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.     

Nuclear pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe strictly requires an intron-contained, conserved sequence element.

It has recently been argued that pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe may be more similar to splicing in metazoan species than in the budding yeast Saccharomyces cerevisiae. In this report we show that, contrary to this assumption, the conserved sequence element 5'-CTPu APy-3' found in all S. pombe introns 6-18 nucleotides upstream of the 3' splice site is, like the TACTAAC box in S. cerevisiae, indispensable for efficient splicing. The conserved adenine residue of this sequence is used for branch formation and point mutations introduced into the CTPuAPy sequence abolish splicing and seem not to result in the recruitment of cryptic branch sites. We also show that an S. cerevisiae intron is correctly excised in S. pombe whereby the TACTAAC box is used in branch formation.     

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.     

An unstable mutant gene of the am locus of Neurospora results from a small duplication

We have cloned the unstable am mutant gene, am126, as well as the am gene from an am126 revertant. The mutation is a result of a 33-bp duplication that repeats a sequence starting 13 bp upstream of the 3' splice junction between intron 1 and exon 2 and extends 20 bp into exon 2. In addition, there is a G----A transition 2 bp upstream of the first copy of the duplicated sequence. In the revertant gene the wild-type sequence is precisely recovered, involving both the loss of the duplication and a reversion (A----G) of the associated transition. Our data suggest that only the more 5' of the two 3' splice junctions present in the duplicated version of the gene is used. This favors a 5'----3' scanning mechanism for exon splicing.     

Introns and splicing elements of five diverse fungi

Genomic sequences and expressed sequence tag data for a diverse group of fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, Neurospora crassa, and Cryptococcus neoformans) provided the opportunity to accurately characterize conserved intronic elements. An examination of large intron data sets revealed that fungal introns in general are short, that 98% or more of them belong to the canonical splice site (ss) class (5'GU...AG3'), and that they have polypyrimidine tracts predominantly in the region between the 5' ss and the branch point. Information content is high in the 5' ss, branch site, and 3' ss regions of the introns but low in the exon regions adjacent to the introns in the fungi examined. The two yeasts have broader intron length ranges and correspondingly higher intron information content than the other fungi. Generally, as intron length increases in the fungi, so does intron information content. Homologs of U2AF spliceosomal proteins were found in all species except for S. cerevisiae, suggesting a nonconventional role for U2AF in the absence of canonical polypyrimidine tracts in the majority of introns. Our observations imply that splicing in fungi may be different from that in vertebrates and may require additional proteins that interact with polypyrimidine tracts upstream of the branch point. Theoretical protein homologs for Nam8p and TIA-1, two proteins that require U-rich regions upstream of the branch point to function, were found. There appear to be sufficient differences between S. cerevisiae and S. pombe introns and the introns of two filamentous members of the Ascomycota and one member of the Basidiomycota to warrant the development of new model organisms for studying the splicing mechanisms of fungi.     

Identification of splicing signals in introns of yeast mitochondrial split genes: mutational alterations in intron bI1 and secondary structures in related introns.

Four mitochondrial mutations are known to block excision of intron I1 of the cob gene in S.cerevisiae. The nucleotide sequence alteration of one of them, M4873, has been determined. It is a deletion of 1 bp in a run of five G's at a distance of 30 to 34 bp upstream to the 3' splice point. Reversion is found to occur by restoration of the run of five G's either by insertion of 1 G (wild type reversion) or by transition A leads to G next to this run of G's (pseudo-wild type reversion). The effect of mutation and reversion on RNA splicing indicates that the run of five G's is of critical importance for intron I1 excision, possibly in participating in the formation of a splice signal with a helical structure. This presumption is confirmed by the observation that this sequence is part of a larger sequence of some 80 bp next to the 3' splice point which is conserved to some extend in the four mitochondrial introns (bI1, aI1, aI2, aI5) that survive after excision as circular RNAs. Most striking is the conservation of this sequence at the level of secondary structure.     

