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It has been previously observed that the intrinsically weak variant GC donor sites, in order to be recognized by the U2-type spliceosome, possess strong consensus sequences maximized for base pair formation with U1 and U5/U6 snRNAs. However, variability in signal strength is a fundamental mechanism for splice site selection in alternative splicing. Here we report human alternative GC-AG introns (for the first time from any species), and show that while constitutive GC-AG introns do possess strong signals at their donor sites, a large subset of alternative GC-AG introns possess weak consensus sequences at their donor sites. Surprisingly, this subset of alternative isoforms shows strong consensus at acceptor exon positions 1 and 2. The improved consensus at the acceptor exon can facilitate a strong interaction with U5 snRNA, which tethers the two exons for ligation during the second step of splicing. Further, these isoforms nearly always possess alternative acceptor sites and exhibit particularly weak polypyrimidine tracts characteristic of AG-dependent introns. The acceptor exon nucleotides are part of the consensus required for the U2AF35-mediated recognition of AG in such introns. Such improved consensus at acceptor exons is not found in either normal or alternative GT-AG introns having weak donor sites or weak polypyrimidine tracts. The changes probably reflect mechanisms that allow GC-AG alternative intron isoforms to cope with two conflicting requirements, namely an apparent need for differential splice strength to direct the choice of alternative sites and a need for improved donor signals to compensate for the central mismatch base pair (C-A) in the RNA duplex of U1 snRNA and the pre-mRNA. The other important findings include (i) one in every twenty alternative introns is a GC-AG intron, and (ii) three of every five observed GC-AG introns are alternative isoforms.

Alternative 5′ splice site selection is one of the major pathways resulting in mRNA diversification. Regulation of this type of alternative splicing depends on the presence of regulatory elements that activate or repress the use of competing splice sites, usually leading to the preferential use of the proximal splice site. However, the mechanisms involved in proximal splice site selection and the thermodynamic advantage realized by proximal splice sites are not well understood. Here, we have carried out a systematic analysis of alternative 5′ splice site usage using in vitro splicing assays. We show that observed rates of splicing correlate well with their U1 snRNA base pairing potential. Weak U1 snRNA interactions with the 5′ splice site were significantly rescued by the proximity of the downstream exon, demonstrating that the intron definition mode of splice site recognition is highly efficient. In the context of competing splice sites, the proximity to the downstream 3′ splice site was more influential in dictating splice site selection than the actual 5′ splice site/U1 snRNA base pairing potential. Surprisingly, the kinetic analysis also demonstrated that an upstream competing 5′ splice site enhances the rate of proximal splicing. These results reveal the discovery of a new splicing regulatory element, an upstream 5′ splice site functioning as a splicing enhancer.

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.

Activation of pre-messenger RNA (pre-mRNA) splicing requires 5′ splice site recognition by U1 small nuclear RNA (snRNA), which is replaced by U5 and U6 snRNA. Here we use crosslinking to investigate snRNA interactions with the 5′ exon adjacent to the 5′ splice site, prior to the first step of splicing. U1 snRNA was found to interact with four different 5′ exon positions using one specific sequence adjacent to U1 snRNA helix 1. This novel interaction of U1 we propose occurs before U1-5′ splice site base pairing. In contrast, U5 snRNA interactions with the 5′ exon of the pre-mRNA progressively shift towards the 5′ end of U5 loop 1 as the crosslinking group is placed further from the 5′ splice site, with only interactions closest to the 5′ splice site persisting to the 5′ exon intermediate and the second step of splicing. A novel yeast U2 snRNA interaction with the 5′ exon was also identified, which is ATP dependent and requires U2-branchpoint interaction. This study provides insight into the nature and timing of snRNA interactions required for 5′ splice site recognition prior to the first step of pre-mRNA splicing.

