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.     

Gene Expression,Intron Density,and Splice Site Strength in <Emphasis Type="Italic">Drosophila</Emphasis> and <Emphasis Type="Italic">Caenorhabditis</Emphasis>

In this paper we investigate the relationships among intron density (number of introns per kilobase of coding sequence), gene expression level, and strength of splicing signals in two species: Drosophila melanogaster and Caenorhabditis elegans. We report a negative correlation between intron density and gene expression levels, opposite to the effect previously observed in human. An increase in splice site strength has been observed in long introns in D. melanogaster. We show this is also true of C. elegans. We also examine the relationship between intron density and splice site strength. There is an increase in splice site strength as the intron structure becomes less dense. This could suggest that introns are not recognized in isolation but could function in a cooperative manner to ensure proper splicing. This effect remains if we control for the effects of alternative splicing on splice site strength. Reviewing Editor: Dr. Nicolas Galtier     

A genetic screen in C. elegans reveals roles for KIN17 and PRCC in maintaining 5’ splice site identity

Pre-mRNA splicing is an essential step of eukaryotic gene expression carried out by a series of dynamic macromolecular protein/RNA complexes, known collectively and individually as the spliceosome. This series of spliceosomal complexes define, assemble on, and catalyze the removal of introns. Molecular model snapshots of intermediates in the process have been created from cryo-EM data, however, many aspects of the dynamic changes that occur in the spliceosome are not fully understood. Caenorhabditis elegans follow the GU-AG rule of splicing, with almost all introns beginning with 5’ GU and ending with 3’ AG. These splice sites are identified early in the splicing cycle, but as the cycle progresses and “custody” of the pre-mRNA splice sites is passed from factor to factor as the catalytic site is built, the mechanism by which splice site identity is maintained or re-established through these dynamic changes is unclear. We performed a genetic screen in C. elegans for factors that are capable of changing 5’ splice site choice. We report that KIN17 and PRCC are involved in splice site choice, the first functional splicing role proposed for either of these proteins. Previously identified suppressors of cryptic 5’ splicing promote distal cryptic GU splice sites, however, mutations in KIN17 and PRCC instead promote usage of an unusual proximal 5’ splice site which defines an intron beginning with UU, separated by 1nt from a GU donor. We performed high-throughput mRNA sequencing analysis and found that mutations in PRCC, and to a lesser extent KIN17, changed alternative 5’ splice site usage at native sites genome-wide, often promoting usage of nearby non-consensus sites. Our work has uncovered both fine and coarse mechanisms by which the spliceosome maintains splice site identity during the complex assembly process.     

The presence of GC-AG introns in Neurospora crassa and other euascomycetes determined from analyses of complete genomes: implications for automated gene prediction

A combination of experimental and computational approaches was employed to identify introns with noncanonical GC-AG splice sites (GC-AG introns) within euascomycete genomes. Evaluation of 2335 cDNA-confirmed introns from Neurospora crassa revealed 27 such introns (1.2%). A similar frequency (1.0%) of GC-AG introns was identified in Fusarium graminearum, in which 3 of 292 cDNA-confirmed introns contained GC-AG splice sites. Computational analyses of the N. crassa genome using a GC-AG intron consensus sequence identified an additional 20 probable GC-AG introns in this fungus. For 8 of the 47 GC-AG introns identified in N. crassa a GC donor site is also present in a homolog from Magnaporthe grisea, F. graminearum, or Aspergillus nidulans. In most cases, however, homologs in these fungi contain a GT-AG intron or no intron at the corresponding position. These findings have important implications for fungal genome annotation, as the automated annotations of euascomycete genomes incorrectly identified intron boundaries for all of the confirmed and probable GC-AG introns reported here.     

