Intron Density Research Articles

Where the minor things are: a pan-eukaryotic survey suggests neutral processes may explain much of minor intron evolution.

Spliceosomal introns are gene segments removed from RNA transcripts by ribonucleoprotein machineries called spliceosomes. In some eukaryotes a second 'minor' spliceosome is responsible for processing a tiny minority of introns. Despite its seemingly modest role, minor splicing has persisted for roughly 1.5 billion years of eukaryotic evolution. Identifying minor introns in over 3000 eukaryotic genomes, we report diverse evolutionary histories including surprisingly high numbers in some fungi and green algae, repeated loss, as well as general biases in their positional and genic distributions. We estimate that ancestral minor intron densities were comparable to those of vertebrates, suggesting a trend of long-term stasis. Finally, three findings suggest a major role for neutral processes in minor intron evolution. First, highly similar patterns of minor and major intron evolution contrast with both functionalist and deleterious model predictions. Second, observed functional biases among minor intron-containing genes are largely explained by these genes' greater ages. Third, no association of intron splicing with cell proliferation in a minor intron-rich fungus suggests that regulatory roles are lineage-specific and thus cannot offer a general explanation for minor splicing's persistence. These data constitute the most comprehensive view of minor introns and their evolutionary history to date, and provide a foundation for future studies of these remarkable genetic elements.

Intron losses and gains in the nematodes

BackgroundThe evolution of spliceosomal introns has been widely studied among various eukaryotic groups. Researchers nearly reached the consensuses on the pattern and the mechanisms of intron losses and gains across eukaryotes. However, according to previous studies that analyzed a few genes or genomes, Nematoda seems to be an eccentric group.ResultsTaking advantage of the recent accumulation of sequenced genomes, we extensively analyzed the intron losses and gains using 104 nematode genomes across all the five Clades of the phylum. Nematodes have a wide range of intron density, from less than one to more than nine per kbp coding sequence. The rates of intron losses and gains exhibit significant heterogeneity both across different nematode lineages and across different evolutionary stages of the same lineage. The frequency of intron losses far exceeds that of intron gains. Five pieces of evidence supporting the model of cDNA-mediated intron loss have been observed in ten Caenorhabditis species, the dominance of the precise intron losses, frequent loss of adjacent introns, high-level expression of the intron-lost genes, preferential losses of short introns, and the preferential losses of introns close to 3′-ends of genes. Like studies in most eukaryotic groups, we cannot find the source sequences for the limited number of intron gains detected in the Caenorhabditis genomes.ConclusionsThese results indicate that nematodes are a typical eukaryotic group rather than an outlier in intron evolution.

Analysis of Fungal Genomes Reveals Commonalities of Intron Gain or Loss and Functions in Intron-Poor Species.

Previous evolutionary reconstructions have concluded that early eukaryotic ancestors including both the last common ancestor of eukaryotes and of all fungi had intron-rich genomes. By contrast, some extant eukaryotes have few introns, underscoring the complex histories of intron–exon structures, and raising the question as to why these few introns are retained. Here, we have used recently available fungal genomes to address a variety of questions related to intron evolution. Evolutionary reconstruction of intron presence and absence using 263 diverse fungal species supports the idea that massive intron reduction through intron loss has occurred in multiple clades. The intron densities estimated in various fungal ancestors differ from zero to 7.6 introns per 1 kb of protein-coding sequence. Massive intron loss has occurred not only in microsporidian parasites and saccharomycetous yeasts, but also in diverse smuts and allies. To investigate the roles of the remaining introns in highly-reduced species, we have searched for their special characteristics in eight intron-poor fungi. Notably, the introns of ribosome-associated genes RPL7 and NOG2 have conserved positions; both intron-containing genes encoding snoRNAs. Furthermore, both the proteins and snoRNAs are involved in ribosome biogenesis, suggesting that the expression of the protein-coding genes and noncoding snoRNAs may be functionally coordinated. Indeed, these introns are also conserved in three-quarters of fungi species. Our study shows that fungal introns have a complex evolutionary history and underappreciated roles in gene expression.

