Interactions between the terminal bases of mammalian introns are retained in inosine-containing pre-mRNAs.

Nuclear pre-mRNA splicing has a fundamentally similar two-step mechanism to that employed by group II self-splicing introns. It is believed that nuclear pre-mRNA splicing involves a network of RNA-RNA interactions which form the catalytic core of the active spliceosome. We show here a non-Watson-Crick interaction between the first and last guanosine residues of a mammalian intron. As in Saccharomyces cerevisiae, substitution of the conserved guanosines at the 5' and 3' splice sites by A and C respectively, specifically suppresses step 2 splicing defects resulting from the individual mutations. No other combination of terminal nucleotides was able to restore splicing. We additionally provide independent evidence for an indirect interaction between other nucleotides of the consensus splice sites during step 2 of splicing. Substitution of the nucleotide in the +3 position of the 5' splice site affects competition between closely spaced AG dinucleotides at the 3' splice site, although the interaction is not via direct differential base pairing. Finally, we show that complete substitution of guanosine residues by inosine in a pre-mRNA has only a modest effect upon step 2 of splicing, although earlier spliceosome assembly steps are impaired. Predictions can thus be made about the precise configuration of the non-Watson-Crick interaction between the terminal residues.     

Identification of a U2/U6 helix la mutant that influences 3' splice site selection during nuclear pre-mRNA splicing

Base substitutions in U2/U6 helix I, a conserved base-pairing interaction between the U6 and U2 snRNAs, have previously been found to specifically block the second catalytic step of nuclear pre-mRNA splicing. To further assess the role of U2/U6 helix I in the second catalytic step, we have screened mutations in U2/U6 helix I to identify those that influence 3' splice site selection using a derivative of the yeast actin pre-mRNA. In these derivatives, the spacing between the branch site adenosine and 3' splice site has been reduced from 43 to 12 nt and this results in enhanced splicing of mutants in the conserved 3' terminal intron residue. In this context, mutation of the conserved 3' intron terminal G to a C also results in the partial activation of a nearby cryptic 3' splice site with U as the 3' terminal intron nucleotide. Using this highly sensitive mutant substrate, we have identified a mutation in the U6 snRNA (U57A) that significantly increases the selection of the cryptic 3' splice site over the normal 3' splice site and augments its utilization relative to that observed with the wild-type U2 or U6 snRNAs. In a previous study, we found that the same U6 mutation suppressed the effects of an A-to-G branch site mutation in an allele-specific fashion. The ability of U6-U57 mutants to influence the fidelity of both branch site and 3' splice site recognition suggests that this nucleotide may participate in the formation of the active site(s) of the spliceosome.     

Unexpected metal ion requirements specific for catalysis of the branching reaction in a group II intron

The splicing process catalyzed by group II intron ribozymes follows the same two-step pathway as nuclear pre-mRNA splicing. In vivo, the first splicing step of wild-type introns is a transesterification reaction giving rise to a branched lariat intron-3'-exon intermediate characteristic of this splicing mode. In the wild-type introns, the ribozyme core and the substrate intron-exon junctions are carried by the same precursor molecule, making it difficult to distinguish between RNA folding and catalysis under normal splicing reactions. To characterize the catalytic step of the first transesterification reaction, we studied the reversal of this reaction, reverse branching. In this reverse reaction, the excised lariat intron and the substrate 5'-exon can be preincubated and folded separately, allowing the measure of the catalytic rate of the reaction. To measure the catalytic rate of the second splicing step, purified lariat intron-3'-exon intermediate molecules were preincubated and folded prior to the addition of 5'-exon. Conditions could be found where chemistry appeared rate limiting for both catalytic steps. Study of the metal ion requirements under these conditions resulted in the unexpected finding that, for the intron studied, substitution of magnesium ions by manganese ions enhanced the rate of the first transesterification reaction by two orders of magnitude but had virtually no effect on the second transesterification reaction or the 5' splice site cleavage by hydrolysis. Finally, the catalytic rates measured under optimal conditions for both splicing steps were faster by three orders of magnitude in the branching pathway than in the hydrolytic pathway.     