Intronic alternative splicing regulators identified by comparative genomics in nematodes

Many alternative splicing events are regulated by pentameric and hexameric intronic sequences that serve as binding sites for splicing regulatory factors. We hypothesized that intronic elements that regulate alternative splicing are under selective pressure for evolutionary conservation. Using a Wobble Aware Bulk Aligner genomic alignment of Caenorhabditis elegans and Caenorhabditis briggsae, we identified 147 alternatively spliced cassette exons that exhibit short regions of high nucleotide conservation in the introns flanking the alternative exon. In vivo experiments on the alternatively spliced let-2 gene confirm that these conserved regions can be important for alternative splicing regulation. Conserved intronic element sequences were collected into a dataset and the occurrence of each pentamer and hexamer motif was counted. We compared the frequency of pentamers and hexamers in the conserved intronic elements to a dataset of all C. elegans intron sequences in order to identify short intronic motifs that are more likely to be associated with alternative splicing. High-scoring motifs were examined for upstream or downstream preferences in introns surrounding alternative exons. Many of the high- scoring nematode pentamer and hexamer motifs correspond to known mammalian splicing regulatory sequences, such as (T)GCATG, indicating that the mechanism of alternative splicing regulation is well conserved in metazoans. A comparison of the analysis of the conserved intronic elements, and analysis of the entire introns flanking these same exons, reveals that focusing on intronic conservation can increase the sensitivity of detecting putative splicing regulatory motifs. This approach also identified novel sequences whose role in splicing is under investigation and has allowed us to take a step forward in defining a catalog of splicing regulatory elements for an organism. In vivo experiments confirm that one novel high-scoring sequence from our analysis, (T)CTATC, is important for alternative splicing regulation of the unc-52 gene.     

Accumulation of GC donor splice signals in mammals

Abstract

The GT dinucleotide in the first two intron positions is the most conserved element of the U2 donor splice signals. However, in a small fraction of donor sites, GT is replaced by GC. A substantial enrichment of GC in donor sites of alternatively spliced genes has been observed previously in human, nematode and Arabidopsis, suggesting that GC signals are important for regulation of alternative splicing. We used parsimony analysis to reconstruct evolution of donor splice sites and inferred 298 GT > GC conversion events compared to 40 GC > GT conversion events in primate and rodent genomes. Thus, there was substantive accumulation of GC donor splice sites during the evolution of mammals. Accumulation of GC sites might have been driven by selection for alternative splicing.

Reviewers

This article was reviewed by Jerzy Jurka and Anton Nekrutenko. For the full reviews, please go to the Reviewers' Reports section.     

A Genetic Screen for Suppressors of a Mutated 5′ Splice Site Identifies Factors Associated With Later Steps of Spliceosome Assembly

