A U1 snRNA:pre-mRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5'' cleavage site.

We analyzed the effects of suppressor mutations in the U1 snRNA (SNR19) gene from Saccharomyces cerevisiae on the splicing of mutant pre-mRNA substrates. The results indicate that pairing between U1 snRNA and the highly conserved position 5 (GTATGT) of the intron occurs early in spliceosome assembly in vitro. This pairing is important for efficient splicing both in vitro and in vivo. However, pairing at position 5 does not appear to influence 5' splice site selection in vivo, indicating that the previously described U1 snRNA:5' splice junction base pairing interaction is not sufficient to define the 5' cleavage site.     

Defining a 5' splice site by functional selection in the presence and absence of U1 snRNA 5' end

Pre-mRNA splicing in metazoans is mainly specified by sequences at the termini of introns. We have selected functional 5' splice sites from randomized intron sequences through repetitive rounds of in vitro splicing in HeLa cell nuclear extract. The consensus sequence obtained after one round of selection in normal extract closely resembled the consensus of natural occurring 5' splice sites, suggesting that the selection pressures in vitro and in vivo are similar. After three rounds of selection under competitive splicing conditions, the base pairing potential to the U1 snRNA increased, yielding a G100%U100%R94%A67%G89%U76%R83% intronic consensus sequence. Surprisingly, a nearly identical consensus sequence was obtained when the selection was performed in nuclear extract containing U1 snRNA with a deleted 5' end, suggesting that other factors than the U1 snRNA are involved in 5' splice site recognition. The importance of a consecutive complementarity between the 5' splice site and the U1 snRNA was analyzed systematically in the natural range for in vitro splicing efficiency and complex formation. Extended complementarity was inhibitory to splicing at a late step in spliceosome assembly when pre-mRNA substrates were incubated in normal extract, but favorable for splicing under competitive splicing conditions or in the presence of truncated U1 snRNA where transition from complex A to complex B occurred more rapidly. This suggests that stable U1 snRNA binding is advantageous for assembly of commitment complexes, but inhibitory for the entry of the U4/U6.U5 tri-snRNP, probably due to a delayed release of the U1 snRNP.     

5' cleavage site in eukaryotic pre-mRNA splicing is determined by the overall 5' splice region, not by the conserved 5' GU

We have generated all possible single point mutations of the invariant 5' GT of the large beta-globin intron and determined their effect on splicing in vitro. None of the mutants prevented cleavage in the 5' splice region, but many reduced or abolished exon joining. The mutations GT----TT and GT----CT resulted in a shift of the 5' cleavage site on nucleotide upstream; in the case of the mutation GT----TT, this shift was reverted by a second site mutation within the 5' splice region. Our results suggest that the 5' cleavage site is determined not by the conserved GU sequence but by the 5' splice region as a whole, most probably via base-pairing to the 5' end of the U1 snRNA.     

Extensive interactions of PRP8 protein with the 5'' and 3'' splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA.

Precursor RNAs containing 4-thiouridine at specific sites were used with UV-crosslinking to map the binding sites of the yeast protein splicing factor PRP8. PRP8 protein interacts with a region of at least eight exon nucleotides at the 5' splice site and a minimum of 13 exon nucleotides and part of the polypyrimidine tract in the 3' splice site region. Crosslinking of PRP8 to mutant and duplicated 3' splice sites indicated that the interaction is not sequence specific, nor does it depend on the splice site being functional. Binding of PRP8 to the 5' exon was established before step 1 and to the 3' splice site region after step 1 of splicing. These interactions place PRP8 close to the proposed catalytic core of the spliceosome during both transesterification reactions. To date, this represents the most extensive mapping of the binding site(s) of a splicing factor on the substrate RNA. We propose that the large binding sites of PRP8 stabilize the intrinsically weaker interactions of U5 snRNA with both exons at the splice sites for exon alignment by the U5 snRNP.     

Mutations in yeast U5 snRNA alter the specificity of 5' splice-site cleavage

Recognition of 5' splice sites in pre-mRNA splicing is achieved in part by base pairing with U1 snRNA. We have used interactive suppression in the yeast Saccharomyces cerevisiae to look for other factors involved in 5' splice-site recognition. This approach identified an extragenic suppressor that activates a cryptic 5' splice site. The suppressor is a gene for U5 snRNA (snR7) with a single base mutation in a strictly conserved 9 base sequence. This suggests that U5 snRNA can play a part in determining the position of 5' splice-site cleavage. Consistent with this, we have been able to isolate other mutations in the 9 base element in U5 snRNA that specifically activate a second cryptic 5' splice site nearby.     

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.     

The 5''-terminal sequence of U1 RNA complementary to the consensus 5'' splice site of hnRNA is single-stranded in intact U1 snRNP particles.

