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.     

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).     

Requirements for U2 snRNP addition to yeast pre-mRNA.

The in vitro spliceosome assembly pathway is conserved between yeast and mammals as U1 and U2 snRNPs associate with the pre-mRNA prior to U5 and U4/U6 snRNPs. In yeast, U1 snRNP-pre-mRNA complexes are the first splicing complexes visualized on native gels, and association with U1 snRNP apparently commits pre-mRNA to the spliceosome assembly pathway. The current study addresses U2 snRNP addition to commitment complexes. We show that commitment complex formation is relatively slow and does not require ATP, whereas U2 snRNP adds to the U1 snRNP complexes in a reaction that is relatively fast and requires ATP or hydrolyzable ATP analogs. In vitro spliceosome assembly was assayed in extracts derived from strains containing several U1 sRNA mutations. The results were consistent with a critical role for U1 snRNP in early complex formation. A mutation that disrupts the base-pairing between the 5' end of U1 snRNA and the 5' splice site allows some U2 snRNP addition to bypass the ATP requirement, suggesting that ATP may be used to destabilize certain U1 snRNP:pre-mRNA interactions to allow subsequent U2 snRNP addition.     

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.     

Selection of alternative 5' splice sites: role of U1 snRNP and models for the antagonistic effects of SF2/ASF and hnRNP A1

The first component known to recognize and discriminate among potential 5' splice sites (5'SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5'SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5'SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5'SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5'SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5'SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5'SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.     

A short 5' splice site RNA oligo can participate in both steps of splicing in mammalian extracts.

A short 5' splice site RNA oligonucleotide (5'SS RNA oligo) undergoes both steps of splicing when a second RNA containing the 3' splice site region (3'SS RNA) is added in trans. This trans-splicing reaction displays the same 5' and 3' splice site sequence requirements as cis-splicing of full-length pre-mRNA. The analysis of RNA-snRNP complexes formed on each of the two splice site RNAs is consistent with the formation of partial complexes, which then associate to form the complete spliceosome. Specifically, U2 snRNP bound to the 3'SS RNA associates with U4/U5/U6 snRNP bound to the 5'SS RNA oligo. Thus, as expected, trans-splicing depends on the integrity of U2, U4, and U6 snRNAs. However, unlike cis-splicing, trans-splicing is enhanced when the 5' end of U1 snRNA is blocked or removed or when the U1 snRNP is depleted. Thus, the early regulatory requirement for U1 snRNP, which is essential in cis-splicing, is bypassed in this trans-splicing system. This simplified trans-splicing reaction offers a unique model system in which to study the mechanistic details of pre-mRNA splicing.     

Probing the structure and function of U2 snRNP with antisense oligonucleotides made of 2'-OMe RNA

We have used oligonucleotides made of 2'-OMe RNA to analyze the role of separate domains of U2 snRNA in the splicing process. We show that antisense 2'-OMe RNA oligonucleotides bind efficiently and specifically to U2 snRNP and demonstrate that masking of two separate regions of U2 snRNA can inhibit splicing by affecting different steps in the spliceosome assembly pathway. Masking the 5' terminus of U2 snRNA does not prevent U2 snRNP binding to pre-mRNA but blocks subsequent assembly of a functional spliceosome. By contrast, masking of U2 sequences complementary to the pre-mRNA branch site completely inhibits binding of pre-mRNA. Hybrid formation at the branch site complementary region also triggers a specific change which affects the 5' terminus of U2 snRNA.     

Identification and characterization of a yeast homolog of U1 snRNP-specific protein C.

U1C is one of the three human U1 small nuclear ribonucleoprotein (snRNP)-specific proteins and is important for efficient complex formation between U1 snRNP and the pre-mRNA 5' splice site. We identified a hypothetical open reading frame in Saccharomyces cerevisiae as the yeast homolog of the human U1C protein. The gene is essential, and its product, YU1C, is associated with U1 snRNP. YU1C depletion gives rise to normal levels of U1 snRNP and does not have any detectable effect on U1 snRNP assembly. YU1C depletion and YU1C ts mutants affect pre-mRNA splicing in vivo, and extracts from these strains form low levels of commitment complexes and spliceosomes in vitro. These experiments indicate a role for YU1C in snRNP function. Structure probing with RNases shows that only the U1 snRNA 5' arm is hypersensitive to RNase I digestion when YU1C is depleted. Similar results were obtained with YU1C ts mutants, indicating that U1C contributes to a proper 5' arm structure prior to its base pairing interaction with the pre-mRNA 5' splice site.     