Analysis of metagenome-assembled viral genomes from the human gut reveals diverse putative CrAss-like phages with unique genomic features

CrAssphage is the most abundant human-associated virus and the founding member of a large group of bacteriophages, discovered in animal-associated and environmental metagenomes, that infect bacteria of the phylum Bacteroidetes. We analyze 4907 Circular Metagenome Assembled Genomes (cMAGs) of putative viruses from human gut microbiomes and identify nearly 600 genomes of crAss-like phages that account for nearly 87% of the DNA reads mapped to these cMAGs. Phylogenetic analysis of conserved genes demonstrates the monophyly of crAss-like phages, a putative virus order, and of 5 branches, potential families within that order, two of which have not been identified previously. The phage genomes in one of these families are almost twofold larger than the crAssphage genome (145-192 kilobases), with high density of self-splicing introns and inteins. Many crAss-like phages encode suppressor tRNAs that enable read-through of UGA or UAG stop-codons, mostly, in late phage genes. A distinct feature of the crAss-like phages is the recurrent switch of the phage DNA polymerase type between A and B families. Thus, comparative genomic analysis of the expanded assemblage of crAss-like phages reveals aspects of genome architecture and expression as well as phage biology that were not apparent from the previous work on phage genomics.

The C- and G-value paradox with polyploidy, repeatomes, introns, phenomes and cell economy.

The apparent disconnection between biological complexity and both genome size (C-value) and gene number (G-value) is one of the long-standing biological puzzles. Gene-dense genomic sequences in prokaryotes or simple eukaryotes are highly constrained during selection, whereas gene-sparse genomic sequences in higher eukaryotes have low selection constraints. This review discusses the correlations of the C-value and G-value with genome architecture, polyploidy, repeatomes, introns, cell economy and phenomes. Eukaryotic chromosomes carry an assortment of various repeated DNA sequences (repeatomes). Expansion of copies of repeatomes together with polyploidization or whole-genome duplication (WGD) are major players in genome size (C-value) bloating, but genomes are equipped with counterbalancing systems such as diploidization, illegitimate recombination, and nonhomologous end joining (NHEJ) after double-strand breaks (DSBs). The lack of these efficient purging systems allowed the accumulation of repeat DNA, which resulted in extremely large genomes in several species. However, the correlation between chromosome number and genome size is not clear due to inconsistent results with different sets of species. Positive correlations between genome size and intron size and density were reported in early studies, but these proposals were refuted by the results with increased numbers of species, in which genome-wide features of introns (size, density, gene contents, repeats) were weakly associated with genome size. The assumption of the correlations between C-value and gene number (G-value) and organismal complexity is acceptable in general, but this assumption is often violated in specific lineages or species, suggesting C- and G-value paradoxes. The C-value paradox is partly explained by noncoding repeatomes. The G-value paradox can also be explained by several genomic features: (1) one gene can produce many mature mRNAs by alternative splicing, and eukaryotic gene expression is highly regulated at both the transcriptional and translational levels; (2) many proteins exert multiple functions during development; (3) gene expansion/contraction are frequent events in the gene family among evolutionarily close species; and (4) sets of homeotic genes regulate development such that organismal complexity is sometimes not clear among organisms. A large genome must be burdensome in terms of cell economy, such that a large genome constraint results in the distribution of genome sizes skewed to small genomes. Moreover, the C-value can affect the phenome. A strong positive correlation has been recognized between genome size and cell size, but the relationship is weak or null with higher-level traits. Additional analyses of the relationship between the C-value and phenome should be carried out, because natural selection acts on the phenotype rather than the genotype. Dramatic advancement in genomics has given some answers to the C-value and G-value paradoxes. We know the mechanisms by which the current genomes have been constructed. However, basic questions have not yet been fully resolved. Why have some species retained small genomes yet some closely related species have large genomes? Random genetic drift and mutational pressure might have affected for genome size in the limited population size during evolution; thus, genome size may be quasiadaptable rather than the best adaptive trait.

