A novel U1/U5 interaction indicates proximity between U1 and U5 snRNAs during an early step of mRNA splicing.

The five spliceosomal snRNAs (U1, U2, U4, U5, and U6) undergo an ordered sequence of conformational changes as mRNA splicing progresses. We have shown that an antisense RNA oligonucleotide complementary to U5 snRNA induces a novel U1/U4/U5 complex that may be a transitional stage in the displacement of U1 from the 5' splice site by U5. Here we identify a novel site-specific crosslink between the 5' end of U1 and the invariant loop of U5 snRNA. This crosslink can be induced in nuclear extract by an antisense oligonucleotide directed against U5 snRNA, but can also be detected during an early step of the splicing reaction in the absence of oligonucleotide. Our data indicate proximity between U1 and U5 snRNPs before the first catalytic step of splicing, and may suggest that U1 helps to direct U5 to the 5' splice site.     

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).     

Evidence for nuclear factors involved in recognition of 5'' splice sites.

To investigate soluble factors involved in pre-messenger RNA splicing we have fractionated nuclear extract by simple centrifugation to produce a supernatant pellet pair. Factors larger than 15S including U2, U4, U5, and U6 snRNPs fractionate with the pellet; U1 snRNPs distribute equally in pellet and supernatant. Each fraction is individually incompetent for splicing and spliceosome assembly; mixing restores wild type activity and assembly. The pellet fraction directs an aberrant assembly pathway in which proper 3', but improper 5' splice site recognition occurs. Complexes formed with the pellet fraction are distinguishable from wild-type complexes using native gel electrophoresis. Pellet complexes contain U1 snRNP antigens and their formation requires ATP, U1 snRNPs, U2 snRNPs, and sequences at the 3' end of the intron - properties shared with the initial steps of normal assembly and directed by sequences at the 3' end of the intron. In contrast, pellet complex assembly shows no dependence on the presence of a 5' splice junction within precursor RNA. Furthermore, binding of factors to the 5' splice junction is deficient in pellet assemblies. Thus, the pellet lacks a factor required for proper recognition of 5' splice sites. This factor can be supplied by the supernatant. Complementation occurs when supernatant U1 RNA is destroyed, suggesting that the supernatant factor recognizing 5' splice sites is not U1 snRNPs.     

U1-U2 snRNPs interaction induced by an RNA complementary to the 5'' end sequence of U1 snRNA.

Several lines of evidences indicate that U1 and U2 snRNPs become interacting during pre-mRNA splicing. Here we present data showing that an U1-U2 snRNPs interaction can be mediated by an RNA only containing the consensus 5' splice site of all of the sequences characteristic of pre-mRNAs. Using monospecific antibodies (anti-(U1) RNP and anti-(U2) RNP), we have found that a tripartite complex comprising U1 and U2 snRNPs is immunoprecipitated in the presence of a consensus 5' splice site containing RNA, either from a crude extract or from an artificial mixture enriched in U1 and U2 snRNPs. This complex does not appear in the presence of an RNA lacking the sequence complementary to the 5' terminus of U1 snRNA. Moreover, RNAse T1 protection coupled to immunoprecipitation experiments have demonstrated that only the 5' end sequence of U1 snRNA contacts the consensus 5' splice site containing RNA, arguing that U2 snRNP binding in the tripartite complex is mediated by U1 snRNP.     

Interactions between small nuclear ribonucleoprotein particles in formation of spliceosomes

Electrophoretic separation of ribonucleoprotein particles in a nondenaturing gel was used to analyze the splicing of mRNA precursors. Early in the reaction, a complex formed consisting of the U2 small nuclear ribonucleoprotein particle (snRNP) bound to sequences upstream of the 3' splice site. This complex is modeled as a precursor of a larger complex, the spliceosome, which contains U2, U4/6, and U5 snRNPs. Conversion of the U2 snRNP-precursor RNA complex to the spliceosome probably involves binding of a single multi-snRNP particle containing U4/6 and U5 snRNPs. The excised intron was released in a complex containing U5, U6, and probably U2 snRNPs. Surprisingly, U4 snRNP was not part of the intron-containing complex, suggesting that U4/6 snRNP disassembles and assembles during splicing. Subsequently, the reassembled U4/6 snRNP would associate with U5 snRNP and participate in de novo spliceosome formation. U1 snRNP was not detected in any of the splicing complexes.     