Molecular consequences of specific intron mutations on yeast mRNA splicing in vivo and in vitro

We have altered the TACTAAC sequence in the yeast CYH2m gene intron to TACTACC. This mutation changes the nucleotide at the normal position of the branch in intron RNA lariats produced during pre-mRNA splicing, and it prevents splicing in vivo. In a yeast pre-mRNA splicing system, CYH2m pre-mRNA carrying the TACTACC mutation is not specifically cut or rearranged in any way. Substitution of an A for the first G of the CYH2m intron, converting the highly conserved GTATGT 5' splice site sequence to ATATGT, also blocks intron excision in vivo and in vitro: pre-mRNA carrying this mutation was still cut normally at the mutant 5' splice site in vitro, to give authentic exon 1 and an intron-exon 2 lariat RNA with an A-A 2'-5' phosphodiester linkage at the branch point. This lariat RNA is a dead-end product. The subsequent cleavage at the 3' splice site is therefore sensitive to the sequence of the 5' end of the intron attached at the branch point.     

Intron splicing: a conserved internal signal in introns of Drosophila pre-mRNAs.

The introns of Drosophila pre-mRNAs have been analysed for conserved internal sequence elements near the 3' intron boundary similar to the T-A-C-T-A-A-C in yeast introns and the C/T-T-A/G-A-C/T in introns of other organisms. Such conserved internal elements are the 3' splice signals recognized in intron splicing. In the lariat splicing mechanism, the G at the 5' end of an intron joins covalently to the last A of a 3' splice signal to form a branch point in a splicing intermediate. Analysis of 39 published sequences of Drosophila introns reveals that potential 3' splice signals with the consensus C/T-T-A/G-A-C/T are present in 18 cases. In 17 of the remaining cases signals are present which vary from this consensus just in the middle or last position. In Drosophila introns the 3' splice signal is usually located in a discrete region between 18 and 35 nucleotides upstream from the 3' splice point. We note that the Drosophila small nuclear U2-RNA has sequences complementary to C-T-G-A-T, one variant of the signal, and to C-A-G, one variant of the 3' terminus of an intron. We also note that the absence of any A-G between -3 and -19 from the 3' splice point may be an essential feature of a strong 3' boundary.     

Splicing of a C. elegans myosin pre-mRNA in a human nuclear extract.

Splicing of mammalian introns requires that the intron possess at least 80 nucleotides. This length requirement presumably reflects the constraints of accommodating multiple snRNPs simultaneously in the same intron. In the free-living nematode, C. elegans, introns typically are 45 to 55 nucleotides in length. In this report, we determine whether C. elegans introns can obviate the mammalian length requirement by virtue of their structure or sequence. We demonstrate that a 53 nucleotide intron from the unc-54 gene of C. elegans does not undergo splicing in a mammalian (HeLa) nuclear extract. However, insertion of 31 nucleotides of foreign, prokaryotic sequence into the same intron results in efficient splicing. The observed splicing proceeds by the same two-step mechanism observed with mammalian introns, and exploits the same 3' and 5' splice sites as are used in C. elegans. The branch point used lies in the inserted sequence. We conclude that C. elegans splicing components are either fewer in number or smaller than their mammalian counterparts.     

The structure of the gene encoding chain c of the hemoglobin of the earthworm, Lumbricus terrestris

The complete nucleotide sequence of the gene for chain c of hemoglobin of the earthworm Lumbricus terrestris has been determined. The sequence of 4037 base pairs (bp) includes about 310 bp of 5'-flanking sequence and 110 bp 3' to the poly(A) site. Comparison of cDNA and genomic sequences shows four silent differences in codons that suggest the presence of at least two genes. The coding sequence is split by two introns of 1344 and 1169 bp at highly conserved positions (Jhiang, S. M., Garey, J. R., and Riggs, A. F. (1988) Science 240, 334-336). The first intron possesses the unusual 5' splice junction sequence GC instead of GT. Many tandem triplet repeats based on (GAT) and (CCT) are present in the first intron. The second intron has nine tandem repeats based on the consensus sequence AAGGAAGGAGGTC. Each intron has several exact inverted repeats of 9-10 bp that might result in loops of 78-140 nucleotides in the RNA prior to splicing. The sequences in the second intron, at positions 2423-2644 are about 65% identical with parts of several genes found in yeast mitochondria and in DNA from several other organisms.     

Pheromone 4 gene of Euplotes octocarinatus.

