Both the polypyrimidine tract and the 3' splice site function prior to the first step of splicing in fission yeast.

While it is known that several trans -acting splicing factors are highly conserved between Schizosaccharomyces pombe and mammals, the roles of cis -acting signals have received comparatively little attention. In Saccharomyces cerevisiae, sequences downstream from the branch point are not required prior to the first transesterification reaction, whereas in mammals the polypyrimidine tract and, in some introns, the 3' AG dinucleotide are critical for initial recognition of an intron. We have investigated the contribution of these two sequence elements to splicing in S.pombe. To determine the stage at which the polypyrimidine tract functions, we analyzed the second intron of the cdc2 gene (cdc 2-Int2), in which pyrimidines span the entire interval between the branch point and 3' splice site. Our data indicate that substitution of a polypurine tract results in accumulation of linear pre-mRNA, while expanding the polypyrimidine tract enhances splicing efficiency, as in mammals. To examine the role of the AG dinucleotide in cdc 2-Int2 splicing, we mutated the 3' splice junction in both the wild-type and pyrimidine tract variant RNAs. These changes block the first transesterification reaction, as in a subset of mammalian introns. However, in contrast to the situation in mammals, we were unable to rescue the first step of splicing in a 3' splice site mutant by expanding the polypyrimidine tract. Mutating the terminal G in the third intron of the nda 3 gene (nda 3-Int3) also blocks the first transesterification reaction, suggesting that early recognition of the 3' splice site is a general property of fission yeast introns. Counter to earlier work with an artificial intron, it is not possible to restore the first step of splicing in cdc 2-Int2 and nda 3-Int3 3' splice site mutants by introducing compensatory changes in U1 snRNA. These results highlight the diversity and probable redundancy of mechanisms for identifying the 3' ends of introns.     

Mutational analysis of the U12-dependent branch site consensus sequence

Highly conserved sequences at the 5′ splice site and branch site of U12-dependent introns are important determinants for splicing by U12-dependent spliceosomes. This study investigates the in vivo splicing phenotypes of mutations in the branch site consensus sequence of the U12-dependent intron F from a human NOL1 (P120) minigene. Intron F contains a fully consensus branch site sequence (UUCCUUAAC). Mutations at each position were analyzed for their effects on U12-dependent splicing in vivo. Mutations at most positions resulted in a significant reduction of correct U12-dependent splicing. Defects observed included increased unspliced RNA levels, the activation of cryptic U2-dependent 5′ and 3′ splice sites, and the activation of cryptic U12-dependent branch/3′ splice sites. A strong correlation was observed between the predicted thermodynamic stability of the branch site: U12 snRNA interaction and correct U12-dependent splicing. The lack of a polypyrimidine tract between the branch site and 3′ splice site of U12-dependent introns and the observed reliance on base-pairing interactions for correct U12-dependent splicing emphasize the importance of RNA/RNA interactions during U12-dependent intron recognition and proper splice site selection.     

mRNA processing in Dictyostelium: sequence requirements for termination and splicing

Criteria for the identification of termination regions in Dictyostelium discoideum genes have been established and the sequence requirements for termination in 33 genes have been analyzed. A canonical hexamer signal AATAAA was present 15-30 nucleotides upstream of the cleavage site, usually a TA, and was embedded in a particularly A-rich environment. T- or GT-rich downstream elements characteristic of animal cells could not be identified. In a sample of 102 introns we have established the consensus AG/GTAAGT and ATAG/ for the 5' and 3' splice sites, respectively. Most introns are 75-150 nucleotides long and the A+T content is high (90%). A putative branch point was identified in half of the introns 20-60 nucleotides upstream of the 3' splice site and the consensus TACTAAY was derived. A polypyrimidine tract required for branching in vertebrates was not identified, but weak preference for pyrimidine was found 10-45 nucleotides upstream of the 3' splice site.     

Functional analysis of the polypyrimidine tract in pre-mRNA splicing.

The polypyrimidine tract is one of the important cis-acting sequence elements directing intron removal in pre-mRNA splicing. Progressive deletions of the polypyrimidine tract have been found to abolish correct lariat formation, spliceosome assembly and splicing. In addition, the polypyrimidine tract can alter 3'-splice site selection by promoting alternative branch site selection. However, there appears to be great flexibility in the specific sequence of a given tract. Not only the optimal composition of the polypyrimidine tract, but also the role of the tract in introns with no apparent polypyrimidine tracts or where changes in the tract are apparently harmless are uncertain. Accordingly, we have designed a series of cis-competition splicing constructs to test the functional competitive efficiency of a variety of systematically mutated polypyrimidine tracts. An RT/PCR assay was used to detect spliced product formation as a result of differential branch point selection dependent on direct competition between two opposing polypyrimidine tracts. We found that pyrimidine tracts containing 11 continuous uridines are the strongest pyrimidine tracts. In such cases, the position of the uridine stretch between the branch point and 3'-splice site AG is unimportant. In contrast, decreasing the continuous uridine stretch to five or six residues requires that the tract be located immediately adjacent to the AG for optimal competitive efficiency. The block to splicing with decreasing polypyrimidine tract strength is primarily prior to the first step of splicing. While lengthy continuous uridine tracts are the most competitive, tracts with decreased numbers of consecutive uridines and even tracts with alternating purine/pyrimidine residues can still function to promote branch point selection, but are far less effective competitors in 3'-splice site selection assays.     