The removal of introns from pre-messenger RNA is mediated by the spliceosome, a large complex composed of many proteins and five small nuclear RNAs (snRNAs). Of the snRNAs, the U6 and U2 snRNAs are the most conserved in sequence, as they interact extensively with each other and also with the intron, in several base pairings that are necessary for splicing. We have isolated and sequenced the genes encoding both U6 and U2 snRNAs from the intracellularly parasitic microsporidian Nosema locustae . Both genes are expressed. Both RNAs can be folded into secondary structures typical of other known U6 and U2 snRNAs. In addition, the N.locustae U6 and U2 snRNAs have the potential to base pair in the functional intermolecular interactions that have been characterized by extensive analyses in yeast and mammalian systems. These results indicate that the N.locustae U6 and U2 snRNAs may be functional components of an active spliceosome, even though introns have not yet been found in microsporidian genes.

U12 snRNA is analogous to U2 snRNA of the U2-dependent spliceosome and is essential for the splicing of U12-dependent introns in metazoan cells. The essential region of U12 snRNA, which base pairs to the branch site of minor class introns is well characterized. However, other regions which are outside of the branch site base pairing region are not yet characterized and the requirement of these structures in U12-dependent splicing is not clear. U12 snRNA is predicted to form an intricate secondary structure containing several stem–loops and single-stranded regions. Using a previously characterized branch site genetic suppression assay, we generated second-site mutations in the suppressor U12 snRNA to investigate the in vivo requirement of structural elements in U12-dependent splicing. Our results show that stem–loop IIa is essential and required for in vivo splicing. Interestingly, an evolutionarily conserved stem–loop IIb is dispensable for splicing. We also show that stem–loop III, which binds to a p65 RNA binding protein of the U11-U12 di.snRNP complex, is essential for in vivo splicing. The data validate the existence of proposed stem–loops of U12 snRNA and provide experimental support for individual secondary structures.

Nematodes are the only group of organisms in which both cis- and trans-splicing of nuclear mRNAs are known to occur. Most Caenorhabditis elegans introns are exceptionally short, often only 50 bases long. The consensus donor and acceptor splice site sequences found in other animals are used for both cis- and trans-splicing. In order to identify the machinery required for these splicing events, we have characterized the C. elegans snRNAs. They are similar in sequence and structure to those characterized in other organisms, and several sequence variations discovered in the nematode snRNAs provide support for previously proposed structure models. The C. elegans snRNAs are encoded by gene families. We report here the sequences of many of these genes. We find a highly conserved sequence, the proximal sequence element (PSE), about 65 bp upstream of all 21 snRNA genes thus far sequenced, including the SL RNA genes, which specify the snRNAs that provide the 5' exons in trans-splicing. The sequence of the C. elegans PSE is distinct from PSE's from other organisms.

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U6 small nuclear RNA (snRNA) is the most highly conserved of the five spliceosomal snRNAs that participate in nuclear mRNA splicing. The proposal that U6 snRNA plays a key catalytic role in splicing [D. Brow and C. Guthrie, Nature (London) 337:14-15, 1989] is supported by the phylogenetic conservation of U6, the sensitivity of U6 to mutation, cross-linking of U6 to the vicinity of the 5' splice site, and genetic evidence for extensive base pairing between U2 and U6 snRNAs. We chose to mutate the phylogenetically invariant 41-ACAGAGA-47 and 53-AGC-55 sequences of human U6 because certain point mutations within the homologous regions of Saccharomyces cerevisiae U6 selectively block the first or second step of mRNA splicing. We found that both sequences are more tolerant to mutation in human cells (assayed by transient expression in vivo) than in S. cerevisiae (assayed by effects on growth or in vitro splicing). These differences may reflect different rate-limiting steps in the particular assays used or differential reliance on redundant RNA-RNA or RNA-protein interactions. The ability of mutations in U6 nucleotides A-45 and A-53 to selectively block step 2 of splicing in S. cerevisiae had previously been construed as evidence that these residues might participate directly in the second chemical step of splicing; an indirect, structural role seems more likely because the equivalent mutations have no obvious phenotype in the human transient expression assay.