In vivo commitment to splicing in yeast involves the nucleotide upstream from the branch site conserved sequence and the Mud2 protein.

Pre-mRNA splicing is a stepwise nuclear process involving intron recognition and the assembly of the spliceosome followed by intron excision. We previously developed a pre-mRNA export assay that allows the discrimination between early steps of spliceosome formation and splicing per se. Here we present evidence that these two assays detect different biochemical defects for point mutations. Mutations at the 5' splice site lead to pre-mRNA export, whereas 3' splice site mutations do not. A genetic screen applied to mutants in the branch site region shows that all positions in the conserved TACTAAC sequence are important for intron recognition. An exhaustive analysis of pre-mRNA export and splicing defects of these mutants shows that the in vivo recognition of the branch site region does not involve the base pairing of U2 snRNA with the pre-mRNA. In addition, the nucleotide preceding the conserved TACTAAC sequence contributes to the recognition process. We show that a T residue at this position allows for optimal intron recognition and that in natural introns, this nucleotide is also used preferentially. Moreover, the Mud2 protein is involved in the recognition of this nucleotide, thus establishing a role for this factor in the in vivo splicing pathway.     

Identification of a human protein that recognizes the 3'' splice site during the second step of pre-mRNA splicing.

Accurate splicing of precursor mRNAs (pre-mRNAs) requires recognition of the 5' and 3' splice sites at the intron boundaries. Interactions between several splicing factors and the 5' splice site, which occur prior to the first step of splicing, have been well described. In contrast, recognition of the 3' splice site, which is cleaved during the second catalytic step, is poorly understood, particularly in higher eukaryotes. Here, using site-specific photo-crosslinking, we find that the conserved AG dinucleotide at the 3' splice site is contacted specifically by a 70 kDa polypeptide (p70). The p70-3' splice site crosslink has kinetics and biochemical requirements similar to those of splicing, was detected only in the mature spliceosome and occurs subsequent to the first step. Thus, p70 has all the properties expected of a factor that functionally interacts with the 3' splice site during the second step of splicing. Using antisera to various known splicing factors, we find that p70 corresponds to a previously reported 69 kDa protein of unknown function associated with the Sm core domain of spliceosomal small nuclear ribonucleoproteins.     

A conformational rearrangement in the spliceosome is dependent on PRP16 and ATP hydrolysis.

PRP16 is an RNA-dependent ATPase that is required for the second catalytic step of pre-mRNA splicing. We have previously shown that PRP16 protein binds stably to spliceosomes that have completed 5' splice site cleavage and lariat formation. PRP16 then promotes 3' splice site cleavage and exon ligation in an ATP-dependent fashion. We now demonstrate that PRP16 can hydrolyse all nucleoside triphosphates and corresponding deoxynucleotides; complementation of the second catalytic step shows the same broad nucleotide specificity. These results link the nucleotide requirement of step 2 to PRP16. Interestingly, we find that PRP16 promotes a conformational change in the spliceosome which results in the protection of the 3' splice site against oligo-directed RNase H cleavage. This structural rearrangement is dependent on the hydrolysis of ATP, since ATP gamma S, a competitive inhibitor of the PRP16 ATPase activity, does not promote the protection of the 3' splice site and formation of mRNA.     

Alterations of RNase H sensitivity of the 3'' splice site region during the in vitro splicing reaction.