The 5'-terminal region of U1 snRNA is highly complementary to the consensus exon-intron regions of hnRNA and it has been suggested that U1 snRNP might play a role in the splicing of the pre-mRNA by intermolecular base-pairing between these regions. Here the secondary structure of the 5' terminus of U1 RNA in the isolated native U1 snRNP particle has been investigated by site-directed enzymatic cleavage of the RNA. Individual oligodeoxynucleotides complementary to various sequences within the first 15 nucleotides of the 5' terminus of U1 RNA have been tested for their ability to form stable DNA X RNA hybrids, with subsequent cleavage of the U1 RNA by RNase H. Our results show unequivocally that the 9 nucleotides at the 5' terminus which are complementary to a consensus 5' splice site are indeed single-stranded in the intact U1 snRNP particle, and are not protected by snRNP proteins. However, they also indicate that the U1 sequence complementary to an intron's consensus 3' end is not readily available for intermolecular base-pairing, either in the intact U1 snRNP particle or in the deproteinized U1 RNA molecule. Therefore our data favour the possibility that U1 snRNP plays a role only in the recognition of a 5' splice site of hnRNA, rather than being involved in the alignment of both ends of an intron for splicing.     

U5 snRNA interacts with exon sequences at 5' and 3' splice sites.

U5 snRNA is an essential pre-mRNA splicing factor whose function remains enigmatic. Specific mutations in a conserved single-stranded loop sequence in yeast U5 snRNA can activate cleavage of G1----A mutant pre-mRNAs at aberrant 5' splice sites and facilitate processing of dead-end lariat intermediates to mRNA. Activation of aberrant 5' cleavage sites involves base pairing between U5 snRNA and nucleotides upstream of the cleavage site. Processing of dead-end lariat intermediates to mRNA correlates with base pairing between U5 and the first two bases in exon 2. The loop sequence in U5 snRNA may therefore by intimately involved in the transesterification reactions at 5' and 3' splice sites. This pattern of interactions is strikingly reminiscent of exon recognition events in group II self-splicing introns and is consistent with the notion that U5 snRNA may be related to a specific functional domain from a group II-like self-splicing ancestral intron.     

Genetic interaction between U6 snRNA and the first intron nucleotide in Saccharomyces cerevisiae.

Nuclear pre-mRNA splicing necessitates specific recognition of the pre-mRNA splice sites. It is known that 5' splice site selection requires base pairing of U6 snRNA with intron positions 4-6. However, no factor recognizing the highly conserved 5' splice site GU has yet been identified. We have tested if the known U6 snRNA-pre-mRNA interaction could be extended to include the first intron nucleotides and the conserved 50GAG52 sequence of U6 snRNA. We observe that some combinations of 5' splice site and U6 snRNA mutations produce a specific synthetic block to the first splicing step. In addition, the U6-G52U allele can switch between two competing 5' splice sites harboring different nucleotides following the cleavage site. These results indicate that U6 snRNA position 52 interacts with the first nucleotide of the intron before 5' splice site cleavage. Some combinations of U6 snRNA and pre-mRNA mutations also blocked the second splicing step, suggesting a role for the corresponding nucleotides in a proofreading step before exon ligation. From studies in diverse organisms, various functions have been ascribed to the conserved U6 snRNA 47ACAGAG52 sequence. Our results suggest that these discrepancies might reflect variations between different experimental systems and point to an important conserved role of this sequence in the splicing reaction.     

A role for U2/U6 helix Ib in 5' splice site selection.

Selection of pre-mRNA splice sites is a highly accurate process involving many trans-acting factors. Recently, we described a role for U6 snRNA position G52 in selection of the first intron nucleotide (+1G). Because some U2 alleles suppress U6-G52 mutations, we investigated whether the corresponding U2 snRNA region also influenced 5' splice site selection. Our results demonstrate that U2 snRNAs mutated at position U23, but not adjacent nucleotides, specifically affect 5' splice site cleavage. Furthermore, all U2 position U23 mutations are synthetic lethal with the thermosensitive U6-G52U allele. Interestingly, the U2-U23C substitution has an unprecedented hyperaccurate splicing phenotype in which cleavage of introns with a +1G substitution is reduced, whereas the strain grows with wild-type kinetics. U2 position U23 forms the first base pair with U6 position A59 in U2/U6 helix Ib. Restoration of the helical structure suppresses 5' splice site cleavage defects, showing an important role for the helix Ib structure in 5' splice site selection. U2/U6 helix Ib and helix II have recently been described as being functionally redundant. This report demonstrates a unique role for helix Ib in 5' splice site selection that is not shared with helix II.     

