EST analysis of Ostreococcus lucimarinus, the most compact eukaryotic genome, shows an excess of introns in highly expressed genes

Background

The genome of the pico-eukaryotic (bacterial-sized) prasinophyte green alga Ostreococcus lucimarinus has one of the highest gene densities known in eukaryotes, yet it contains many introns. Phylogenetic studies suggest this unusually compact genome (13.2 Mb) is an evolutionarily derived state among prasinophytes. The presence of introns in the highly reduced O. lucimarinus genome appears to be in opposition to simple explanations of genome evolution based on unidirectional tendencies, either neutral or selective. Therefore, patterns of intron retention in this species can potentially provide insights into the forces governing intron evolution.

Methodology/Principal Findings

Here we studied intron features and levels of expression in O. lucimarinus using expressed sequence tags (ESTs) to annotate the current genome assembly. ESTs were assembled into unigene clusters that were mapped back to the O. lucimarinus Build 2.0 assembly using BLAST and the level of gene expression was inferred from the number of ESTs in each cluster. We find a positive correlation between expression levels and both intron number (R = +0.0893, p = <0.0005) and intron density (number of introns/kb of CDS; R = +0.0753, p = <0.005).

Conclusions/Significance

In a species with a genome that has been recently subjected to a great reduction of non-coding DNA, these results imply the existence of selective/functional roles for introns that are principally detectable in highly expressed genes. In these cases, introns are likely maintained by balancing the selective forces favoring their maintenance with other mutational and/or selective forces acting on genome size.     

Evidence for Transitional Stages in the Evolution of Euglenid Group II Introns and Twintrons in the Monomorphina aenigmatica Plastid Genome

Background

Photosynthetic euglenids acquired their plastid by secondary endosymbiosis of a prasinophyte-like green alga. But unlike its prasinophyte counterparts, the plastid genome of the euglenid Euglena gracilis is riddled with introns that interrupt almost every protein-encoding gene. The atypical group II introns and twintrons (introns-within-introns) found in the E. gracilis plastid have been hypothesized to have been acquired late in the evolution of euglenids, implying that massive numbers of introns may be lacking in other taxa. This late emergence was recently corroborated by the plastid genome sequences of the two basal euglenids, Eutreptiella gymnastica and Eutreptia viridis, which were found to contain fewer introns.

Methodology/Principal Findings

To gain further insights into the proliferation of introns in euglenid plastids, we have characterized the complete plastid genome sequence of Monomorphina aenigmatica, a freshwater species occupying an intermediate phylogenetic position between early and late branching euglenids. The M. aenigmatica UTEX 1284 plastid genome (74,746 bp, 70.6% A+T, 87 genes) contains 53 intron insertion sites, of which 41 were found to be shared with other euglenids including 12 of the 15 twintron insertion sites reported in E. gracilis.

Conclusions

The pattern of insertion sites suggests an ongoing but uneven process of intron gain in the lineage, with perhaps a minimum of two bursts of rapid intron proliferation. We also identified several sites that represent intermediates in the process of twintron evolution, where the external intron is in place, but not the internal one, offering a glimpse into how these convoluted molecular contraptions originate.     

Evolutionarily emerged G tracts between the polypyrimidine tract and 3′ AG are splicing silencers enriched in genes involved in cancer

Background

The 3′ splice site (SS) at the end of pre-mRNA introns has a consensus sequence (Y)_nNYAG for constitutive splicing of mammalian genes. Deviation from this consensus could change or interrupt the usage of the splice site leading to alternative or aberrant splicing, which could affect normal cell function or even the development of diseases. We have shown that the position “N” can be replaced by a CA-rich RNA element called CaRRE1 to regulate the alternative splicing of a group of genes.

Results

Taking it a step further, we searched the human genome for purine-rich elements between the -3 and -10 positions of the 3′ splice sites of annotated introns. This identified several thousand such 3′SS; more than a thousand of them contain at least one copy of G tract. These sites deviate significantly from the consensus of constitutive splice sites and are highly associated with alterative splicing events, particularly alternative 3′ splice and intron retention. We show by mutagenesis analysis and RNA interference that the G tracts are splicing silencers and a group of the associated exons are controlled by the G tract binding proteins hnRNP H/F. Species comparison of a group of the 3′SS among vertebrates suggests that most (~87%) of the G tracts emerged in ancestors of mammals during evolution. Moreover, the host genes are most significantly associated with cancer.

Conclusion

We call these elements together with CaRRE1 regulatory RNA elements between the Py and 3′AG (REPA). The emergence of REPA in this highly constrained region indicates that this location has been remarkably permissive for the emergence of de novo regulatory RNA elements, even purine-rich motifs, in a large group of mammalian genes during evolution. This evolutionary change controls alternative splicing, likely to diversify proteomes for particular cellular functions.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1143) contains supplementary material, which is available to authorized users.     

Detection of variants in 15 genes in 87 unrelated Chinese patients with Leber congenital amaurosis

Background

Leber congenital amaurosis (LCA) is the earliest onset and most severe form of hereditary retinal dystrophy. So far, full spectrum of variations in the 15 genes known to cause LCA has not been systemically evaluated in East Asians. Therefore, we performed comprehensive detection of variants in these 15 genes in 87 unrelated Han Chinese patients with LCA.

Methodology/Principal Findings

The 51 most frequently mutated exons and introns in the 15 genes were selected for an initial scan using cycle sequencing. All the remaining exons in 11 of the 15 genes were subsequently sequenced. Fifty-three different variants were identified in 44 of the 87 patients (50.6%), involving 78 of the 88 alleles (11 homozygous and 56 heterozygous variants). Of the 53 variants, 35 (66%) were novel pathogenic mutations. In these Chinese patients, variants in GUCY2D are the most common cause of LCA (16.1% cases), followed by CRB1 (11.5%), RPGRIP1 (8%), RPE65 (5.7%), SPATA7 (4.6%), CEP290 (4.6%), CRX (3.4%), LCA5 (2.3%), MERTK (2.3%), AIPL1 (1.1%), and RDH12 (1.1%). This differs from the variation spectrum described in other populations. An initial scan of 55 of 215 PCR amplicons, including 214 exons and 1 intron, detected 83.3% (65/78) of the mutant alleles ultimately found in these 87 patients. In addition, sequencing only 9 exons would detect over 50% of the identified variants and require less than 5% of the labor and cost of comprehensive sequencing for all exons.

Conclusions/Significance

Our results suggest that specific difference in the variation spectrum found in LCA patients from the Han Chinese and other populations are related by ethnicity. Sequencing exons in order of decreasing risk is a cost-effective way to identify causative mutations responsible for LCA, especially in the context of genetic counseling for individual patients in a clinical setting.