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Chromatin organization affects alternative splicing and previous studies have shown that exons have increased nucleosome occupancy compared with their flanking introns. To determine whether alternative splicing affects chromatin organization we developed a system in which the alternative splicing pattern switched from inclusion to skipping as a function of time. Changes in nucleosome occupancy were correlated with the change in the splicing pattern. Surprisingly, strengthening of the 5′ splice site or strengthening the base pairing of U1 snRNA with an internal exon abrogated the skipping of the internal exons and also affected chromatin organization. Over-expression of splicing regulatory proteins also affected the splicing pattern and changed nucleosome occupancy. A specific splicing inhibitor was used to show that splicing impacts nucleosome organization endogenously. The effect of splicing on the chromatin required a functional U1 snRNA base pairing with the 5′ splice site, but U1 pairing was not essential for U1 snRNA enhancement of transcription. Overall, these results suggest that splicing can affect chromatin organization.

SUMMARY

Pairing between U2 snRNA and the branch site of spliceosomal introns is essential for spliceosome assembly and is thought to be required for the first catalytic step of splicing. We have identified an RNA comprising the 5’ end of U2 snRNA and the 3’ exon of the ACT1-CUP1 reporter gene, resulting from a trans-splicing reaction in which a 5’ splice site-like sequence in the universally conserved branch site-binding region of U2 is used in trans as a 5’ splice site for both steps of splicing in vivo. Formation of this product occurs in functional spliceosomes assembled on reporter genes whose 5’ splice sites are predicted to bind poorly at the spliceosome catalytic centre. Multiple spatially disparate splice sites in U2 can be used, calling into question both the fate of its pairing to the branch site and the details of its role in splicing catalysis.

U4atac snRNA forms a base-paired complex with U6atac snRNA. Both snRNAs are required for the splicing of the minor U12-dependent class of eukaryotic nuclear introns. We have developed a new genetic suppression assay to investigate the in vivo roles of several regions of U4atac snRNA in U12-dependent splicing. We show that both the stem I and stem II regions, which have been proposed to pair with U6atac snRNA, are required for in vivo splicing. Splicing activity also requires U4atac sequences in the 5′ stem–loop element that bind a 15.5 kDa protein that also binds to a similar region of U4 snRNA. In contrast, mutations in the region immediately following the stem I interaction region, as well as a deletion of the distal portion of the 3′ stem–loop element, were active for splicing. Complete deletion of the 3′ stem–loop element abolished in vivo splicing function as did a mutation of the Sm protein binding site. These results show that the in vivo sequence requirements of U4atac snRNA are similar to those described previously for U4 snRNA using in vitro assays and provide experimental support for models of the U4atac/U6atac snRNA interaction.

We showed previously that a branch site mutation in simian virus 40 early pre-mRNA that prevented small t antigen mRNA splicing could be efficiently suppressed by a compensatory mutation in a coexpressed U2 small nuclear (sn) RNA gene. We have now generated second-site mutations in this suppressor gene to investigate regions of U2 RNA required for function. A number of mutations in a putative stem at the 5' end of the molecule inhibited splicing, indicating that bases in this region are important for activity. However, several lines of evidence suggested that formation of the entire stem is not essential for splicing. Indeed, mutations that strengthen the stem actually inhibited splicing, and evidence that this prevents a required base-pairing interaction with U6 snRNA is presented. These results suggest that the relative stabilities of competing intra- and intermolecular base-pairing interactions play an important role in the splicing reaction. Mutations in a conserved single-stranded region immediately 3' to the branch site recognition sequence all inhibited splicing, indicating that this region is required for U2 function, although its exact role remains unknown. Finally, two mutations in the loop of stem IV at the 3' end of the molecule, which destroy the binding site of U2 sn ribonucleoprotein B", prevented small t splicing; this finding contrasts with previous studies which utilized different assay systems. Analysis of the accumulation and subcellular localization of all of the mutant RNAs showed that they were similar to those of the parental suppressor U2 RNA, indicating that the effects observed indeed reflect defects in splicing.