Evaluating the performance of sequence encoding schemes and machine learning methods for splice sites recognition

Identification of splice sites is imperative for prediction of gene structure. Machine learning-based approaches (MLAs) have been reported to be more successful than the rule-based methods for identification of splice sites. However, the strings of alphabets should be transformed into numeric features through sequence encoding before using them as input in MLAs. In this study, we evaluated the performances of 8 different sequence encoding schemes i.e., Bayes kernel, density and sparse (DS), distribution of tri-nucleotide and 1st order Markov model (DM), frequency difference distance measure (FDDM), paired-nucleotide frequency difference between true and false sites (FDTF), 1st order Markov model (MM1), combination of both 1st and 2nd order Markov model (MM1 + MM2) and 2nd order Markov model (MM2) in respect of predicting donor and acceptor splice sites using 5 supervised learning methods (ANN, Bagging, Boosting, RF and SVM). The encoding schemes and machine learning methods were first evaluated in 4 species i.e., A. thaliana, C. elegans, D. melanogaster and H. sapiens, and then performances were validated with another four species i.e., Ciona intestinalis, Dictyostelium discoideum, Phaeodactylum tricornutum and Trypanosoma brucei. In terms of ROC (receiver-operating-characteristics) and PR (precision-recall) curves, FDTF encoding approach achieved higher accuracy followed by either MM2 or FDDM. Further, SVM was found to achieve higher accuracy (in terms of ROC and PR curves) followed by RF across encoding schemes and species. In terms of prediction accuracy across species, the SVM-FDTF combination was optimum than other combinations of classifiers and encoding schemes. Further, splice site prediction accuracies were observed higher for the species with low intron density. To our limited knowledge, this is the first attempt as far as comprehensive evaluation of sequence encoding schemes for prediction of splice sites is concerned. We have also developed an R-package EncDNA (https://cran.r-project.org/web/packages/EncDNA/index.html) for encoding of splice site motifs with different encoding schemes, which is expected to supplement the existing nucleotide sequence encoding approaches. This study is believed to be useful for the computational biologists for predicting different functional elements on the genomic DNA.

Evolution and Diversity of Pre-mRNA Splicing in Highly Reduced Nucleomorph Genomes.

Eukaryotic genes are interrupted by introns that are removed in a conserved process known as pre-mRNA splicing. Though well-studied in select model organisms, we are only beginning to understand the variation and diversity of this process across the tree of eukaryotes. We explored pre-mRNA splicing and other features of transcription in nucleomorphs, the highly reduced remnant nuclei of secondary endosymbionts. Strand-specific transcriptomes were sequenced from the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans, whose plastids are derived from red and green algae, respectively. Both organisms exhibited elevated nucleomorph antisense transcription and gene expression relative to their respective nuclei, suggesting unique properties of gene regulation and transcriptional control in nucleomorphs. Marked differences in splicing were observed between the two nucleomorphs: the few introns of the G. theta nucleomorph were largely retained in mature transcripts, whereas the many short introns of the B. natans nucleomorph are spliced at typical eukaryotic levels (>90%). These differences in splicing levels could be reflecting the ancestries of the respective plastids, the different intron densities due to independent genome reduction events, or a combination of both. In addition to extending our understanding of the diversity of pre-mRNA splicing across eukaryotes, our study also indicates potential links between splicing, antisense transcription, and gene regulation in reduced genomes.

Sesquiterpene Synthase-3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase Fusion Protein Responsible for Hirsutene Biosynthesis in Stereum hirsutum.

