Exon junction sequences as cryptic splice sites: implications for intron origin

Introns are flanked by a partially conserved coding sequence that forms the immediate exon junction sequence following intron removal from pre-mRNA. Phylogenetic evidence indicates that these sequences have been targeted by numerous intron insertions during evolution, but little is known about this process. Here, we test the prediction that exon junction sequences were functional splice sites that existed in the coding sequence of genes prior to the insertion of introns. To do this, we experimentally identified nine cryptic splice sites within the coding sequence of actin genes from humans, Arabidopsis, and Physarum by inactivating their normal intron splice sites. We found that seven of these cryptic splice sites correspond exactly to the positions of exon junctions in actin genes from other species. Because actin genes are highly conserved, we could conclude that at least seven actin introns are flanked by cryptic splice sites, and from the phylogenetic evidence, we could also conclude that actin introns were inserted into these cryptic splice sites during evolution. Furthermore, our results indicate that these insertion events were dependent upon the splicing machinery. Because most introns are flanked by similar sequences, our results are likely to be of general relevance.     

Information contents and dinucleotide compositions of plant intron sequences vary with evolutionary origin

The DNA sequence composition of 526 dicot and 345 monocot intron sequences have been characterized using computational methods. Splice site information content and bulk intron and exon dinucleotide composition were determined. Positions 4 and 5 of 5 splice sites contain different statistically significant levels of information in the two groups. Basal levels of information in introns are higher in dicots than in monocots. Two dinucleotide groups, WW (AA, AU, UA, UU) and SS (CC, CG, GC, GG) have significantly different frequencies in exons and introns of the two plant groups. These results suggest that the mechanisms of splice-site recognition and binding may differ between dicot and monocot plants.     

A combinatorial role for exon, intron and splice site sequences in splicing in maize

Plant introns are typically AU-rich or U-rich, and this feature has been shown to be important for splicing. In maize, however, about 20% of the introns exceed 50% GC, and most of them are efficiently spliced. A series of constructs has been designed to analyze the cis requirements for splicing of the GC-rich Bz2 maize intron and two other GC-rich intron derivatives. By manipulating exon, intron and splice site sequences it is shown that exons can play an important role in intron definition: changes in exon sequences can increase splicing efficiency of a GC-rich intron from 17% to 86%. The relative difference, or base compositional contrast, in GC and U content between exon and intron sequences in the vicinity of splice sites, rather than the absolute base-content of the intron or exons, correlates with splicing efficiency. It is also shown that GC-rich intron constructs that are poorly spliced can be partially rescued by an improved 3' splice site.     

Conserved quartets near 5' intron junctions in primate nuclear pre-mRNA

Analysis of a 1000 nucleotide span around 664 primate 5' exon/intron junctions revealed frequent recurrences of G-rich runs downstream of the 5' splice sites. In particular, AGGG, GGGA, GGGG, GGGT and TGGG are frequent at this site. Some C-rich quarters are frequent upstream of the 5' splice site. Similar behaviour of these G- and C-rich quartets is indicated for the 587 rodent introns and for a combined eukaryotic file containing 1688 introns. (A)GGG(A) is also frequent in the introns 60 nucleotides upstream of the 3' splice site, and (A)CCC(A) is frequently found in the exons downstream of the 3' site. The same consistent behaviour of the 3' splice sites is obtained as for the 5' sites, for the primates, rodents and combined eukaryotic file. These results suggest that in addition to the well-conserved 5' and 3' splice sequences, exon as well as intron sequences may play a role in nuclear pre-mRNA splicing.     

Expression of maize Adh1 intron mutants in tobacco nuclei

In vivo and in vitro gene transfer experiments have suggested that the elements mediating intron recognition differ in mammalian, yeast and plant nuclei. Differences in the sequence dependencies, which also exist between dicotyledonous and monocotyledonous nuclei, have prevented some monocot introns from being spliced in dicot nuclei. To locate elements which modulate efficient recognition of introns in dicot nuclei, the maize Adh1 gene has been expressed in full-length and single intron constructs in Nicotiana benthamiana nuclei using an autonomously replicating plant expression vector. Quantitative PCR-Southern analyses indicate that the inefficient splicing of the maize Adh1 intron 1 (57% AU) in these dicot nuclei can be dramatically enhanced by increasing the degree of U1 snRNA complementarity at the 5′ splice site. This indicates that the 5′ splice site plays a significant role in defining the splicing efficiency of an intron in dicot nuclei and that, most importantly, the remainder of this monocot intron contains no elements which inhibit its accurate recognition in dicot nuclei. Deletions in intron 3 (66% AU) which effectively move the 3′ boundary between AU-rich intron and GC-rich exon sequences strongly activate a cryptic upstream splice site; those which do not reposition this boundary activate a downstream cryptic splice site. This suggests that 3′ splice site selection in dicot nuclei is extremely flexible and not dependent on strict sequence requirements but rather on the transition points between introns and exons. Our results are consistent with a model in which potential splice sites are selected if they are located upstream (5′ splice site) or downstream (3′ splice site) of AU transition points and not if they are embedded within AU-rich sequences.     