Many alleles of human disease genes have mutations within splicing consensus sequences that activate cryptic splice sites. In Caenorhabditis elegans, the unc-73(e936) allele has a G-to-U mutation at the first base of the intron downstream of exon 15, which results in an uncoordinated phenotype. This mutation triggers cryptic splicing at the −1 and +23 positions and retains some residual splicing at the mutated wild-type (wt) position. We previously demonstrated that a mutation in sup-39, a U1 snRNA gene, suppresses e936 by increasing splicing at the wt splice site. We report here the results of a suppressor screen in which we identify three proteins that function in cryptic splice site choice. Loss-of-function mutations in the nonessential splicing factor smu-2 suppress e936 uncoordination through changes in splicing. SMU-2 binds SMU-1, and smu-1(RNAi) also leads to suppression of e936. A dominant mutation in the conserved C-terminal domain of the C. elegans homolog of the human tri-snRNP 27K protein, which we have named SNRP-27, suppresses e936 uncoordination through changes in splicing. We propose that SMU-2, SMU-1, and SNRP-27 contribute to the fidelity of splice site choice after the initial identification of 5′ splice sites by U1 snRNP.PRE-mRNA splicing takes place in a large ribonucleoprotein complex called the spliceosome (Burge et al. 1999). Components of this splicing machinery assemble at conserved signal sequences within the pre-mRNA. The 5′ splice site consensus sequence M₋₃A₋₂G₋₁ | G₊₁U₊₂R₊₃A₊₄G₊₅U₊₆ and the 3′ splice site consensus sequence Y₋₃A₋₂G₋₁ | R₊₁ (M is either A or C; R is a purine, and Y is a pyrimidine) define the limits of the intron. Base-pairing interactions between the 5′ end of the U1 snRNA and the 5′ splice site consensus sequence occur early in spliceosome assembly. It is the nearly invariable GU dinucleotide at the first two positions of the 5′ end of the intron that defines the beginning of the intron. The 5′ consensus sequence is essential but insufficient for splice site selection, as 5′ splice sites with weaker consensus matches may require additional determinants for proper activation (Sanford et al. 2005).Mutations that disrupt the 5′ consensus splice signal can lead to genetic disease in humans (Nelson and Green 1990; Cohen et al. 1994). Approximately 15% of point mutations that cause genetic diseases affect pre-mRNA splicing consensus sequences (Krawczak et al. 1992). For some specific disease genes, as many as 50% of the known heritable alleles alter splicing (Teraoka et al. 1999; Ars et al. 2000; Roca et al. 2003; Pagenstecher et al. 2006). Among all the positions of the 5′ splice site consensus sequence, the highest proportion of human disease mutations occur at the +1G position (Buratti et al. 2007). The fidelity of pre-mRNA splice site choice is largely disrupted by this defect, since this mutation causes splicing at this site to be either abolished or outcompeted by the activation of nearby cryptic 5′ splice sites (Nelson and Green 1990; Cohen et al. 1994). Cryptic splice sites are used only when the wild-type splice donor is disrupted by mutation, as they tend to have very weak splice donor consensus sequences outside of a 5′-GU dinucleotide that defines the beginning of the intron (Roca et al. 2003). Suppression of mutations to the 5′ splice site consensus sequence in vivo has been achieved through the expression of U1 snRNAs containing compensatory base substitutions (Zhuang and Weiner 1986); however, suppression of mutations to the +1 position of the intron using reverse genetic approaches has not been successful (Newman et al. 1985; Nelson and Green 1990; Cohen et al. 1994).We have used a specific allele of the Caenorhabditis elegans unc-73 gene, e936, which contains a G-to-U mutation at the first nucleotide of intron 16 (Steven et al. 1998), as a model for studying cryptic splice site choice (Roller et al. 2000; Zahler et al. 2004). unc-73 encodes a RAC guanine nucleotide exchange factor that is expressed in neurons and is important for axon guidance (Steven et al. 1998). The e936 allele induces the use of three different cryptic 5′ splice sites (Figure 1A). Two of these 5′ splice sites, located at the −1 and +23 positions, define introns beginning with GU. The third 5′ splice site used is at the mutated wild-type (wt) position and is referred to as “wt” since splicing at this site still produces wild-type unc-73 mRNA and protein, even though the intron begins with UU (Roller et al. 2000). Use of either the −1 or the +23 cryptic site causes a shift in the reading frame and loss of gene function. In e936 animals, 90% of the stable messages of unc-73 are out-of-frame, yet the phenotype is not as severe as for other alleles in this gene. This indicates that the 10% of steady-state messages that are in frame have some functional role.Open in a separate windowFigure 1.—(A) Diagram of the unc-73 gene between exons 15 and 16. The positions of the −1 and +23 cryptic 5′ splice sites are indicated by arrows. The intronic e936 (+1G → U) point mutation is highlighted. (B) γ-³²P-labeled RT–PCR results across the cryptic splicing region of unc-73(e936) for different strains. Lanes 1, 2, and 3 are loaded with RT–PCR reactions from wild type (N2), unc-73(e936);sup-39(je5), and unc-73(e936) RNA, respectively. The lines carrying the suppressor alleles and e936 follow in lanes 4–10 as indicated. (C) The unc-73 genomic sequence from exon 15 (uppercase letters) and intron 15 (lowercase letters). The locations of the az23 and e936 mutational substitutions are indicated below. The position of the −9 cryptic splice donor activated in e936az23 is indicated by an arrow above.In a previous genetic screen for extragenic suppressors of e936 movement defects, Way and colleagues identified sup-39 (Run et al. 1996). It was subsequently shown that mutations in sup-39 alter cryptic splice site choice of e936 (Roller et al. 2000). sup-39 encodes a U1 snRNA gene with a compensatory mutation at the position that normally base pairs with the +1G. This allows sup-39 to base pair with an intron with a +1U (Zahler et al. 2004). This dominant suppressor increases usage of the mutated splice site and improves the fraction of in-frame messages from e936 from 10 to 33%, with a dramatic improvement in coordination. A similar mutant U1 snRNA suppressor with a different compensatory substitution, sup-6(st19), was found to suppress the intronic +1G to A transition of unc-13(e309) to allow for splicing at the mutated wild-type site, even though the intron begins with AU instead of GU (Zahler et al. 2004).We are interested in identifying additional factors that play a role in cryptic 5′ splice site choice. To do this, we took advantage of unc-73(e936), in which modest increases in the use of the wt splice site lead to dramatic increases in coordination, as a sensitive screen for changes in cryptic splice site choice. In this article we report that the proteins SMU-1 and SMU-2, which are nonessential factors previously shown to have a role in alternative splicing (Spartz et al. 2004), have a role in selection of cryptic 5′ splice sites. We also report the identification of a new dominant suppressor of cryptic splicing, snrp-27, which encodes a C. elegans homolog of the human tri-snRNP 27K protein.     