The wood-rotting mushroom Stereum hirsutum is a known producer of a large number of namesake hirsutenoids, many with important bioactivities. Hirsutenoids form a structurally diverse and distinct class of sesquiterpenoids. No genes involved in hirsutenoid biosynthesis have yet been identified or their enzymes characterized. Here, we describe the cloning and functional characterization of a hirsutene synthase as an unexpected fusion protein of a sesquiterpene synthase (STS) with a C-terminal 3-hydroxy-3-methylglutaryl-coenzyme A (3-hydroxy-3-methylglutaryl-CoA) synthase (HMGS) domain. Both the full-length fusion protein and truncated STS domain are highly product-specific 1,11-cyclizing STS enzymes with kinetic properties typical of STSs. Complementation studies in Saccharomyces cerevisiae confirmed that the HMGS domain is also functional in vivo Phylogenetic analysis shows that the hirsutene synthase domain does not form a clade with other previously characterized sesquiterpene synthases from Basidiomycota. Comparative gene structure analysis of this hirsutene synthase with characterized fungal enzymes reveals a significantly higher intron density, suggesting that this enzyme may be acquired by horizontal gene transfer. In contrast, the HMGS domain is clearly related to other fungal homologs. This STS-HMGS fusion protein is part of a biosynthetic gene cluster that includes P450s and oxidases that are expressed and could be cloned from cDNA. Finally, this unusual fusion of a terpene synthase to an HMGS domain, which is not generally recognized as a key regulatory enzyme of the mevalonate isoprenoid precursor pathway, led to the identification of additional HMGS duplications in many fungal genomes, including the localization of HMGSs in other predicted sesquiterpenoid biosynthetic gene clusters.IMPORTANCE Hirsutenoids represent a structurally diverse class of bioactive sesquiterpenoids isolated from fungi. Identification of their biosynthetic pathways will provide access to this chemodiversity for the discovery and synthesis of molecules with new bioactivities. The identification and successful cloning of the previously elusive hirsutene synthase from the S. hirsutum provide important insights and strategies for biosynthetic gene discovery in Basidiomycota. The finding of a terpene synthase-HMGS fusion, the discovery of other sesquiterpenoid biosynthetic gene clusters with dedicated HMGS genes, and HMGS gene duplications in fungal genomes give new importance to the role of HMGS as a key regulatory enzyme in isoprenoid and sterol biosynthesis that should be exploited for metabolic engineering.

Multispecies reconstructions uncover widespread conservation, and lineage-specific elaborations in eukaryotic mRNA metabolism.

The degree of conservation and evolution of cytoplasmic mRNA metabolism pathways across the eukaryotes remains incompletely resolved. In this study, we describe a comprehensive genome and transcriptome-wide analysis of proteins involved in mRNA maturation, translation, and mRNA decay across representative organisms from the six eukaryotic super-groups. We demonstrate that eukaryotes share common pathways for mRNA metabolism that were almost certainly present in the last eukaryotic common ancestor, and show for the first time a correlation between intron density and a selective absence of some Exon Junction Complex (EJC) components in eukaryotes. In addition, we identify pathways that have diversified in individual lineages, with a specific focus on the unique gene gains and losses in members of the Excavata and SAR groups that contribute to their unique gene expression pathways compared to other organisms.

Identifying RNA splicing factors using IFT genes in Chlamydomonas reinhardtii.

Intraflagellar transport moves proteins in and out of flagella/cilia and it is essential for the assembly of these organelles. Using whole-genome sequencing, we identified splice site mutations in two IFT genes, IFT81 (fla9) and IFT121 (ift121-2), which lead to flagellar assembly defects in the unicellular green alga Chlamydomonas reinhardtii. The splicing defects in these ift mutants are partially corrected by mutations in two conserved spliceosome proteins, DGR14 and FRA10. We identified a dgr14 deletion mutant, which suppresses the 3′ splice site mutation in IFT81, and a frameshift mutant of FRA10, which suppresses the 5′ splice site mutation in IFT121. Surprisingly, we found dgr14-1 and fra10 mutations suppress both splice site mutations. We suggest these two proteins are involved in facilitating splice site recognition/interaction; in their absence some splice site mutations are tolerated. Nonsense mutations in SMG1, which is involved in nonsense-mediated decay, lead to accumulation of aberrant transcripts and partial restoration of flagellar assembly in the ift mutants. The high density of introns and the conservation of noncore splicing factors, together with the ease of scoring the ift mutant phenotype, make Chlamydomonas an attractive organism to identify new proteins involved in splicing through suppressor screening.