Identification of motifs that function in the splicing of non-canonical introns

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.     

A class of human exons with predicted distant branch points revealed by analysis of AG dinucleotide exclusion zones

Background

The three consensus elements at the 3' end of human introns - the branch point sequence, the polypyrimidine tract, and the 3' splice site AG dinucleotide - are usually closely spaced within the final 40 nucleotides of the intron. However, the branch point sequence and polypyrimidine tract of a few known alternatively spliced exons lie up to 400 nucleotides upstream of the 3' splice site. The extended regions between the distant branch points (dBPs) and their 3' splice site are marked by the absence of other AG dinucleotides. In many cases alternative splicing regulatory elements are located within this region.

Results

We have applied a simple algorithm, based on AG dinucleotide exclusion zones (AGEZ), to a large data set of verified human exons. We found a substantial number of exons with large AGEZs, which represent candidate dBP exons. We verified the importance of the predicted dBPs for splicing of some of these exons. This group of exons exhibits a higher than average prevalence of observed alternative splicing, and many of the exons are in genes with some human disease association.

Conclusion

The group of identified probable dBP exons are interesting first because they are likely to be alternatively spliced. Second, they are expected to be vulnerable to mutations within the entire extended AGEZ. Disruption of splicing of such exons, for example by mutations that lead to insertion of a new AG dinucleotide between the dBP and 3' splice site, could be readily understood even though the causative mutation might be remote from the conventional locations of splice site sequences.     

Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions

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.     

Different effects of intron nucleotide composition and secondary structure on pre-mRNA splicing in monocot and dicot plants.

We have found previously that the sequences important for recognition of pre-mRNA introns in dicot plants differ from those in the introns of vertebrates and yeast. Neither a conserved branch point nor a polypyrimidine tract, found in yeast and vertebrate introns respectively, are required. Instead, AU-rich sequences, a characteristic feature of dicot plant introns, are essential. Here we show that splicing in protoplasts of maize, a monocot, differs significantly from splicing in a dicot, Nicotiana plumbaginifolia. As in the case of dicots, a conserved branch point and a polypyrimidine tract are not required for intron processing in maize. However, unlike in dicots, AU-rich sequences are not essential, although their presence facilitates splicing if the splice site sequences are not optimal. The lack of an absolute requirement for AU-rich stretches in monocot introns in reflected in the occurrence of GC-rich introns in monocots but not in dicots. We also show that maize protoplasts are able to process a mammalian intron and short introns containing stem--loops, neither of which are spliced in N.plumbaginifolia protoplasts. The ability of maize, but not of N.plumbaginifolia to process stem--loop-containing or GC-rich introns suggests that one of the functions of AU-rich sequences during splicing of dicot plant pre-mRNAs may be to minimize secondary structure within the intron.     

(A)GGG(A), (A)CCC(A) and other potential 3′ splice signals in primate nuclear pre-mRNA sequences

Several 3′ splice signals in nuclear precursor mRNAs have already been known for some time: the AG doublet on the left-hand side of the splice and a run of pyrimidines just upstream of it. More recently it has been noted that the YNYTRAY sequence (where Y is a pyrimidine, R a purine and N any base) is a branching-sequence participating in formation of a lariat structure. Keller and Noon have shown the existence of several putative consensus sequences at this site. In this work, extensive computations of the distributions of 256 quartets in all primate nuclear pre-mRNA intron sequences present in GenBank have been carried out. Several putative signals upstream and downstream of the 3′ splice have been detected. These have been compared with the results obtained in analogous computations carried out on all nuclear pre-mRNA introns present in a combined eukaryotic file containing mammal, non-mammalian vertebrate, invertebrate and plant sequences. The distributions of the more interesting oligomers are shown here. Of particular interest are the putative (A)GGG(A) signal 60 nucleotides upstream of the 3′ splice site and (A)CCC(A) 3–40 nucleotides downstream of it. A possible splicing model explaining these data and involving formation of alterantive hairpin loop structures is proposed.     

Mutational analysis of a plant branchpoint and polypyrimidine tract required for constitutive splicing of a mini-exon

The branchpoint sequence and associated polypyrimidine tract are firmly established splicing signals in vertebrates. In plants, however, these signals have not been characterized in detail. The potato invertase mini-exon 2 (9 nt) requires a branchpoint sequence positioned around 50 nt upstream of the 5' splice site of the neighboring intron and a U11 element found adjacent to the branchpoint in the upstream intron (Simpson et al., RNA, 2000, 6:422-433). Utilizing the sensitivity of this plant splicing system, these elements have been characterized by systematic mutation and analysis of the effect on inclusion of the mini-exon. Mutation of the branchpoint sequence in all possible positions demonstrated that branchpoints matching the consensus, CURAY, were most efficient at supporting splicing. Branchpoint sequences that differed from this consensus were still able to permit mini-exon inclusion but at greatly reduced levels. Mutation of the downstream U11 element suggested that it functioned as a polypyrimidine tract rather than a UA-rich element, common to plant introns. The minimum sequence requirement of the polypyrimidine tract for efficient splicing was two closely positioned groups of uridines 3-4 nt long (<6 nt apart) that, within the context of the mini-exon system, required being close (<14 nt) to the branchpoint sequence. The functional characterization of the branchpoint sequence and polypyrimidine tract defines these sequences in plants for the first time, and firmly establishes polypyrimidine tracts as important signals in splicing of at least some plant introns.     