Discovery and analysis of evolutionarily conserved intronic splicing regulatory elements

Knowledge of the functional cis-regulatory elements that regulate constitutive and alternative pre-mRNA splicing is fundamental for biology and medicine. Here we undertook a genome-wide comparative genomics approach using available mammalian genomes to identify conserved intronic splicing regulatory elements (ISREs). Our approach yielded 314 ISREs, and insertions of ~70 ISREs between competing splice sites demonstrated that 84% of ISREs altered 5′ and 94% altered 3′ splice site choice in human cells. Consistent with our experiments, comparisons of ISREs to known splicing regulatory elements revealed that 40%–45% of ISREs might have dual roles as exonic splicing silencers. Supporting a role for ISREs in alternative splicing, we found that 30%–50% of ISREs were enriched near alternatively spliced (AS) exons, and included almost all known binding sites of tissue-specific alternative splicing factors. Further, we observed that genes harboring ISRE-proximal exons have biases for tissue expression and molecular functions that are ISRE-specific. Finally, we discovered that for Nova1, neuronal PTB, hnRNP C, and FOX1, the most frequently occurring ISRE proximal to an alternative conserved exon in the splicing factor strongly resembled its own known RNA binding site, suggesting a novel application of ISRE density and the propensity for splicing factors to auto-regulate to associate RNA binding sites to splicing factors. Our results demonstrate that ISREs are crucial building blocks in understanding general and tissue-specific AS regulation and the biological pathways and functions regulated by these AS events.     

Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns

As part of the exploratory sequencing program Génolevures, visual scrutinisation and bioinformatic tools were used to detect spliceosomal introns in seven hemiascomycetous yeast species. A total of 153 putative novel introns were identified. Introns are rare in yeast nuclear genes (<5% have an intron), mainly located at the 5′ end of ORFs, and not highly conserved in sequence. They all share a clear non-random vocabulary: conserved splice sites and conserved nucleotide contexts around splice sites. Homologues of metazoan snRNAs and putative homologues of SR splicing factors were identified, confirming that the spliceosomal machinery is highly conserved in eukaryotes. Several introns’ features were tested as possible markers for phylogenetic analysis. We found that intron sizes vary widely within each genome, and according to the phylogenetic position of the yeast species. The evolutionary origin of spliceosomal introns was examined by analysing the degree of conservation of intron positions in homologous yeast genes. Most introns appeared to exist in the last common ancestor of present day yeast species, and then to have been differentially lost during speciation. However, in some cases, it is difficult to exclude a possible sliding event affecting a pre-existing intron or a gain of a novel intron. Taken together, our results indicate that the origin of spliceosomal introns is complex within a given genome, and that present day introns may have resulted from a dynamic flux between intron conservation, intron loss and intron gain during the evolution of hemiascomycetous yeasts.     

hnRNP H binding at the 5' splice site correlates with the pathological effect of two intronic mutations in the NF-1 and TSHbeta genes

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

Vitamin A Metabolite,All-trans-retinoic Acid,Mediates Alternative Splicing of Protein Kinase C δVIII (PKCδVIII) Isoform via Splicing Factor SC35