Extended base pair complementarity between U1 snRNA and the 5' splice site does not inhibit splicing in higher eukaryotes, but rather increases 5' splice site recognition

Spliceosome formation is initiated by the recognition of the 5′ splice site through formation of an RNA duplex between the 5′ splice site and U1 snRNA. We have previously shown that RNA duplex formation between U1 snRNA and the 5′ splice site can protect pre-mRNAs from degradation prior to splicing. This initial RNA duplex must be disrupted to expose the 5′ splice site sequence for base pairing with U6 snRNA and to form the active spliceosome. Here, we investigated whether hyperstabilization of the U1 snRNA/5′ splice site duplex interferes with splicing efficiency in human cell lines or nuclear extracts. Unlike observations in Saccharomyces cerevisiae, we demonstrate that an extended U1 snRNA/5′ splice site interaction does not decrease splicing efficiency, but rather increases 5′ splice site recognition and exon inclusion. However, low complementarity of the 5′ splice site to U1 snRNA significantly increases exon skipping and RNA degradation. Although the splicing mechanisms are conserved between human and S.cerevisiae, these results demonstrate that distinct differences exist in the activation of the spliceosome.     

Comparative analysis detects dependencies among the 5' splice-site positions

Human-mouse comparative genomics is an informative tool to assess sequence functionality as inferred from its conservation level. We used this approach to examine dependency among different positions of the 5' splice site. We compiled a data set of 50,493 homologous human-mouse internal exons and analyzed the frequency of changes among different positions of homologous human-mouse 5' splice-site pairs. We found mutual relationships between positions +4 and +5, +5 and +6, -2 and +5, and -1 and +5. We also demonstrated the association between the exonic and the intronic positions of the 5' splice site, in which a stronger interaction of U1 snRNA and the intronic portion of the 5' splice site compensates for weak interaction of U1 snRNA and the exonic portion of the 5' splice site, and vice versa. By using an ex vivo system that mimics the effect of mutation in the 5' splice site leading to familial dysautonomia, we demonstrated that U1 snRNA base-pairing with positions +6 and -1 is the only functional requirement for mRNA splicing of this 5' splice site. Our findings indicate the importance of U1 snRNA base-pairing to the exonic portion of the 5' splice site.     

ATP-dependent interaction of yeast U5 snRNA loop 1 with the 5' splice site.

Pre-messenger RNA splicing is a two-step process by which introns are removed and exons joined together. In yeast, the U5 snRNA loop 1 interacts with the 5' exon before the first step of splicing and with the 5' and 3' exons before the second step. In vitro studies revealed that yeast U5 loop 1 is not required for the first step of splicing but is essential for holding the 5' and 3' exons for ligation during the second step. It is critical, therefore, that loop 1 contacts the 5' exon before the first step of splicing to hold this exon following cleavage from the pre-mRNA. At present it is not known how U5 loop 1 is positioned on the 5' exon prior to the first step of splicing. To address this question, we have used site-specific photoactivated crosslinking in yeast spliceosomes to investigate the interaction of U5 loop 1 with the pre-mRNA prior to the first step of splicing. We have found that the highly conserved uridines in loop 1 make ATP-dependent contacts with an approximately 8-nt region at the 5' splice site that includes the invariant GU. These interactions are dependent on functional U2 and U6 snRNAs. Our results support a model where U5 snRNA loop 1 interacts with the 5' exon in two steps during its targeting to the 5' splice site.     

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.     

A novel approach to describe a U1 snRNA binding site

RNA duplex formation between U1 snRNA and a splice donor (SD) site can protect pre-mRNA from degradation prior to splicing and initiates formation of the spliceosome. This process was monitored, using sub-genomic HIV-1 expression vectors, by expression analysis of the glycoprotein env, whose formation critically depends on functional SD4. We systematically derived a hydrogen bond model for the complementarity between the free 5' end of U1 snRNA and 5' splice sites and numerous mutations following transient transfection of HeLa-T4+ cells with 5' splice site mutated vectors. The resulting model takes into account number, interdependence and neighborhood relationships of predicted hydrogen bond formation in a region spanning the three most 3' base pairs of the exon (-3 to -1) and the eight most 5' base pairs of the intron (+1 to +8). The model is represented by an algorithm classifying U1 snRNA binding sites which can or cannot functionally substitute SD4 with respect to Rev-mediated env expression. In a data set of 5' splice site mutations of the human ATM gene we found a significant correlation between the algorithmic classification and exon skipping (P = 0.018, chi2-test), showing that the applicability of the proposed model reaches far beyond HIV-1 splicing. However, the algorithmic classification must not be taken as an absolute measure of SD usage as it may be modified by upstream sequence elements. Upstream to SD4 we identified a fragment supporting ASF/SF2 binding. Mutating GAR nucleotide repeats within this site decreased the SD4-dependent Rev-mediated env expression, which could be balanced simply by artificially increasing the complementarity of SD4.     