We have cloned and sequenced a 1.7 kb macronuclear chromosome encoding the pheromone 4 gene of Euplotes octocarinatus. The sequence of the secreted pheromone is preceded by a 42 amino acid leader peptide, which ends with a lysine residue. The sequence coding for the leader peptide contains information for a putative signal peptide and is interrupted by a 772 bp intron as shown by comparison with a cDNA clone. A 64 bp intron and a 145 bp intron interrupt the sequence coding for the secreted pheromone. The three introns contain typical 5' and 3' splice junctions and a putative branch point site. The small introns have a low GC content. The large intron has a GC content similar to that of the pheromone 4 gene exons. The amino acid sequence of pheromone 4, deduced from both the genomic DNA and the cDNA of pheromone 4, shows that the secreted pheromone consists of 85 amino acids. One of its amino acids is encoded by a UGA codon. Since it has been shown for pheromone 3 of E. octocarinatus that UGA is translated as cysteine, it is assumed that the UGA codon encodes cysteine in pheromone 4 as well. The 164 bp noncoding region upstream of the leader peptide is AT-rich and contains an inverted repeat capable of forming a stem-loop structure with a stem of 11 bp. The 151 bp noncoding region at the 3' end of the chromosome contains a putative polyadenylation sequence and an inverted repeat. The macronuclear molecule is flanked by telomeres and carries the pentanucleotide motif TTGAA, located at a distance of 17 nucleotides from the telomeres. This motif has been suggested to be involved in the formation of macronuclear chromosomes.     

Genomic mRNA profiling reveals compensatory mechanisms for the requirement of the essential splicing factor U2AF

The large subunit of the U2 auxiliary factor (U2AF) recognizes the polypyrimidine tract (Py-tract) located adjacent to the 3' splice site to facilitate U2 snRNP recruitment. While U2AF is considered essential for pre-mRNA splicing, its requirement for splicing on a genome-wide level has not been analyzed. Using Solexa sequencing, we performed mRNA profiling for splicing in the Schizosaccharomyces pombe U2AF(59) (prp2.1) temperature-sensitive mutant. Surprisingly, our analysis revealed that introns show a range of splicing defects in the mutant strain. While U2AF(59) inactivation (nonpermissive) conditions inhibit splicing of some introns, others are spliced apparently normally. Bioinformatics analysis indicated that U2AF(59)-insensitive introns have stronger 5' splice sites and higher A/U content. Most importantly, features that contribute to U2AF(59) insensitivity of an intron unexpectedly reside in its 5'-most 30 nucleotides. These include the 5' splice site, a guanosine at position 7, and the 5' splice site-to-branch point sequence context. A differential requirement (similar to U2AF(59)) for introns may also apply to other general splicing factors (e.g., prp10). Our combined results indicate that U2AF insensitivity is a common phenomenon and that varied intron features support the existence of unrecognized aspects of spliceosome assembly.     

Mutations at the 3' splice site can be suppressed by compensatory base changes in U1 snRNA in fission yeast.

U1 snRNA is an essential splicing factor known to base pair with 5' splice sites of premessenger RNAs. We demonstrate that pairing between the universally conserved CU just downstream from the 5' junction interaction region and the 3' splice site AG contributes to efficient splicing of Schizosaccharomyces pombe introns that typify the AG-dependent class described in mammals. Strains carrying mutations in the 3' AG of an artificial intron accumulate linear precursor, indicative of a first step block. Lariat formation is partially restored in these mutants by compensatory changes in nucleotides C7 and U8 of U1 snRNA. Consistent with a general role in fission yeast splicing, mutations at C7 are lethal, while U8 mutants are growth impaired and accumulate linear, unspliced precursor to U6 snRNA. U1 RNA-mediated recognition of the 3' splice site may have origins in analogous intramolecular interactions in an ancestral self-splicing RNA.     

Cryptic intron activation within the large exon of the mouse polymeric immunoglobulin receptor gene: cryptic splice sites correspond to protein domain boundaries.

The fourth exon of the mouse polymeric immuno-globulin receptor (pIgR) is 654 nt long and, despite being surrounded by large introns, is constitutively spliced into the mRNA. Deletion of an 84 nt sequence from this exon strongly activated both cryptic 5' and 3' splice sites surrounding a 78 nt cryptic intron. The 84 nt deletion is just upstream of the cryptic 3' splice site; the cryptic 3' splice site was likely activated because the deletion created a better 3' splice site. However, the cryptic 5' splice site was also required to activate the cryptic splice reaction; point mutations in either of the cryptic splice sites that decreased their match to the consensus splice site sequence inactivated the cryptic splice reaction. The activation and inactivation of these cryptic splice sites as a pair suggests that they are being co-recognized by the splicing machinery. Interestingly, the large fourth exon of the pIgR gene encodes two immunoglobulin-like extracellular protein domains; the cryptic 3' splice site coincides with the junction between these protein domains. The cryptic 5' splice site is located between protein subdomains where an intron is found in another gene of the immunoglobulin superfamily.     

Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants

Invertases are responsible for the breakdown of sucrose to fructose and glucose. In all but one plant invertase gene, the second exon is only 9 nt in length and encodes three amino acids of a five-amino-acid sequence that is highly conserved in all invertases of plant origin. Sequences responsible for normal splicing (inclusion) of exon 2 have been investigated in vivo using the potato invertase, invGF gene. The upstream intron 1 is required for inclusion whereas the downstream intron 2 is not. Mutations within intron 1 have identified two sequence elements that are needed for inclusion: a putative branchpoint sequence and an adjacent U-rich region. Both are recognized plant intron splicing signals. The branchpoint sequence lies further upstream from the 3' splice site of intron 1 than is normally seen in plant introns. All dicotyledonous plant invertase genes contain this arrangement of sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequences in exons 1, 2, or 3 do not determine inclusion, suggesting that intron or exon splicing enhancer elements seen in vertebrate mini-exon systems are absent. In addition, mutation of the 3' and 5' splice sites flanking the mini-exon cause skipping of the mini-exon, suggesting that both splice sites are required. The branchpoint/U-rich sequence is able to promote splicing of mini-exons of 6, 3, and 1 nt in length and of a chicken cTNT mini-exon of 6 nt. These sequence elements therefore act as a splicing enhancer and appear to function via interactions between factors bound at the branchpoint/U-rich region and at the 5' splice site of intron 2, activating removal of this intron followed by removal of intron 1. This first example of splicing of a plant mini-exon to be analyzed demonstrates that particular arrangement of standard plant intron splicing signals can drive constitutive splicing of a mini-exon.     

An intron in a ribosomal protein gene from Tetrahymena

We have cloned and sequenced a single copy gene encoding a ribosomal protein from the ciliate Tetrahymena thermophila. The gene product was identified as ribosomal protein S25 by comparison of the migration in two-dimensional polyacrylamide gels of the protein synthesized by translation in vitro of hybrid-selected mRNA and authentic ribosomal proteins. The proteins show strong homology to ribosomal protein S12 from Escherichia coli. The coding region of the gene is interrupted by a 979-bp intron 68 bp downstream of the translation start. This is the first intron in a protein encoding gene of a ciliate to be described at the nucleotide sequence level. The intron obeys the GT/AG rule for splice junctions of nuclear mRNA introns from higher eukaryotes but lacks the pyrimidine stretch usually found in the immediate vicinity of the 3' splice junction. The structure of the intron and the fact that it is found together with the well described self-splicing rRNA intron is discussed in relation to the evolution of RNA splicing.     

Evidence for splice site pairing via intron definition in Schizosaccharomyces pombe

Schizosaccharomyces pombe pre-mRNAs are generally multi-intronic and share certain features with pre-mRNAs from Drosophila melanogaster, in which initial splice site pairing can occur via either exon or intron definition. Here, we present three lines of evidence suggesting that, despite these similarities, fission yeast splicing is most likely restricted to intron definition. First, mutating either or both splice sites flanking an internal exon in the S. pombe cdc2 gene produced almost exclusively intron retention, in contrast to the exon skipping observed in vertebrates. Second, we were unable to induce skipping of the internal microexon in fission yeast cgs2, whereas the default splicing pathway excludes extremely small exons in mammals. Because nearly quantitative removal of the downstream intron in cgs2 could be achieved by expanding the microexon, we propose that its retention is due to steric occlusion. Third, several cryptic 5' junctions in the second intron of fission yeast cdc2 are located within the intron, in contrast to their generally exonic locations in metazoa. The effects of expanding and contracting this intron are as predicted by intron definition; in fact, even highly deviant 5' junctions can compete effectively with the standard 5' splice site if they are closer to the 3' splicing signals. Taken together, our data suggest that pairing of splice sites in S. pombe most likely occurs exclusively across introns in a manner that favors excision of the smallest segment possible.