Splicing signals in Drosophila: intron size, information content, and consensus sequences.

A database of 209 Drosophila introns was extracted from Genbank (release number 64.0) and examined by a number of methods in order to characterize features that might serve as signals for messenger RNA splicing. A tight distribution of sizes was observed: while the smallest introns in the database are 51 nucleotides, more than half are less than 80 nucleotides in length, and most of these have lengths in the range of 59-67 nucleotides. Drosophila splice sites found in large and small introns differ in only minor ways from each other and from those found in vertebrate introns. However, larger introns have greater pyrimidine-richness in the region between 11 and 21 nucleotides upstream of 3' splice sites. The Drosophila branchpoint consensus matrix resembles C T A A T (in which branch formation occurs at the underlined A), and differs from the corresponding mammalian signal in the absence of G at the position immediately preceding the branchpoint. The distribution of occurrences of this sequence suggests a minimum distance between 5' splice sites and branchpoints of about 38 nucleotides, and a minimum distance between 3' splice sites and branchpoints of 15 nucleotides. The methods we have used detect no information in exon sequences other than in the few nucleotides immediately adjacent to the splice sites. However, Drosophila resembles many other species in that there is a discontinuity in A + T content between exons and introns, which are A + T rich.     

Testing the "proto-splice sites" model of intron origin: evidence from analysis of intron phase correlations

A few nucleotide sites of nuclear exons that flank introns are often conserved. A hypothesis has suggested that these sites, called "proto-splice sites," are remnants of recognition signals for the insertion of introns in the early evolution of eukaryotic genes. This notion of proto-splice sites has been an important basis for the insertional theory of introns. This hypothesis predicts that the distribution of proto-splice sites would determine the distribution of intron phases, because the positions of introns are just a subset of the proto-splice sites. We previously tested this prediction by examining the proportions of the phases of proto-splice sites, revealing nothing in these proportion distributions similar to observed proportions of intron phases. Here, we provide a second independent test of the proto-splice site hypothesis, with regard to its prediction that the proto-splice sites would mimic intron phase correlations, using a CDS database we created from GenBank. We tested four hypothetical proto-splice sites G / G, AG / G, AG / GT, and C/AAG / R. Interestingly, while G / G and AG / GT site phase distributions are not consistent with actual introns, we observed that AG / G and C/AAG / R sites have a symmetric phase excess. However, the patterns of the excess are quite different from the actual intron phase distribution. In addition, particular amino acid repeats in proteins were found to partially contribute to the excess of symmetry at these two types of sites. The phase associations of all four sites are significantly different from those of intron phases. Furthermore, a general model of intron insertion into proto-splice sites was simulated by Monte Carlo simulation to investigate the probability that the random insertion of introns into AG / G and C/AAG / R sites could generate the observed intron phase distribution. The simulation showed that (1) no observed correlation of intron phases was statistically consistent with the phase distribution of proto-splice sites in the simulated virtual genes; (2) most conservatively, no simulation in 10,000 Monte Carlo experiments gave a pattern with an excess of symmetric (1, 1) exons larger than those of (0, 0) and (2, 2), a major statistical feature of intron phase distribution that is consistent with the directly observed cases of exon shuffling. Thus, these results reject the null hypothesis that introns are randomly inserted into preexisting proto-splice sites, as suggested by the insertional theory of introns.     

Selection of cryptic 5'' splice sites by group II intron RNAs in vitro.