Pathways for selection of 5' splice sites by U1 snRNPs and SF2/ASF.

We have used protection against ribonuclease H to investigate the mechanisms by which U1 small nuclear ribonucleoprotein particles (snRNPs) determine the use of two alternative 5' splice sites. The initial binding of U1 snRNPs to alternative consensus splice sites was indiscriminate, and on a high proportion of pre-mRNA molecules both sites were occupied simultaneously. When the sites were close, this inhibited splicing. We propose that double occupancy leads to the use of the downstream site for splicing and that this is the cause of the proximity effect seen with strong alternative splice sites. This model predicts that splicing to an upstream site of any strength requires a low affinity of U1 snRNPs for the downstream site. This prediction was tested both by cleaving the 5' end of U1 snRNA and by altering the sequence of the downstream site of an adenovirus E1A gene. The enhancement of downstream 5' splice site use by splicing factor SF2/ASF appears to be mediated by an increase in the strength of U1 snRNP binding to all sites indiscriminately.     

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.     

A novel set of spliceosome-associated proteins and the essential splicing factor PSF bind stably to pre-mRNA prior to catalytic step II of the splicing reaction.

We have isolated and determined the protein composition of the spliceosomal complex C. The pre-mRNA in this complex has undergone catalytic step I, but not step II, of the splicing reaction. We show that a novel set of 14 spliceosome-associated proteins (SAPs) and the essential splicing factor PSF are specifically associated with the C complex, implicating these proteins in catalytic step II. Significantly, immunodepletion and biochemical complementation studies demonstrate directly that PSF is essential for catalytic step II. Purified PSF is known to UV crosslink to pyrimidine tracts, and our data show that PSF UV crosslinks to pre-mRNA in purified C complex. Thus, PSF may replace the 3' splice site binding factor U2AF65 which is destabilized during spliceosome assembly. Finally, we show that SAPs 60 and 90, which are present in both the B and C complexes, are specifically associated with U4 and U6 snRNPs, and thus may have important roles in the functioning of these snRNPs during the splicing reaction.     

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.     

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.     

Structural requirements for the function of a yeast chromosomal replicator

We have investigated the role of small nuclear ribonucleoprotein particles (snRNPs) in the in vitro splicing of messenger RNA precursors by a variety of procedures. Removal of the U-type snRNPs from the nuclear extracts of HeLa cells with protein A-Sepharose-coupled human autoimmune antibodies leads to complete loss of splicing activity. The inhibition of splicing can be prevented by saturating the coupled antibodies with purified nucleoplasmic U snRNPs prior to incubation with nuclear extract. We further demonstrate that an intact 5' terminus of U1 snRNA is required for the functioning of U1 snRNP in the splicing reaction. Antibodies directed against the trimethylated cap structure of the U snRNAs inhibit splicing. Upon removal of the first eight nucleotides of the U1 snRNA in the particles by site-directed hydrolysis with ribonuclease H in the presence of a synthetic complementary oligodeoxynucleotide splicing is completely abolished. These results are in strong support of current models suggesting that a base-pairing interaction between the 5' terminus of the U1 snRNA and the 5' splice site of a mRNA precursor is a prerequisite for proper splicing.     

An intronic splicing enhancer binds U1 snRNPs to enhance splicing and select 5' splice sites

Intronic G triplets are frequently located adjacent to 5' splice sites in vertebrate pre-mRNAs and have been correlated with splicing efficiency and specificity via a mechanism that activates upstream 5' splice sites in exons containing duplicated sites (26). Using an intron dependent upon G triplets for maximal activity and 5' splice site specificity, we determined that these elements bind U1 snRNPs via base pairing with U1 RNA. This interaction is novel in that it uses nucleotides 8 to 10 of U1 RNA and is independent of nucleotides 1 to 7. In vivo functionality of base pairing was documented by restoring activity and specificity to mutated G triplets through compensating U1 RNA mutations. We suggest that the G-rich region near vertebrate 5' splice sites promotes accurate splice site recognition by recruiting the U1 snRNP.     

Exon definition may facilitate splice site selection in RNAs with multiple exons.

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.