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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 Å 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.

A general method based on the statistical technique of discriminant analysis is developed to distinguish boundaries of coding and non-coding regions in nucleic acid sequences. In particular, the method is applied to the prediction of splicing sites in messenger RNA precursors. Information used for discrimination includes consensus sequence patterns around splice junctions, free energy of snRNA and mRNA base pairing, and statistical differences between coding and non-coding regions such as periodic appearance of specific bases in coding regions reflecting the non-random usage of degenerate codons. Given the reading frame of an exon (but not the exon/intron boundaries), the method will predict the following exon, namely, the intron to be excised out. When applied to human sequences in the GenBank database, the method correctly identified 80% of true splice junctions.

Summary

Correct splice site recognition is critical in pre-mRNA splicing. We find that almost all of a diverse panel of exonic splicing silencer (ESS) elements alter splice site choice when placed between competing sites, consistently inhibiting use of intron-proximal 5′ and 3′ splice sites. Supporting a general role for ESSs in splice site definition, we found that ESSs are both abundant and highly conserved between alternative splice site pairs and that mutation of ESSs located between natural alternative splice site pairs consistently shifted splicing toward the intron-proximal site. Some exonic splicing enhancers (ESEs) promoted use of intron-proximal 5′ splice sites, and tethering of hnRNP A1 and SF2/ASF proteins between competing splice sites mimicked the effects of ESS and ESE elements, respectively. Further, we observed that specific subsets of ESSs had distinct effects on a multifunctional intron retention reporter, and that one of these subsets is likely preferred for regulation of endogenous intron retention events. Together, our findings provide a comprehensive picture of the functions of ESSs in the control of diverse types of splicing decisions.

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, χ2-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.

The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests a novel mechanism for intron recognition that compensates for a weakened canonical pre-mRNA splicing motif.

Background

While the current model of pre-mRNA splicing is based on the recognition of four canonical intronic motifs (5' splice site, branchpoint sequence, polypyrimidine (PY) tract and 3' splice site), it is becoming increasingly clear that splicing is regulated by both canonical and non-canonical splicing signals located in the RNA sequence of introns and exons that act to recruit the spliceosome and associated splicing factors. The diversity of human intronic sequences suggests the existence of novel recognition pathways for non-canonical introns. This study addresses the recognition and splicing of human introns that lack a canonical PY tract. The PY tract is a uridine-rich region at the 3' end of introns that acts as a binding site for U2AF65, a key factor in splicing machinery recruitment.

Results

Human introns were classified computationally into low- and high-scoring PY tracts by scoring the likely U2AF65 binding site strength. Biochemical studies confirmed that low-scoring PY tracts are weak U2AF65 binding sites while high-scoring PY tracts are strong U2AF65 binding sites. A large population of human introns contains weak PY tracts. Computational analysis revealed many families of motifs, including C-rich and G-rich motifs, that are enriched upstream of weak PY tracts. In vivo splicing studies show that C-rich and G-rich motifs function as intronic splicing enhancers in a combinatorial manner to compensate for weak PY tracts.

Conclusion

The enrichment of specific intronic splicing enhancers upstream of weak PY tracts suggests that a novel mechanism for intron recognition exists, which compensates for a weakened canonical pre-mRNA splicing motif.

Combinations of different mutations within the 5' splice region of the rabbit beta-globin large intron were analyzed for their effect on in vitro splicing. Based upon the complementarity of the 5' splice region to the 5' terminal region of the U1 snRNA, the exact location of the 5' cleavage site of different mutants could be predicted and was experimentally confirmed. These findings add further strong support to the hypothesis (1) that the exact location of the 5' cleavage site in pre-mRNA splicing of higher eukaryotes is determined by the overall 5' splice region via the complementarity to the 5' end of the U1 snRNA, and not by the strongly conserved GU dinucleotide.