The Evolution Force of Genome Reduction in Carnivorous Plants

The introns are widely present in the genome of eukaryotes and the distribution of intron varies greatly among different organisms or different genes. Generally, introns loss is an important way for uneven distribution of intron during genome evolution. In this study, two closely related carnivorous plants (Genlisea aurea and Utricularia gibba) were chosen, their genome were relatively integrity and high quality, especially, the large difference in genome size between them. We detected intron loss events, then investigated the relationship between the genome size, intron density, intron loss and the mutation rate in the carnivorous plants. Finally, a total of 752 and 124 intron loss positions were identified in G. aurea and U. gibba, respectively. In carnivorous plants, we found that the region around lost site had high mutation rate, the genes of intron loss had high mutation rate. Besides, for the species with more intron losses, the genome size was smaller and the mutation rate was higher. Thus, we propose that the mutation rate was positively correlated with intron losses, but negatively correlated with intron number and genome size. These could be explained by the selection to minimize mutational hazards.

Cut-and-Paste Transposons in Fungi with Diverse Lifestyles.

Transposable elements (TEs) shape genomes via recombination and transposition, lead to chromosomal rearrangements, create new gene neighborhoods, and alter gene expression. They play key roles in adaptation either to symbiosis in Amanita genus or to pathogenicity in Pyrenophora tritici-repentis. Despite growing evidence of their importance, the abundance and distribution of mobile elements replicating in a “cut-and-paste” fashion is barely described so far. In order to improve our knowledge on this old and ubiquitous class of transposable elements, 1,730 fungal genomes were scanned using both de novo and homology-based approaches. DNA TEs have been identified across the whole data set and display uneven distribution from both DNA TE classification and fungal taxonomy perspectives. DNA TE content correlates with genome size, which confirms that many transposon families proliferate simultaneously. In contrast, it is independent from intron density, average gene distance and GC content. TE count is associated with species’ lifestyle and tends to be elevated in plant symbionts and decreased in animal parasites. Lastly, we found that fungi with both RIP and RNAi systems have more total DNA TE sequences but less elements retaining a functional transposase, what reflects stringent control over transposition.

Comparative Analysis of the Exon-Intron Structure in Eukaryotic Genomes

The exon numbers and lengths vary in different eukaryotic species. With increasing completed genomic sequences, it is indispensable to reanalyze the gene organization in diverse eukaryotic genomes. We performed a large-scale comparative analysis of the exon-intron structure in 72 eukaryotic organisms, including plants, fungi and animals. We confirmed that the exon-intron structure varies massively among eukaryotic genomes and revealed some lineage-specific features of eukaryotic genes. These include a teleost-specific exon-intron structure pattern, relatively small introns and large exons in fungi and algae, and a gradual expansion of introns in vertebrates. Furthermore, the conservation analysis of exon-intron boundaries indicates that several bases near splice site junctions are different in introns with variable length among different species. After comparison, we identified a trend showing increases in intron densities and lengths in diverse species from fungi, plants, invertebrates to vertebrates, while it was the opposite in relation to exon lengths. The statistical properties of eukaryotic genomic organization suggest that genome-specific features are preserved by diverse evolutionary processes, which paves way for further research on the diversification of eukaryotic evolution.

Morphological Identification and Single-Cell Genomics of Marine Diplonemids

Recent global surveys of marine biodiversity have revealed that a group of organisms known as "marine diplonemids" constitutes one of the most abundant and diverse planktonic lineages [1]. Though discovered over a decade ago [2, 3], their potential importance was unrecognized, and our knowledge remains restricted to a single gene amplified from environmental DNA, the 18S rRNA gene (small subunit [SSU]). Here, we use single-cell genomics (SCG) and microscopy to characterize ten marine diplonemids, isolated from a range of depths in the eastern North Pacific Ocean. Phylogenetic analysis confirms that the isolates reflect the entire range of marine diplonemid diversity, and comparisons to environmental SSU surveys show that sequences from the isolates range from rare to superabundant, including the single most common marine diplonemid known. SCG generated a total of ∼915 Mbp ofassembled sequence across all ten cells and ∼4,000 protein-coding genes with homologs in theKyoto Encyclopedia of Genes and Genomes (KEGG) orthology database, distributed across categories expected for heterotrophic protists. Models of highly conserved genes indicate a high density of non-canonical introns, lacking conventional GT-AG splice sites. Mapping metagenomic datasets [4] toSCG assemblies reveals virtually no overlap, suggesting that nuclear genomic diversity is too great forrepresentative SCG data to provide meaningful phylogenetic context to metagenomic datasets. This work provides an entry point to the future identification, isolation, and cultivation of these elusive yet ecologically important cells. The high density of nonconventional introns, however, also portends difficulty in generating accurate gene models and highlights the need for the establishment of stable cultures and transcriptomic analyses.

