast and metazoa in early splicing events. We have comprehensively analyzed the composition of yeast U1 snRNPs using a combination of biochemical, mass spectrometric, and genetic methods. We demonstrate the specific association of four novel U1 snRNP proteins, Snu71p, Snu65p, Nam8p, and Snu56p, that have no known metazoan homologues. A fifth protein, Npl3p, is an abundant cellular component that reproducibly co-purifies with the U1 snRNP, but its association is salt-sensitive. Therefore, we are unable to establish conclusively whether it binds specifically to the U1 snRNP. Interestingly, Nam8p and Npl3p were previously assigned functions in (pre-m)RNA-metabolism; however, so far, no association with U1 snRNP has been demonstrated or proposed. We also show that the yeast SmB protein is a U1 snRNP component. Yeast U1 snRNP therefore contains 16 different proteins, including seven snRNP core proteins, three homologues of the metazoan U1 snRNP-specific proteins, and six yeast-specific U1 snRNP proteins. We have simultaneously continued the characterization of additional mutants isolated in a synthetic lethal (MUD) screen for genes that functionally cooperate with U1 snRNA. Consistent with the biochemical results, mud10, mud15, and mud16 are alleles of SNU56, NAM8, and SNU65, respectively. mud10 and mud15 affect the in vivo splicing efficiency of noncanonical introns. Moreover, mud10p strongly affects the in vitro formation of splicing complexes, and extracts from the mud15 strain contain a U1 snRNP that migrates aberrantly on native gels. Finally, we show that Nam8p/Mud15p contributes to the stability of U1 snRNP.     

Mapping U2 snRNP--pre-mRNA interactions using biotinylated oligonucleotides made of 2''-OMe RNA.

Biotinylated 2'-OMe RNA oligonucleotides complementary to two separate regions of human U2 snRNA have been used as affinity probes to study U2 snRNP--pre-mRNA interactions. Both oligonucleotides bind specifically and allow highly selective removal of U2 snRNP from HeLa cell nuclear extracts. Pre-mRNA substrates can also be specifically affinity selected through oligonucleotides binding to U2 snRNP particles in splicing complexes. Stable binding of U2 snRNP to pre-mRNA is blocked by the pre-binding of an oligonucleotide to the branch site complementary region of U2 snRNA, but not by an oligonucleotide binding to the 5' terminus of U2. Both oligonucleotides affinity select the intron product, but not the intron intermediate, when added after spliceosome assembly has taken place. The effect of 2'-OMe RNA oligonucleotides on splicing complex formation has been used to demonstrate that complexes containing U2 snRNP and unspliced pre-mRNA are precursors to functional spliceosomes.     

Trans mRNA splicing in trypanosomes: cloning and analysis of a PRP8-homologous gene from Trypanosoma brucei provides evidence for a U5-analogous RNP.

In trypanosomes all mRNAs are generated through trans mRNA splicing, requiring the functions of the small nuclear RNAs U2, U4 and U6. In the absence of conventional cis mRNA splicing, the structure and function of a U5-analogous snRNP in trypanosomes has remained an open question. In cis splicing, a U5 snRNP-specific protein component called PRP8 in yeast and p220 in man is a highly conserved, essential splicing factor involved in splice-site recognition and selection. We have cloned and sequenced a genomic region from Trypanosoma brucei, that contains a PRP8/p220-homologous gene (p277) coding for a 277 kDa protein. Using an antibody against a C-terminal region of the trypanosomal p277 protein, a small RNA of approximately 65 nucleotides could be specifically co-immunoprecipitated that appears to be identical with a U5 RNA (SLA2 RNA) recently identified by Dungan et al. (1996). Based on sedimentation, immunoprecipitation and Western blot analyses we conclude that this RNA is part of a stable ribonucleoprotein (RNP) complex and associated not only with the p277 protein, but also with the common proteins present in the other trans-spliceosomal snRNPs. Together these results demonstrate that a U5-analogous RNP exists in trypanosomes and suggest that basic functions of the U5 snRNP are conserved between cis and trans splicing.     

A novel U2 and U11/U12 snRNP protein that associates with the pre-mRNA branch site

Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa protein (p14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this protein by microsequencing a 14 kDa protein isolated from U2-type spliceosomes. This protein contains an RNA recognition motif and is highly conserved across species. Antibodies raised against this cDNA-encoded protein precipitated the 14 kDa protein cross-linked to the branch adenosine, confirming the identity of the p14 cDNA. A combination of immunoblotting, protein microsequencing and immunoprecipitation revealed that p14 is a component of both 17S U2 and 18S U11/U12 snRNPs, suggesting that it contributes to the interaction of these snRNPs with the branch sites of U2- and U12-type pre-mRNAs, respectively. p14 was also shown to be a subunit of the heteromeric splicing factor SF3b and to interact directly with SF3b155. Immuno precipitations indicated that p14 is present in U12-type spliceosomes, consistent with the idea that branch point selection is similar in the major and minor spliceosomes.     