The U11/U12 snRNP 65K protein acts as a molecular bridge, binding the U12 snRNA and U11-59K protein

U11 and U12 interact cooperatively with the 5' splice site and branch site of pre-mRNA as a stable preformed di-snRNP complex, thereby bridging the 5' and 3' ends of the intron within the U12-dependent prespliceosome. To identify proteins contributing to di-snRNP formation and intron bridging, we investigated protein-protein and protein-RNA interactions between components of the U11/U12 snRNP. We demonstrate that the U11/U12-65K protein possesses dual binding activity, interacting directly with U12 snRNA via its C-terminal RRM and the U11-associated 59K protein via its N-terminal half. We provide evidence that, in contrast to the previously published U12 snRNA secondary structure model, the 3' half of U12 forms an extended stem-loop with a highly conserved seven-nucleotide loop and that the latter serves as the 65K binding site. Addition of an oligonucleotide comprising the 65K binding site to an in vitro splicing reaction inhibited U12-dependent, but not U2-dependent, pre-mRNA splicing. Taken together, these data suggest that U11/U12-65K and U11-59K contribute to di-snRNP formation and intron bridging in the minor prespliceosome.     

Reconstituted U1 small nuclear ribonucleoprotein complex restores 5' splice site cleavage activity

Functional reconstitution of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was performed using in vitro transcribed U1 snRNA. Hela cell nuclear extract was depleted of its constituent snRNPs by centrifugation at 100,000 X g. The supernatant was devoid of snRNAs and lacked cleavage activity in splicing reactions using in vitro transcribed beta-globin pre-mRNA as substrate. The resulting pellet which contained the snRNAs, retained 5' splice site cleavage activity in a similar splicing reaction. Supplementation of the inactive supernatant fraction with in vitro transcribed U1 snRNA, partially restored 5' splice site cleavage activity thereby demonstrating the specific requirement of U1 snRNP in the initial stage of pre-mRNA splicing.     

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.     

Combinatorial splicing of exon pairs by two-site binding of U1 small nuclear ribonucleoprotein particle.

A two-site model for the binding of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was tested in order to understand how exon partners are selected in complex pre-mRNAs containing alternative exons. In this model, it is proposed that two U1 snRNPs define a functional unit of splicing by base pairing to the 3' boundary of the downstream exon as well as the 5' boundary of the intron to be spliced. Three-exon substrates contained the alternatively spliced exon 4 (E4) region of the preprotachykinin gene. Combined 5' splice site mutations at neighboring exons demonstrate that weakened binding of U1 snRNP at the downstream site and improved U1 snRNP binding at the upstream site result in the failure to rescue splicing of the intron between the mutations. These results indicate the stringency of the requirement for binding a second U1 snRNP to the downstream 5' splice site for these substrates as opposed to an alternative model in which a certain threshold level of U1 snRNP can be provided at either site. Further support for the two-site model is provided by single-site mutations in the 5' splice site of the third exon, E5, that weaken base complementarity to U1 RNA. These mutations block E5 branchpoint formation and, surprisingly, generate novel branchpoints that are specified chiefly by their proximity to a cryptic 5' splice site located at the 3' terminus of the pre-mRNA. The experiments shown here demonstrate a true stimulation of 3' splice site activity by the downstream binding of U1 snRNP and suggest a possible mechanism by which combinatorial patterns of exon selection are achieved for alternatively spliced pre-mRNAs.     

Early commitment of yeast pre-mRNA to the spliceosome pathway.

Pre-mRNA splicing in vitro is preceded by complex formation (spliceosome assembly). U2 small nuclear RNA (snRNA) is found in the earliest form of the spliceosome detected by native gel electrophoresis, both in Saccharomyces cerevisiae and in metazoan extracts. To examine the requirements for the formation of this early complex (band III) in yeast extracts, we cleaved the U2 snRNA by oligonucleotide-directed RNase H digestion. U2 snRNA depletion by this means inhibits both splicing and band III formation. Using this depleted extract, we were able to design a chase experiment which shows that a pre-mRNA substrate is committed to the spliceosome assembly pathway in the absence of functional U2 snRNP. Interactions occurring during the commitment step are highly resistant to the addition of an excess of unlabeled substrate and require little or no ATP. Sequence requirements for this commitment step have been analyzed by competition experiments with deletion mutants: both the 5' splice site consensus sequence and the branch point TACTAAC box sequence are necessary. These experiments strongly suggest that the initial assembly process requires a trans-acting factor(s) (RNA and/or proteins) that recognizes and stably binds to the two consensus sequences of the pre-mRNA prior to U2 snRNP binding.     

