Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing

Although both U1 and U2 snRNPs have been implicated in the splicing process, their respective roles in the earliest stages of intron recognition and spliceosome assembly are uncertain. To address this issue, we developed a new strategy to prepare snRNP-depleted splicing extracts using Saccharomyces cerevisiae cells conditionally expressing U1 or U2 snRNP. Complementation analyses and chase experiments show that a stable complex, committed to the splicing pathway, forms in the absence of U2 snRNP. U1 snRNP and a substrate containing both a 5' splice site and a branchpoint sequence are required for optimal formation of this commitment complex. We developed new gel electrophoresis conditions to identify these committed complexes and to show that they contain U1 snRNA. Chase experiments demonstrated that these complexes are functional intermediates in spliceosome assembly and splicing. Our results have implications for the process of splice site selection.     

Specific accessory sequences in Saccharomyces cerevisiae introns control assembly of pre-mRNAs into spliceosomes.

In experiments involving deletion and rearrangement of intron sequences two small regions of the intron in the yeast CYH2 ribosomal protein gene were found to play important roles in splicing of the pre-mRNA. One element lies downstream of the 5' splice site, and the other is upstream of the branchpoint sequence UACUAAC. Deletion of the element upstream of the branchpoint prevents spliceosome formation and blocks splicing in vivo and in vitro. Deletion of the element downstream of the 5' splice site does not on its own block splicing but rescues spliceosome formation and splicing of pre-mRNA lacking the element upstream of the branchpoint. These elements correspond to two regions of sequence complementarity which are a conserved feature of the introns in yeast pre-mRNAs. Mixing and matching of the elements from the ACT1 and CYH2 gene introns showed that these elements can cooperate in an intron-specific fashion to control spliceosome assembly.     

Species-specific signals for the splicing of a short Drosophila intron in vitro.

The effects of branchpoint sequence, the pyrimidine stretch, and intron size on the splicing efficiency of the Drosophila white gene second intron were examined in nuclear extracts from Drosophila and human cells. This 74-nucleotide intron is typical of many Drosophila introns in that it lacks a significant pyrimidine stretch and is below the minimum size required for splicing in human nuclear extracts. Alteration of sequences of adjacent to the 3' splice site to create a pyrimidine stretch was necessary for splicing in human, but not Drosophila, extracts. Increasing the size of this intron with insertions between the 5' splice site and the branchpoint greatly reduced the efficiency of splicing of introns longer than 79 nucleotides in Drosophila extracts but had an opposite effect in human extracts, in which introns longer than 78 nucleotides were spliced with much greater efficiency. The white-apricot copia insertion is immediately adjacent to the branchpoint normally used in the splicing of this intron, and a copia long terminal repeat insertion prevents splicing in Drosophila, but not human, extracts. However, a consensus branchpoint does not restore the splicing of introns containing the copia long terminal repeat, and alteration of the wild-type branchpoint sequence alone does not eliminate splicing. These results demonstrate species specificity of splicing signals, particularly pyrimidine stretch and size requirements, and raise the possibility that variant mechanisms not found in mammals may operate in the splicing of small introns in Drosophila and possibly other species.     

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.     

Identification of proteins that interact with exon sequences, splice sites, and the branchpoint sequence during each stage of spliceosome assembly.

We have carried out a systematic analysis of the proteins that interact with specific intron and exon sequences during each stage of mammalian spliceosome assembly. This was achieved by site-specifically labeling individual nucleotides within the 5' and 3' splice sites, the branchpoint sequence (BPS), or the exons with 32P and identifying UV-cross-linked proteins in the E, A, B, or C spliceosomal complex. Significantly, two members of the SR family of splicing factors, which are known to promote E-complex assembly, cross-link within exon sequences to a region approximately 25 nucleotides upstream from the 5' splice site. At the 5' splice site, cross-linking of the U5 small nuclear ribonucleoprotein particle protein, U5(200), was detected in both the B and C complexes. As observed in yeast cells, U5(200), also cross-links to intron/exon sequences at the 3' splice site in the C complex and may play a role in aligning the 5' and 3' exons for ligation. With label at the branch site, we detected three distinct proteins, designated BPS72,BpS70, and BPS56, which replace one another in the E, A, and C complexes. Another dynamic exchange was detected with pre-mRNA labeled at the AG dinucleotide of the 3' splice site. In this case, a protein, AG100,cross-links in the A complex and is replaced by another protein, AG75, in the C complex. The observation that these proteins are specifically associated with critical pre-mRNA sequence elements in functional complexes at different stages of spliceosome assembly implicates roles for these factors in key recognition events during the splicing pathway.     

Intron definition in splicing of small Drosophila introns.