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.     

剪接因子U2AF65相关蛋白的研究进展

U2核糖核蛋白小体辅助因子(U2AF)65是参与前体mRNA剪接的重要辅助因子,前体RNA生成之初,U1核糖核蛋白小体(snRNP)结合到内含子的5'剪接位点,U2AF65和U2AF35分别结合到多聚嘧啶序列和3'剪接位点,剪接因子1(SF1)结合到分支位点是剪接体形成的第一步。U2AF的存在又辅助U2snRNP代替SF1结合到分支位点,使剪接反应顺利进行。最近几年,发现基因组中存在一些U2AF65的旁系同源基因序列。这些旁系同源基因由祖先基因经连续复制而横向形成,复制出的基因副本经历了各自的进化途径,最终它们在结构和功能上有相似之处,又各有独特之处。我们简要讨论了U2AF65、PUF60、CAPERα和CAPERβ这4种同源蛋白的发现过程、结构特征、自身的多样性、基因的进化和生物学功能。     

An ordered pathway of snRNP binding during mammalian pre-mRNA splicing complex assembly.

We have studied the assembly, composition and structure of splicing complexes using biotin-avidin affinity chromatography and RNase protection assays. We find that U1, U2, U4, U5 and U6 snRNPs associate with the pre-mRNA and are in the mature, functional complex. Association of U1 snRNP with the pre-mRNA is rapid and ATP independent; binding of all other snRNPs occurs subsequently and is ATP dependent. Efficient binding of U1 and U2 snRNPs requires a 5' splice site or a 3' splice site/branch point region, respectively. Both sequence elements are required for efficient U4, U5 and U6 snRNP binding. Mutant RNA substrates containing only a 5' splice site or a 3' splice site/branch point region are assembled into 'partial' splicing complexes, which contain a subset of these five snRNPs. RNase protection experiments indicate that in contrast to U1 and U2 snRNPs, U4, U5 and U6 snRNPs do not contact the pre-mRNA. Based upon the time course of snRNP binding and the composition of sucrose gradient fractionated splicing complexes we suggest an assembly pathway proceeding from a 20S (U1 snRNP only) through a 40S (U1 and U2 snRNPs) to the functional 60S splicing complex (U1, U2, U4, U5 and U6 snRNPs).     

Characterization of a U2AF-independent commitment complex (E') in the mammalian spliceosome assembly pathway

Early recognition of pre-mRNA during spliceosome assembly in mammals proceeds through the association of U1 small nuclear ribonucleoprotein particle (snRNP) with the 5' splice site as well as the interactions of the branch binding protein SF1 with the branch region and the U2 snRNP auxiliary factor U2AF with the polypyrimidine tract and 3' splice site. These factors, along with members of the SR protein family, direct the ATP-independent formation of the early (E) complex that commits the pre-mRNA to splicing. We report here the observation in U2AF-depleted HeLa nuclear extract of a distinct, ATP-independent complex designated E' which can be chased into E complex and itself commits a pre-mRNA to the splicing pathway. The E' complex is characterized by a U1 snRNA-5' splice site base pairing, which follows the actual commitment step, an interaction of SF1 with the branch region, and a close association of the 5' splice site with the branch region. These results demonstrate that both commitment to splicing and the early proximity of conserved sequences within pre-mRNA substrates can occur in a minimal complex lacking U2AF, which may function as a precursor to E complex in spliceosome assembly.     

Ser/Arg-rich protein-mediated communication between U1 and U2 small nuclear ribonucleoprotein particles

Previous work demonstrated that U1 small nuclear ribonucleoprotein particle (snRNP), bound to a downstream 5' splice site, can positively influence utilization of an upstream 3' splice site via exon definition in both trans- and cis-splicing systems. Although exon definition results in the enhancement of splicing of an upstream intron, the nature of the factors involved has remained elusive. We assayed the interaction of U1 snRNP as well as the positive effect of a downstream 5' splice site on trans-splicing in nematode extracts containing either inactive (early in development) or active (later in development) serine/arginine-rich splicing factors (SR proteins). We have determined that U1 snRNP interacts with the 5' splice site in the downstream exon even in the absence of active SR proteins. In addition, we determined that U1 snRNP-directed loading of U2 snRNP onto the branch site as well as efficient trans-splicing in these inactive extracts could be rescued upon the addition of active SR proteins. Identical results were obtained when we examined the interaction of U1 snRNP as well as the requirement for SR proteins in communication across a cis-spliced intron. Weakening of the 3' splice site uncovered distinct differences, however, in the ability of U1 snRNP to promote U2 addition, dependent upon its position relative to the branch site. These results demonstrate that SR proteins are required for communication between U1 and U2 snRNPs whether this interaction is across introns or exons.