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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.

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We have recently reported a disease-causing substitution (+5G > C) at the donor site of NF-1 exon 3 that produces its skipping. We have now studied in detail the splicing mechanism involved in analyzing RNA–protein complexes at several 5′ splice sites. Characteristic protein patterns were observed by pulldown and band-shift/super-shift analysis. Here, we show that hnRNP H binds specifically to the wild-type GGGgu donor sequence of the NF-1 exon 3. Depletion analyses shows that this protein restricts the accessibility of U1 small nuclear ribonucleoprotein (U1snRNA) to the donor site. In this context, the +5G > C mutation abolishes both U1snRNP base pairing and the 5′ splice site (5′ss) function. However, exon recognition in the mutant can be rescued by disrupting the binding of hnRNP H, demonstrating that this protein enhances the effects of the +5G > C substitution. Significantly, a similar situation was found for a second disease-causing +5G > A substitution in the 5′ss of TSHβ exon 2, which harbors a GGgu donor sequence. Thus, the reason why similar nucleotide substitutions can be either neutral or very disruptive of splicing function can be explained by the presence of specific binding signatures depending on local contexts.

U2 snRNA-intron branchpoint pairing is a critical step in pre-mRNA recognition by the splicing apparatus, but the mechanism by which these two RNAs engage each other is unknown. Here we identify a new U2 snRNA structure, the branchpoint interaction stem-loop (BSL), that presents the U2 nucleotides that will contact the intron. We provide evidence that the BSL forms prior to interaction with the intron, and is disrupted by the DExD/H protein Prp5p during engagement of the snRNA with the intron. In vitro splicing complex assembly in a BSL-destabilized mutant extract suggests that the BSL is required at a previously unrecognized step between commitment complex and prespliceosome formation. The extreme evolutionary conservation of the BSL suggests it represents an ancient structural solution to the problem of intron branchpoint recognition by dynamic RNA elements that must serve multiple functions at other times during splicing.

The Saccharomyces cerevisiae HOP2 gene is required to prevent formation of synaptonemal complex between nonhomologous chromosomes during meiosis. The HOP2 gene is expressed specifically in meiotic cells, with the transcript reaching maximum abundance early in meiotic prophase. The HOP2 coding region is interrupted by an intron located near the 5′ end of the gene. This intron contains a nonconsensus 5′ splice site (GUUAAGU) that differs from the consensus 5′ splice signal (GUAPyGU) by the insertion of a nucleotide and by a single nucleotide substitution. Bases flanking the HOP2 5′ splice site have the potential to pair with sequences in U1 small nuclear RNA, and mutations disrupting this pairing reduce splicing efficiency. HOP2 pre-mRNA is spliced efficiently in the absence of the Mer1 and Nam8 proteins, which are required for splicing the transcripts of two other meiosis-specific genes.

More than 5% of alternatively spliced internal exons in the human genome are derived from Alu elements in a process termed exonization. Alus are comprised of two homologous arms separated by an internal polypyrimidine tract (PPT). In most exonizations, splice sites are selected from within the same arm. We hypothesized that the internal PPT may prevent selection of a splice site further downstream. Here, we demonstrate that this PPT enhanced the selection of an upstream 5′ splice site (5′ss), even in the presence of a stronger 5′ss downstream. Deletion of this PPT shifted selection to the stronger downstream 5′ss. This enhancing effect depended on the strength of the downstream 5′ss, on the efficiency of base-pairing to U1 snRNA, and on the length of the PPT. This effect of the PPT was mediated by the binding of TIA proteins and was dependent on the distance between the PPT and the upstream 5′ss. A wide-scale evolutionary analysis of introns across 22 eukaryotes revealed an enrichment in PPTs within ∼20 nt downstream of the 5′ss. For most metazoans, the strength of the 5′ss inversely correlated with the presence of a downstream PPT, indicative of the functional role of the PPT. Finally, we found that the proteins that mediate this effect, TIA and U1C, and in particular their functional domains, are highly conserved across evolution. Overall, these findings expand our understanding of the role of TIA1/TIAR proteins in enhancing recognition of exons, in general, and Alu exons, in particular.