We have developed a splicing assay system with an immobilized pre-mRNA to study the mechanism of the splicing reaction after spliceosome assembly. Using this system, we have found that the second step of the splicing reaction could be dissected into two stages. After the 5' splice site reaction, at least two factors interact with the pre-formed spliceosome containing intermediate molecules in an ATP-independent manner to convert the spliceosome into a form competent for the 3' splice site reaction. Then, the 3' splice site reaction occurs on this spliceosome, if ATP is supplied to the reaction mixture. We have also investigated the dynamic state of the 3' splice site region in the spliceosomes during the splicing reaction by probing with RNase H sensitivity. Prior to the 5' splice site reaction, the 3' splice site region was protected from RNase H attack. The region became sensitive immediately after the 5' splice site reaction, and subsequently became resistant again as the spliceosome competent for the 3' splice site reaction was formed. These results suggest that the interaction of the 3' splice site region with some spliceosome components changes significantly during the splicing reaction.     

Requirement for SLU7 in yeast pre-mRNA splicing is dictated by the distance between the branchpoint and the 3' splice site.

Yeast pre-mRNA splicing factors SLU7 and PRP16 are required for cleavage of the 3' splice site and exon ligation in vitro. Using natural and model precursor RNAs, we found that SLU7 is dispensable for splicing of RNAs in which the 3' splice site is in close proximity to the branchpoint. SLU7 is only required when the interval between the branchpoint and the 3' splice site is greater than 7 nt. In contrast, PRP16 is essential for splicing of all pre-mRNAs tested. Immunoprecipitation of the products of step 1 by anti-SLU7 antibodies demonstrates that SLU7 is a component of the spliceosome. Recruitment of SLU7 to the spliceosome is greatly enhanced by prior addition of PRP16. PRP16 is liberated from the spliceosome after completion of step 2, whereas SLU7 remains bound to the excised intron and spliced mature RNA until the spliceosome disassembles, in a reaction that requires ATP.     

An LKB1 AT-AC intron mutation causes Peutz-Jeghers syndrome via splicing at noncanonical cryptic splice sites

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disorder associated with gastrointestinal polyposis and an increased cancer risk. PJS is caused by germline mutations in the tumor suppressor gene LKB1. One such mutation, IVS2+1A>G, alters the second intron 5' splice site, which has sequence features of a U12-type AT-AC intron. We report that in patients, LKB1 RNA splicing occurs from the mutated 5' splice site to several cryptic, noncanonical 3' splice sites immediately adjacent to the normal 3' splice site. In vitro splicing analysis demonstrates that this aberrant splicing is mediated by the U12-dependent spliceosome. The results indicate that the minor spliceosome can use a variety of 3' splice site sequences to pair to a given 5' splice site, albeit with tight constraints for maintaining the 3' splice site position. The unusual splicing defect associated with this PJS-causing mutation uncovers differences in splice-site recognition between the major and minor pre-mRNA splicing pathways.     

Phosphorothioate substitution identifies phosphate groups important for pre-mRNA splicing.

Substitution of pre-mRNA in vitro splicing substrates with alpha-phosphorothioate ribonucleotide analogs has multiple effects on the processes of spliceosome formation and splicing. A major effect of substitution is on the splicing cleavage/ligation reactions. Substitution at the 5' splice junction blocks the first cleavage/ligation reaction while substitution at the 3' splice junction blocks the second cleavage/ligation reaction. A second effect of phosphorothioate substitution is the inhibition of spliceosome formation. A substitution/interference assay was used to determine positions where substitution inhibits spliceosome formation or splicing. Substitution in the 3' splice site polypyrimidine tract was found to inhibit spliceosome formation and splicing. This effect was enhanced with multiple substitutions in the region. No sites of substitution within the exons were found which affected spliceosome formation or splicing.     

Repositioning of the reaction intermediate within the catalytic center of the spliceosome

Conformational change within the spliceosome is required between the first catalytic step of pre-mRNA splicing, when the branch site attacks the 5' splice site (SS), and the second step, when the 5' exon attacks the 3'SS. Little is known, however, about repositioning of the reaction substrates during this transition. Whereas the 5'SS is positioned for the first step by pairing with the invariant U6 snRNA-ACAGAG site, we demonstrate that this pairing interaction must be disrupted to allow transition to the second step. We propose that removal of the branch structure from the catalytic center is in competition with binding of the 3'SS substrate for the second step. Changes in the relative occupancy of first and second step substrates at the catalytic center alter efficiency of the two steps of splicing, allowing use of suboptimal intron sequences and thereby altering substrate selectivity.     