Vitamin A metabolite, all-trans-retinoic acid (RA), induces cell growth, differentiation, and apoptosis and has an emerging role in gene regulation and alternative splicing events. Protein kinase Cδ (PKCδ), a serine/threonine kinase, has a role in cell proliferation, differentiation, and apoptosis. We reported an alternatively spliced variant of human PKCδ, PKCδVIII that functions as a pro-survival protein (1). RA regulates the splicing and expression of PKCδVIII via utilization of a downstream 5′ splice site of exon 10 on PKCδ pre-mRNA. Here, we further elucidate the molecular mechanisms involved in RA regulation of alternative splicing of PKCδVIII mRNA. Overexpression and knockdown of the splicing factor SC35 (i.e. SRp30b) indicated that it is involved in PKCδVIII alternative splicing. To identify the cis-elements involved in 5′ splice site selection we cloned a minigene, which included PKCδ exon 10 and its flanking introns in the pSPL3 splicing vector. Alternative 5′ splice site utilization in the minigene was promoted by RA. Further, co-transfection of SC35 with PKCδ minigene promoted selection of 5′ splice site II. Mutation of the SC35 binding site in the PKCδ minigene abolished RA-mediated utilization of 5′ splice splice II. RNA binding assays demonstrated that the enhancer element downstream of PKCδ exon 10 is a SC35 cis-element. We conclude that SC35 is pivotal in RA-mediated PKCδ pre-mRNA alternative splicing. This study demonstrates how a nutrient, vitamin A, via its metabolite RA, regulates alternative splicing and thereby gene expression of the pro-survival protein PKCδVIII.     

Analysis of canonical and non-canonical splice sites in mammalian genomes

A set of 43 337 splice junction pairs was extracted from mammalian GenBank annotated genes. Expressed sequence tag (EST) sequences support 22 489 of them. Of these, 98.71% contain canonical dinucleotides GT and AG for donor and acceptor sites, respectively; 0.56% hold non-canonical GC-AG splice site pairs; and the remaining 0.73% occurs in a lot of small groups (with a maximum size of 0.05%). Studying these groups we observe that many of them contain splicing dinucleotides shifted from the annotated splice junction by one position. After close examination of such cases we present a new classification consisting of only eight observed types of splice site pairs (out of 256 a priori possible combinations). EST alignments allow us to verify the exonic part of the splice sites, but many non-canonical cases may be due to intron sequencing errors. This idea is given substantial support when we compare the sequences of human genes having non-canonical splice sites deposited in GenBank by high throughput genome sequencing projects (HTG). A high proportion (156 out of 171) of the human non-canonical and EST-supported splice site sequences had a clear match in the human HTG. They can be classified after corrections as: 79 GC-AG pairs (of which one was an error that corrected to GC-AG), 61 errors that were corrected to GT-AG canonical pairs, six AT-AC pairs (of which two were errors that corrected to AT-AC), one case was produced from non-existent intron, seven cases were found in HTG that were deposited to GenBank and finally there were only two cases left of supported non-canonical splice sites. If we assume that approximately the same situation is true for the whole set of annotated mammalian non-canonical splice sites, then the 99.24% of splice site pairs should be GT-AG, 0.69% GC-AG, 0.05% AT-AC and finally only 0.02% could consist of other types of non-canonical splice sites. We analyze several characteristics of EST-verified splice sites and build weight matrices for the major groups, which can be incorporated into gene prediction programs. We also present a set of EST-verified canonical splice sites larger by two orders of magnitude than the current one (22 199 entries versus approximately 600) and finally, a set of 290 EST-supported non-canonical splice sites. Both sets should be significant for future investigations of the splicing mechanism.     

The Peculiarities of Large Intron Splicing in Animals

In mammals a considerable 92% of genes contain introns, with hundreds and hundreds of these introns reaching the incredible size of over 50,000 nucleotides. These “large introns” must be spliced out of the pre-mRNA in a timely fashion, which involves bringing together distant 5′ and 3′ acceptor and donor splice sites. In invertebrates, especially Drosophila, it has been shown that larger introns can be spliced efficiently through a process known as recursive splicing—a consecutive splicing from the 5′-end at a series of combined donor-acceptor splice sites called RP-sites. Using a computational analysis of the genomic sequences, we show that vertebrates lack the proper enrichment of RP-sites in their large introns, and, therefore, require some other method to aid splicing. We analyzed over 15,000 non-redundant, large introns from six mammals, 1,600 from chicken and zebrafish, and 560 non-redundant large introns from five invertebrates. Our bioinformatic investigation demonstrates that, unlike the studied invertebrates, the studied vertebrate genomes contain consistently abundant amounts of direct and complementary strand interspersed repetitive elements (mainly SINEs and LINEs) that may form stems with each other in large introns. This examination showed that predicted stems are indeed abundant and stable in the large introns of mammals. We hypothesize that such stems with long loops within large introns allow intron splice sites to find each other more quickly by folding the intronic RNA upon itself at smaller intervals and, thus, reducing the distance between donor and acceptor sites.