Recognition of 5' splice points by group I and group II self-splicing introns involves the interaction of exon sequences--directly preceding the 5' splice site--with intronic sequence elements. We show here that the exon binding sequences (EBS) of group II intron aI5c can accept various substitutes of the authentic intron binding sites (IBS) provided in cis or in trans. The efficiency of cleavages at these cryptic 5' splice sites was enhanced by deletion of the authentic IBS2 element. All cryptic 5' cleavage sites studied here were preceded by an IBS1 like sequence; indicating that the IBS1/EBS1 pairing alone is sufficient for proper 5' splice site selection by the intronic EBS element. The results are discussed in terms of minimal requirements for 5' cleavages and position effects of IBS sites relative to the intron.     

Association of intron phases with conservation at splice site sequences and evolution of spliceosomal introns.

How exon-intron structures of eukaryotic genes evolved under various evolutionary forces remains unknown. The phases of spliceosomal introns (the placement of introns with respect to reading frame) provide an opportunity to approach this question. When a large number of nuclear introns in protein-coding genes were analyzed, it was found that most introns were of phase 0, which keeps codons intact. We found that the phase distribution of spliceosomal introns is strongly correlated with the sequence conservation of splice signals in exons; the relatively underrepresented phase 2 introns are associated with the lowest conservation, the relatively overrepresented phase 0 introns display the highest conservation, and phase 1 introns are intermediate. Given the detrimental effect of mutations in exon sequences near splice sites as found in molecular experiments, the underrepresentation of phase 2 introns may be the result of deleterious-mutation-driven intron loss, suggesting a possible genetic mechanism for the evolution of intron-exon structures.     

In vitro splicing of cardiac troponin T precursors. Exon mutations disrupt splicing of the upstream intron.

A single cardiac troponin T (cTNT) gene generates two mRNAs by including or excluding the 30-nucleotide exon 5 during pre-mRNA processing. Transfection analysis of cTNT minigenes has previously demonstrated that both mRNAs are expressed from unmodified minigenes, and mutations within exon 5 can lead to complete skipping of the exon. These results suggested a role for exon sequence in splice site recognition. To investigate this potential role, an in vitro splicing system using cTNT precursors has been established. Two-exon precursors containing the alternative exon and either the upstream exon or downstream exon were spliced accurately and efficiently in vitro. The mutations within the alternative exon that resulted in exon skipping in vivo specifically blocked splicing of the upstream intron in vitro and had no effect on removal of the downstream intron. In addition, the splicing intermediates of these two precursors have been characterized, and the branch sites utilized on the introns flanking the alternative exon have been determined. Potential roles of exon sequence in splice site selection are discussed. These results establish a system that will be useful for the biochemical characterization of the role of exon sequence in splice site selection.     

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.     

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.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Investigating the intron recognition mechanism in eukaryotes

Recent studies indicate that many introns, as well as the complex spliceosomal mechanism to remove them, were present early in eukaryotic evolution. This study examines intron and exon characteristics from annotations of whole genomes to investigate the intron recognition mechanism. Exon definition uses the exon as the unit of recognition, placing length constraints on the exon but not on the intron (allowing it a greater range of lengths). In contrast, intron definition uses the intron itself as the unit of recognition and thus removes constraints on internal exon length forced by the use of an exon definition mechanism. Thus, intron and exon lengths within a genome can reflect the constraints imposed by its splicing. This study shows that it is possible firstly to recover valid intron and exon information from genome annotation. We then compare internal intron and exon information from a range of eukaryotic genomes and investigate possible evolutionary length constraints on introns and exons and how they can impact on the intron recognition mechanism. Results indicate that exon definition-based mechanisms may predominate in vertebrates although the exact system in fish is expected to show some differences with the better characterized system from mammals. We also raise the possibility that the last common ancestor of plants and animals contained some type of exon definition and that this mechanism was replaced in some genes and lineages by intron definition, possibly as a result of intron loss and/or intron shortening.     

Nonsense-Mediated Decay Enables Intron Gain in Drosophila

Intron number varies considerably among genomes, but despite their fundamental importance, the mutational mechanisms and evolutionary processes underlying the expansion of intron number remain unknown. Here we show that Drosophila, in contrast to most eukaryotic lineages, is still undergoing a dramatic rate of intron gain. These novel introns carry significantly weaker splice sites that may impede their identification by the spliceosome. Novel introns are more likely to encode a premature termination codon (PTC), indicating that nonsense-mediated decay (NMD) functions as a backup for weak splicing of new introns. Our data suggest that new introns originate when genomic insertions with weak splice sites are hidden from selection by NMD. This mechanism reduces the sequence requirement imposed on novel introns and implies that the capacity of the spliceosome to recognize weak splice sites was a prerequisite for intron gain during eukaryotic evolution.