Mechanisms of fidelity in pre-mRNA splicing

The pre-mRNA splicing machinery consists of five small nuclear RNAs (U1, U2, U4, U5 and U6) and more than fifty proteins. Over the past year, important advances have been made in understanding how these factors function to achieve fidelity in splicing. Of particular note were the discoveries that the splicing factor U2AF(35) recognizes the AG dinucleotide at the 3' splice site early in spliceosome assembly, that a DEAD-box ATPase, Prp28, triggers specific rearrangements of the spliceosome, and that the splicing factor hSlu7 functions in the fidelity of AG choice during catalytic step II of splicing.     

RS domain-splicing signal interactions in splicing of U12-type and U2-type introns

Serine-arginine (SR) proteins are general metazoan splicing factors that contain an essential arginine/serine-rich (RS) domain. On typical U2-type introns, RS domains contact the branchpoint and 5' splice site to promote base-pairing with U small nuclear RNAs (snRNAs). Here we analyze the role of SR proteins in splicing of U12-type introns and in the second step of U2-type intron splicing. We show that RS domains contact the branchpoint and 5' splice site of a U12-type intron. On a U2-type intron, we find that the RS domain contacts the site of the U6 snRNA-5' splice site interaction during the first step of splicing and shifts to contact the site of the U5 snRNA-exon 1 interaction during the second step. Our results reveal alternative interactions between the RS domain and 5' splice site region that coincide with remodeling of the spliceosome between the two catalytic steps.     

Antisense-targeted immuno-EM localization of the pre-mRNA path in the spliceosomal C complex

A first step in understanding the architecture of the spliceosome is elucidating the positions of individual spliceosomal components and functional centers. Catalysis of the first step of pre-mRNA splicing leads to the formation of the spliceosomal C complex, which contains the pre-mRNA intermediates--the cleaved 5' exon and the intron-3' exon lariat. To topographically locate the catalytic center of the human C complex, we first determined, by DNA oligonucleotide-directed RNAse H digestions, accessible pre-mRNA regions closest to nucleotides of the cleaved 5' splice site (i.e., the 3' end of exon 1 and the 5' end of the intron) and the intron lariat branch point, which are expected to be at/near the catalytic center in complex C. For electron microscopy (EM) localization studies, C complexes were allowed to form, and biotinylated 2'-OMe RNA oligonucleotides were annealed to these accessible regions. To allow localization by EM of the bound oligonucleotide, first antibiotin antibodies and then protein A-coated colloidal gold were additionally bound. EM analyses allowed us to map the position of exon and intron nucleotides near the cleaved 5' splice site, as well as close to the anchoring site just upstream of the branch adenosine. The identified positions in the C complex EM map give first hints as to the path of the pre-mRNA splicing intermediates in an active spliceosomal C complex and further define a possible location for its catalytic center.     

A spontaneous duplication in U6 spliceosomal RNA uncouples the early and late functions of the ACAGA element in vivo.

U6 RNA enters the spliceosome base paired with U4 RNA, but dissociates from U4 RNA before the catalytic steps of splicing. We have identified a cold-sensitive lethal mutation in U4 RNA (U4-cs1) that blocks the splicing pathway after U4/U6 complex formation, but before the first catalytic step of splicing. Remarkably, selection for suppressors of the cold-sensitive growth of the U4-cs1 strain yielded a tandem duplication of the highly conserved ACAGA sequence of U6 RNA (U6-Dup). The ACAGA sequence plays an essential role in spliceosome assembly and in the second catalytic step of pre-mRNA splicing; one or both of these roles involves direct base pairing to the pre-mRNA 5' splice site. In a U4-cs1/U6-Dup double-mutant strain grown at low temperature, the upstream ACAGA sequence of U6 RNA is required for suppression of the U4 mutation, whereas the downstream ACAGA sequence is required for other essential functions. Based on the sequence requirements for function of the upstream ACAGA element of U6-Dup, we propose that it pairs with the pre-mRNA 5' splice site during incorporation of the U4/U6 complex into the spliceosome and that the subsequent dissociation of U4 RNA exposes the downstream ACAGA sequence, which functions in the catalytic steps. The properties of this mutant U4/U6 complex provide compelling in vivo evidence that U6 RNA normally base pairs with the 5' splice site before disruption of its pairing with U4 RNA.     

Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing.

In order to assess the role of Prp22 in yeast pre-mRNA splicing, we have purified the 130 kDa Prp22 protein and developed an in vitro depletion/reconstitution assay. We show that Prp22 is required for the second step of actin pre-mRNA splicing. Prp22 can act on pre-assembled spliceosomes that are arrested after step 1 in an ATP-independent fashion. The requirement for Prp22 during step 2 depends on the distance between the branchpoint and the 3' splice site, suggesting a previously unrecognized role for Prp22 in splice site selection. We characterize the biochemical activities of Prp22, a member of the DExH-box family of proteins, and we show that purified recombinant Prp22 protein is an RNA-dependent ATPase and an ATP-dependent RNA helicase. Prp22 uses the energy of ATP hydrolysis to effect the release of mRNA from the spliceosome. Thus, Prp22 has two distinct functions in yeast pre-mRNA splicing: an ATP-independent role during the second catalytic step and an ATP-requiring function in disassembly of the spliceosome.     

Effects of secondary structure on pre-mRNA splicing: hairpins sequestering the 5'' but not the 3'' splice site inhibit intron processing in Nicotiana plumbaginifolia.

We have performed a systematic study of the effect of artificial hairpins on pre-mRNA splicing in protoplasts of a dicot plant, Nicotiana plumbaginifolia. Hairpins with a potential to form 18 or 24 bp stems strongly inhibit splicing when they sequester the 5' splice site or are placed in the middle of short introns. However, similar 24 bp hairpins sequestering the 3' splice site do not prevent this site from being used as an acceptor. Utilization of the stem-located 3' site requires that the base of the stem is separated from the upstream 5' splice site by a minimum of approximately 45 nucleotides and that another 'helper' 3' splice site is present downstream of the stem. The results indicate that the spliceosome or factors associated with it may have a potential to unfold secondary structure present in the downstream portion of the intron, prior to or at the step of the 3' splice site selection. The finding that the helper 3' site is required for utilization of the stem-located acceptor confirms and extends previous observations, obtained with HeLa cell in vitro splicing systems, indicating that the 3' splice site may be recognized at least twice during spliceosome assembly.     

The spliceosome catalyzes debranching in competition with reverse of the first chemical reaction

Splicing of nuclear pre-mRNA occurs via two steps of the transesterification reaction, forming a lariat intermediate and product. The reactions are catalyzed by the spliceosome, a large ribonucleoprotein complex composed of five small nuclear RNAs and numerous protein factors. The spliceosome shares a similar catalytic core structure with that of fungal group II introns, which can self-splice using the same chemical mechanism. Like group II introns, both catalytic steps of pre-mRNA splicing can efficiently reverse on the affinity-purified spliceosome. The spliceosome also catalyzes a hydrolytic spliced-exon reopening reaction as observed in group II introns, indicating a strong link in their evolutionary relationship. We show here that, by arresting splicing after the first catalytic step, the purified spliceosome can catalyze debranching of lariat-intron-exon 2. The debranching reaction, although not observed in group II introns, has similar monovalent cation preferences as those for splicing catalysis of group II introns. The debranching reaction is in competition with the reverse Step 1 reaction influenced by the ionic environment and the structure of components binding near the catalytic center, suggesting that the catalytic center of the spliceosome can switch between different conformations to direct different chemical reactions.     

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.