Approximately half of the introns in Drosophila melanogaster are too small to function in a vertebrate and often lack the pyrimidine tract associated with vertebrate 3' splice sites. Here, we report the splicing and spliceosome assembly properties of two such introns: one with a pyrimidine-poor 3' splice site and one with a pyrimidine-rich 3' splice site. The pyrimidine-poor intron was absolutely dependent on its small size for in vivo and in vitro splicing and assembly. As such, it had properties reminiscent of those of yeast introns. The pyrimidine-rich intron had properties intermediate between those of yeasts and vertebrates. This 3' splice site directed assembly of ATP-dependent complexes when present as either an intron or exon and supported low levels of in vivo splicing of a moderate-length intron. We propose that splice sites can be recognized as pairs across either exons or introns, depending on which distance is shorter, and that a pyrimidine-rich region upstream of the 3' splice site facilitates the exon mode.     

Functional group substitutions of the branchpoint adenosine in a nuclear pre-mRNA and a group II intron.

Splicing of nuclear mRNA precursors (pre-mRNAs) takes place in the spliceosome, a large and complex ribonucleoprotein. Nuclear pre-mRNA splicing and group II intron self-splicing occur by a chemically identical pathway involving recognition of a specific branchpoint adenosine and nucleophilic activation of its 2'-hydroxyl group. The chemical similarity between these two splicing reactions, as well as other considerations, have suggested that the catalytic core of the spliceosome and group II introns may be related. Here we test this hypothesis by analyzing splicing and RNA branch formation of a pre-mRNA and a group II intron in which the branchpoint adenosine was substituted with purine base analogues. We find that replacement of the branchpoint adenosine with either of two modified adenosine analogues or guanosine leads to remarkably similar patterns of splicing and RNA branch formation in the two systems.     

A pathway of sequential arginine-serine-rich domain-splicing signal interactions during mammalian spliceosome assembly

Serine-arginine (SR) proteins are general splicing factors and can function through binding to exonic splicing enhancers (ESEs). SR proteins and several other mammalian splicing factors contain an arginine-serine-rich (RS) domain required to promote splicing. We have recently found that the ESE bound RS domain functions by contacting the branchpoint. Here, we perform RNA-protein crosslinking experiments to show that the branchpoint is sequentially contacted first in complex E by the RS domain of the essential splicing factor U2AF(65) and then in the prespliceosome by the ESE bound RS domain. Although the ESE bound RS domain can promote formation of the prespliceosome, at least one additional SR protein is required for complete spliceosome assembly. We show that the RS domain of this additional SR protein contacts the 5' splice site specifically in the mature spliceosome. We propose that direct contact with splicing signals is a general mechanism by which RS domains promote splicing.     

Requirements for intron-mediated enhancement of gene expression in Arabidopsis

To explore possible mechanisms of intron-mediated enhancement of gene expression, the features of PAT1 intron 1 required to elevate mRNA accumulation were systematically tested in transgenic Arabidopsis. This intron is remarkably resilient, retaining some ability to increase mRNA accumulation when splicing was prevented by mutation of 5' and 3' splice sites, branchpoint sequences, or when intron U-richness was reduced. Enhancement was abolished by simultaneously eliminating branchpoints and the 5' splice site, structures involved in the first two steps of spliceosome assembly. Although this suggests that the splicing machinery is required, intron splicing is clearly not enough to enhance mRNA accumulation. Five other introns were all efficiently spliced but varied widely in their ability to increase mRNA levels. Furthermore, PAT1 intron 1 was spliced but lost the ability to elevate mRNA accumulation when moved to the 3' UTR. These findings demonstrate that splicing per se is neither necessary nor sufficient for an intron to enhance mRNA accumulation, and suggest a mechanism that requires intron recognition by the splicing machinery but also involves nonconserved intron sequences.     

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.     

The branchpoint residue is recognized during commitment complex formation before being bulged out of the U2 snRNA-pre-mRNA duplex.

We have analyzed the mechanism of branchpoint nucleotide selection during the first step of pre-mRNA splicing. It has previously been proposed that the branchpoint is selected as an adenosine residue bulged out of an RNA helix formed by the U2 snRNA-pre-mRNA base pairing. Although compatible with this bulge hypothesis, available data from both yeast and mammalian systems did not rule out alternative structures for the branch nucleotide. Mutating the residue preceding the branchpoint nucleotide in our reporter construct conferred a splicing defect that was suppressed in vivo by the complementary U2 snRNA mutants. In contrast, substitutions on the 3' side of the branchpoint could be suppressed by complementary U2 snRNA mutants only in a weakened intron context. To test why the identity of the branch nucleotide was important for its selection, we analyzed the effect of substitutions at this position on spliceosome assembly. We observed that these mutations block the formation of one of the two commitment complexes. Our results demonstrate that yeast branchpoint selection occurs in multiple steps. The nature of the branch residue is recognized, in the absence of U2 snRNA, during commitment complex formation. Then, base pairing with U2 snRNA constrains this residue into a bulge conformation.     