Activation of a cryptic 5' splice site by U1 snRNA

In the course of analyzing 5' splice site mutations in the second intron of Schizosaccharomyces pombe cdc2, we identified a cryptic 5' junction containing a nonconsensus nucleotide at position +2. An even more unusual feature of this cryptic 5' junction was its pattern of activation. By analyzing the profile of splicing products for an extensive series of cdc2 mutants in the presence and absence of compensatory U1 alleles, we have obtained evidence that the natural 5' splice site participates in activation of the cryptic 5' splice site, and that it does so via base pairing to U1 snRNA. Furthermore, the results of follow-up experiments strongly suggest that base pairing between U1 snRNA and the cryptic 5' junction itself plays a dominant role in its activation. Most remarkably, a mutant U1 can activate the cryptic 5' splice site even in the presence of a wild-type sequence at the natural 5' junction, providing unambiguous evidence that this snRNA redirects splicing via base pairing. Although previous work has demonstrated that U5 and U6 snRNAs can activate cryptic 5' splice sites through base pairing interactions, this is the first example in which U1 snRNA has been implicated in the final selection of a cryptic 5' junction.     

Proximity of the U12 snRNA with both the 5' splice site and the branch point during early stages of spliceosome assembly

U12 snRNA is required for branch point recognition in the U12-dependent spliceosome. Using site-specific cross-linking, we have captured an unexpected interaction between the 5' end of the U12 snRNA and the -2 position upstream of the 5' splice site of P120 and SCN4a splicing substrates. The U12 snRNA nucleotides that contact the 5' exon are the same ones that form the catalytically important helix Ib with U6atac snRNA in the spliceosome catalytic core. However, the U12/5' exon interaction is transient, occurring prior to the entry of the U4atac/U6atac.U5 tri-snRNP to the spliceosome. This suggests that the helix Ib region of U12 snRNA is positioned near the 5' splice site early during spliceosome assembly and only later interacts with U6atac to form helix Ib. We also provide evidence that U12 snRNA can simultaneously interact with 5' exon sequences near 5' splice site and the branch point sequence, suggesting that the 5' splice site and branch point sequences are separated by <40 to 50 A in the complex A of the U12-dependent spliceosome. Thus, no major rearrangements are subsequently needed to position these sites for the first step of catalysis.     

snRNA interactions at 5' and 3' splice sites monitored by photoactivated crosslinking in yeast spliceosomes.

Splice site recognition and catalysis of the transesterification reactions in the spliceosome are accompanied by a dynamic series of interactions involving conserved or invariant sequences in the spliceosomal snRNAs. We have used site-specific photoactivated crosslinking in yeast spliceosomes to monitor interactions between snRNAs and exon sequences near the 5' and 3' splice sites. The last nucleotide of the 5' exon can be crosslinked to an invariant loop sequence in U5 SnRNA before and after 5' splice site cleavage. The first nucleotide of the 3' exon can also be crosslinked to the same U5 loop sequence, but this contact is only detectable after the first transesterification. These results are in close agreement with earlier data from mammalian splicing extracts, and they are consistent with a model in which U5 snRNA aligns the 5' and 3' exons for the second transesterification. After the first catalytic step of splicing, the first nucleotide of the 3' exon can also crosslink to nt U23 in U2 snRNA. This is one of a cluster of residues in U2-U6 helix I implicated by mutational analysis in the second catalytic step of splicing. The crosslinking data suggest that these residues in U2-U6 helix I are in close proximity to the scissile phosphodiester bond at the 3' splice site prior to the second transesterification. These results constitute the first biochemical evidence for a direct interaction between the 3' splice site and U2 snRNA.     

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 RNA switch at the 5' splice site requires ATP and the DEAD box protein Prp28p

Pre-mRNA splicing requires dramatic RNA rearrangements hypothesized to be catalyzed by ATP-dependent RNA unwindases of the DExD/H box family. In a rearrangement critical for the fidelity of 5' splice site recognition, a base-pairing interaction between the 5' splice site and U1 snRNA must be switched for a mutually exclusive interaction between the 5' splice site and U6 snRNA. By lengthening the U1:5' splice site duplex, we impeded this switch in a temperature-dependent manner and prevented formation of the spliceosome's catalytic core. Using genetics, we identified the DExD/H box protein Prp28p as a potential mediator of the switch. In vitro, the switch requires both Prp28p and ATP. We propose that Prp28p directs isomerization of RNA at the 5' splice site and promotes fidelity in splicing.