Yeast U1 snRNP-pre-mRNA complex formation without U1snRNA-pre-mRNA base pairing

Base pairing between the 5' end of U1 snRNA and the conserved 5' splice site of pre-mRNA is important for commitment complex formation in vitro. However, the biochemical mechanisms by which pre-mRNA is initially recognized by the splicing machinery is not well understood. To evaluate the role of this base pairing interaction, we truncated U1 snRNA to eliminate the RNA-RNA interaction and surprisingly found that U1 snRNP can still form a nearly normal RNA-protein complex and maintain sequence specificity. We propose that some feature of U1 snRNP, perhaps one or more protein factors, is more important than the base pairing for initial 5' splice site recognition. In addition, at least five sets of interactions contribute to complex formation or stability. Only one of these is base pairing between the 5' splice site and the 5' end of U1 snRNA, without which the U1 snRNP-pre-mRNA complex is less stable and has a somewhat altered conformation.     

RBFOX2 promotes protein 4.1R exon 16 selection via U1 snRNP recruitment

The erythroid differentiation-specific splicing switch of protein 4.1R exon 16, which encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability, is modulated by the differentiation-induced splicing factor RBFOX2. We have now characterized the mechanism by which RBFOX2 regulates exon 16 splicing through the downstream intronic element UGCAUG. Exon 16 possesses a weak 5' splice site (GAG/GTTTGT), which when strengthened to a consensus sequence (GAG/GTAAGT) leads to near-total exon 16 inclusion. Impaired RBFOX2 binding reduces exon 16 inclusion in the context of the native weak 5' splice site, but not the engineered strong 5' splice site, implying that RBFOX2 achieves its effect by promoting utilization of the weak 5' splice site. We further demonstrate that RBFOX2 increases U1 snRNP recruitment to the weak 5' splice site through direct interaction between its C-terminal domain (CTD) and the zinc finger region of U1C and that the CTD is required for the effect of RBFOX2 on exon 16 splicing. Our data suggest a novel mechanism for exon 16 5' splice site activation in which the binding of RBFOX2 to downstream intronic splicing enhancers stabilizes the pre-mRNA-U1 snRNP complex through interactions with U1C.     

The U1 snRNP base pairs with the 5' splice site within a penta-snRNP complex

Recognition of the 5' splice site is an important step in mRNA splicing. To examine whether U1 approaches the 5' splice site as a solitary snRNP or as part of a multi-snRNP complex, we used a simplified in vitro system in which a short RNA containing the 5' splice site sequence served as a substrate in a binding reaction. This system allowed us to study the interactions of the snRNPs with the 5' splice site without the effect of other cis-regulatory elements of precursor mRNA. We found that in HeLa cell nuclear extracts, five spliceosomal snRNPs form a complex that specifically binds the 5' splice site through base pairing with the 5' end of U1. This system can accommodate RNA-RNA rearrangements in which U5 replaces U1 binding to the 5' splice site, a process that occurs naturally during the splicing reaction. The complex in which U1 and the 5' splice site are base paired sediments in the 200S fraction of a glycerol gradient together with all five spliceosomal snRNPs. This fraction is functional in mRNA spliceosome assembly when supplemented with soluble nuclear proteins. The results argue that U1 can bind the 5' splice site in a mammalian preassembled penta-snRNP complex.     

A protein that specifically recognizes the 3' splice site of mammalian pre-mRNA introns is associated with a small nuclear ribonucleoprotein

Using a protein blotting method for the detection of nucleic acid binding proteins, we have identified in HeLa cell nuclear extracts an intron binding protein (IBP) that selectively recognizes the 3' splice site region of mammalian pre-mRNAs. The binding site was accurately delineated using oligonucleotides complementary to human beta-globin pre-mRNA. It spans the 3' splice site AG dinucleotide and the crucial polypyrimidine stretch upstream, but includes neither the branchpoint nor the lariat structure. Although the technique used here shows that the binding specificity is an intrinsic property of IBP and does not depend on snRNA-pre-mRNA interactions, it comigrates with U5 snRNP and is immunoprecipitated by anti-Sm antibody. This strongly suggests that IBP belongs to U5 snRNP. We propose that it is involved in one of the earliest steps of the splicing reaction by mediating the interaction of U5 snRNP with the 3' splice site.