The Drosophila SR protein RBP1 contributes to the regulation of doublesex alternative splicing by recognizing RBP1 RNA target sequences.

The SR proteins represent a family of splicing factors several of which have been implicated in the regulation of sex-specific alternative splicing of doublesex (dsx) pre-mRNA in Drosophila. The dsx gene is involved in Drosophila sex determination. We have identified two RNA target sequence motifs recognized by the SR protein RBP1 from Drosophila using an in vitro selection approach. Several copies of these RBP1 target sequences were found within two regions of the dsx pre-mRNA which are important for the regulation of dsx alternative splicing, the repeat region and the purine-rich polypyrimidine tract of the regulated female-specific 3' splice site. We show that RBP1 target sequences within the dsx repeat region are required for the efficient splicing of dsx pre-mRNA. Moreover, our studies reveal that RBP1 contributes to the activation of female-specific dsx splicing in vivo by recognizing the RBP1 target sequences within the purine-rich polypyrimidine tract of the female-specific 3' splice site.     

Mutation of putative branchpoint consensus sequences in plant introns reduces splicing efficiency

Intron lariat formation between the 5' end of an intron and a branchpoint adenosine is a fundamental aspect of the first step in animal and yeast nuclear pre-mRNA splicing. Despite similarities in intron sequence requirements and the components of splicing, differences exist between the splicing of plant and vertebrate introns. The identification of AU-rich sequences as major functional elements in plant introns and the demonstration that a branchpoint consensus sequence was not required for splicing have led to the suggestion that the transition from AU-rich intron to GC-rich exon is a major potential signal by which plant pre-mRNA splice sites are recognized. The role of putative branchpoint sequences as an internal signal in plant intron recognition/definition has been re-examined. Single nucleotide mutations in putative branchpoint adenosines contained within CUNAN sequences in four different plant introns all significantly reduced splicing efficiency. These results provide the most direct evidence to date for preferred branchpoint sequences being required for the efficient splicing of at least some plant introns in addition to the important role played by AU sequences in dicot intron recognition. The observed patterns of 3' splice site selection in the introns studied are consistent with the scanning model described for animal intron 3' splice site selection. It is suggested that, despite the clear importance of AU sequences for plant intron splicing, the fundamental processes of splice site selection and splicing in plants are similar to those in animals.     

Overlapping splicing regulatory motifs—combinatorial effects on splicing

Regulation of splicing in eukaryotes occurs through the coordinated action of multiple splicing factors. Exons and introns contain numerous putative binding sites for splicing regulatory proteins. Regulation of splicing is presumably achieved by the combinatorial output of the binding of splicing factors to the corresponding binding sites. Although putative regulatory sites often overlap, no extensive study has examined whether overlapping regulatory sequences provide yet another dimension to splicing regulation. Here we analyzed experimentally-identified splicing regulatory sequences using a computational method based on the natural distribution of nucleotides and splicing regulatory sequences. We uncovered positive and negative interplay between overlapping regulatory sequences. Examination of these overlapping motifs revealed a unique spatial distribution, especially near splice donor sites of exons with weak splice donor sites. The positively selected overlapping splicing regulatory motifs were highly conserved among different species, implying functionality. Overall, these results suggest that overlap of two splicing regulatory binding sites is an evolutionary conserved widespread mechanism of splicing regulation. Finally, over-abundant motif overlaps were experimentally tested in a reporting minigene revealing that overlaps may facilitate a mode of splicing that did not occur in the presence of only one of the two regulatory sequences that comprise it.     

Reconstruction of ancestral protosplice sites

Most of the eukaryotic protein-coding genes are interrupted by multiple introns. A substantial fraction of introns occupy the same position in orthologous genes from distant eukaryotes, such as plants and animals, and consequently are inferred to have been inherited from the common ancestor of these organisms. In contrast to these conserved introns, many other introns appear to have been gained during evolution of each major eukaryotic lineage. The mechanism(s) of insertion of new introns into genes remains unknown. Because the nucleotides that flank splice junctions are nonrandom, it has been proposed that introns are preferentially inserted into specific target sequences termed protosplice sites. However, it remains unclear whether the consensus nucleotides flanking the splice junctions are remnants of the original protosplice sites or if they evolved convergently after intron insertion. Here, we directly address the existence of protosplice sites by examining the context of introns inserted within codons that encode amino acids conserved in all eukaryotes and accordingly are not subject to selection for splicing efficiency. We show that introns are either predominantly inserted into specific protosplice sites, which have the consensus sequence (A/C)AG/Gt, or that they are inserted randomly but are preferentially fixed at such sites.