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.     

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.     

An intronic (A/U)GGG repeat enhances the splicing of an alternative intron of the chicken beta-tropomyosin pre-mRNA.

Computer analysis of human intron sequences have revealed a 50 nucleotide (nt) GC-rich region downstream of the 5' splice site; the trinucleotide GGG occurs almost four times as frequently as it would in a random sequence. The 5' part of a beta-tropomyosin intron exhibits six repetitions of the motif (A/U)GGG. In order to test whether these motifs play a role in the splicing process we have mutated some or all of them. Mutated RNAs show a lower in vitro splicing efficiency when compared with the wild-type, especially when all six motifs are mutated (> 70% inhibition). Assembly of the spliceosome complex B and, to a lesser extent, of the pre-spliceosome complex A also appears to be strongly affected by this mutation. A 55 kDa protein within HeLa cell nuclear extract is efficiently cross-linked to the G-rich region. This protein is present in the splicing complexes and its cross-linking to the pre-mRNA requires the presence of one or several snRNP. Altogether our results suggest that the G-rich sequences present in the 5' part of introns may act as an enhancer of the splicing reaction at the level of spliceosome assembly.     

The yeast PRP8 protein interacts directly with pre-mRNA.

The PRP8 protein of Saccharomyces cerevisiae is required for nuclear pre-mRNA splicing. Previously, immunological procedures demonstrated that PRP8 is a protein component of the U5 small nuclear ribonucleoprotein particle (U5 snRNP), and that PRP8 protein maintains a stable association with the spliceosome during both step 1 and step 2 of the splicing reaction. We have combined immunological analysis with a UV-crosslinking assay to investigate interaction(s) of PRP8 protein with pre-mRNA. We show that PRP8 protein interacts directly with splicing substrate RNA during in vitro splicing reactions. This contact event is splicing-specific in that it is ATP-dependent, and does not occur with mutant RNAs that contain 5' splice site or branchpoint mutations. The use of truncated RNA substrates demonstrated that the assembly of PRP8 protein into splicing complexes is not, by itself, sufficient for the direct interaction with the RNA; PRP8 protein only becomes UV-crosslinked to RNA substrates capable of participating in step 1 of the splicing reaction. We propose that PRP8 protein may play an important structural and/or regulatory role in the spliceosome.     

The yeast branchpoint sequence is not required for the formation of a stable U1 snRNA-pre-mRNA complex and is recognized in the absence of U2 snRNA.

Commitment complexes contain U1 snRNP as well as pre-mRNA and are the earliest functional complexes that have been described during in vitro spliceosome assembly. We have used a gel retardation assay to analyze the role of the yeast pre-mRNA cis-acting sequences in commitment complex formation. The results suggest that only a proper 5' splice site sequence is required for efficient U1 snRNA-pre-mRNA complex formation. A role for the highly conserved UACUAAC branchpoint sequence is indicated, however, by competition experiments and by the direct analysis of branchpoint mutant substrates, which cannot form one of the two commitment complex species observed with wild-type substrates. The results suggest that the formation of a U1 snRNP-pre-mRNA complex is not dependent upon the presence of a branchpoint sequence but that the branchpoint sequence is recognized prior to U2 snRNP addition during in vitro spliceosome assembly.     

Characterization of human RNA splice signals by iterative functional selection of splice sites

An iterative in vitro splicing strategy was employed to select for optimal 3' splicing signals from a pool of pre-mRNAs containing randomized regions. Selection of functional branchpoint sequences in HeLa cell nuclear extract yielded a sequence motif that evolved from UAA after one round of splicing toward a UACUAAC consensus after seven rounds. A significant part of the selected sequences contained a conserved AAUAAAG motif that proved to be functional both as a polyadenylation signal and a branch site in a competitive manner. Characterization of the branchpoint in these clones to either the upstream or downstream adenosines of the AAUAAAG sequence revealed that the branching process proceeded efficiently but quite promiscuously. Surprisingly, the conserved guanosine, adjacent to the common AAUAAA polyadenylation motif, was found to be required only for polyadenylation. In an independent experiment, sequences surrounding an optimal branchpoint sequence were selected from two randomized 20-nt regions. The clones selected after six rounds of splicing revealed an extended polypyrimidine tract with a high frequency of UCCU motifs and a highly conserved YAG sequence in the extreme 3' end of the randomized insert. Mutating the 3' terminal guanosine of the intron strongly affects complex A formation, implying that the invariant AG is recognized early in spliceosome assembly.     

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.