Author Summary

Human genes are composed of functional regions, termed exons, separated by non-functional regions, termed introns. Intronic sequences may gradually accumulate mutations and subsequently become recognized by the splicing machinery as exons, a process termed exonization. Alu elements are prone to undergo exonization: more than 5% of alternatively spliced internal exons in the human genome originate from Alu elements. A typical Alu element is ∼300 nucleotides long, consisting of two arms separated by a polypyrimdine tract (PPT). Interestingly, in most cases, exonization occurs almost exclusively within either the right arm or the left, not both. Here we found that the PPT between the two arms serves as a binding site for TIA proteins and prevents the exon selection process from expanding into downstream regions. To obtain a wider overview of TIA function, we performed a cross-evolutionary analysis within 22 eukaryotes of this protein and of U1C, a protein known to interact with it, and found that functional regions of both these proteins were highly conserved. These findings highlight the pivotal role of TIA proteins in 5′ splice-site selection of Alu exons and exon recognition in general.

We have investigated electrostatic and surface features of an essential region of the catalytic core of the spliceosome, the eukaryotic precursor messenger (pre-m)RNA splicing apparatus. The nucleophile for the first of two splicing reactions is the 2′-hydroxyl (OH) of the ribose of a specific adenosine within the intron. During assembly of the spliceosome's catalytic core, this adenosine is positioned by pairing with a short region of the U2 small nuclear (sn)RNA to form the pre-mRNA branch site helix. The solution structure of the spliceosomal pre-mRNA branch site [Newby,M.I. and Greenbaum,N.L. (2002) Nature Struct. Biol., 9, 958–965] showed that a phylogenetically conserved pseudouridine (ψ) residue in the segment of U2 snRNA that pairs with the intron induces a markedly different structure compared with that of its unmodified counterpart. In order to achieve a more detailed understanding of the factors that contribute to recognition of the spliceosome's branch site helix and activation of the nucleophile for the first step of pre-mRNA splicing, we have calculated surface areas and electrostatic potentials of ψ-modified and unmodified branch site duplexes. There was no significant difference between the total accessible area or ratio of total polar:nonpolar groups between modified and unmodified duplexes. However, there was substantially greater exposure of nonpolar area of the adenine base, and less exposure of the 2′-OH, in the ψ-modified structure. Electrostatic potentials computed using a hybrid boundary element and finite difference nonlinear Poisson–Boltzmann approach [Boschitsch, A.H. and Fenley, M.O. (2004) J. Comput. Chem., 25, 935–955] revealed a region of exceptionally negative potential in the major groove surrounding the 2′-OH of the branch site adenosine. These surface and electrostatic features may contribute to the overall recognition of the pre-mRNA branch site region by other components of the splicing reaction.

It has been proposed that recognition of the 3' splice site in many group I introns involves base pairing between the start of the 3' exon and a region of the intron known as the internal guide sequence (R. W. Davies, R. B. Waring, J. Ray, T. A. Brown, and C. Scazzocchio, Nature [London] 300:719-724, 1982). We have examined this hypothesis, using the self-splicing rRNA intron from Tetrahymena thermophila. Mutations in the 3' exon that weaken this proposed pairing increased use of a downstream cryptic 3' splice site. Compensatory mutations in the guide sequence that restore this pairing resulted in even stronger selection of the normal 3' splice site. These changes in 3' splice site usage were more pronounced in the background of a mutation (414A) which resulted in an adenine instead of a guanine being the last base of the intron. These results show that the proposed pairing (P10) plays an important role in ensuring that cryptic 3' splice sites are selected against. Surprisingly, the 414A mutation alone did not result in activation of the cryptic 3' splice site.

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