Determinants of the Usage of Splice-Associated cis-Motifs Predict the Distribution of Human Pathogenic SNPs.

Where in genes do pathogenic mutations tend to occur and does this provide clues as to the possible underlying mechanisms by which single nucleotide polymorphisms (SNPs) cause disease? As splice-disrupting mutations tend to occur predominantly at exon ends, known also to be hot spots of cis-exonic splice control elements, we examine the relationship between the relative density of such exonic cis-motifs and pathogenic SNPs. In particular, we focus on the intragene distribution of exonic splicing enhancers (ESE) and the covariance between them and disease-associated SNPs. In addition to showing that disease-causing genes tend to be genes with a high intron density, consistent with missplicing, five factors established as trends in ESE usage, are considered: relative position in exons, relative position in genes, flanking intron size, splice sites usage, and phase. We find that more than 76% of pathogenic SNPs are within 3–69 bp of exon ends where ESEs generally reside, this being 13% more than expected. Overall from enrichment of pathogenic SNPs at exon ends, we estimate that approximately 20–45% of SNPs affect splicing. Importantly, we find that within genes pathogenic SNPs tend to occur in splicing-relevant regions with low ESE density: they are found to occur preferentially in the terminal half of genes, in exons flanked by short introns and at the ends of phase (0,0) exons with 3′ non-“AGgt” splice site. We suggest the concept of the “fragile” exon, one home to pathogenic SNPs owing to its vulnerability to splice disruption owing to low ESE density.

LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes.

The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced.

Why Selection Might Be Stronger When Populations Are Small: Intron Size and Density Predict within and between-Species Usage of Exonic Splice Associated cis-Motifs.

The nearly neutral theory predicts that small effective population size provides the conditions for weakened selection. This is postulated to explain why our genome is more “bloated” than that of, for example, yeast, ours having large introns and large intergene spacer. If a bloated genome is also an error prone genome might it, however, be the case that selection for error-mitigating properties is stronger in our genome? We examine this notion using splicing as an exemplar, not least because large introns can predispose to noisy splicing. We thus ask whether, owing to genomic decay, selection for splice error-control mechanisms is stronger, not weaker, in species with large introns and small populations. In humans much information defining splice sites is in cis-exonic motifs, most notably exonic splice enhancers (ESEs). These act as splice-error control elements. Here then we ask whether within and between-species intron size is a predictor of the commonality of exonic cis-splicing motifs. We show that, as predicted, the proportion of synonymous sites that are ESE-associated and under selection in humans is weakly positively correlated with the size of the flanking intron. In a phylogenetically controlled framework, we observe, also as expected, that mean intron size is both predicted by Ne.μ and is a good predictor of cis-motif usage across species, this usage coevolving with splice site definition. Unexpectedly, however, across taxa intron density is a better predictor of cis-motif usage than intron size. We propose that selection for splice-related motifs is driven by a need to avoid decoy splice sites that will be more common in genes with many and large introns. That intron number and density predict ESE usage within human genes is consistent with this, as is the finding of intragenic heterogeneity in ESE density. As intronic content and splice site usage across species is also well predicted by Ne.μ, the result also suggests an unusual circumstance in which selection (for cis-modifiers of splicing) might be stronger when population sizes are smaller, as here splicing is noisier, resulting in a greater need to control error-prone splicing.

MitoCOGs: clusters of orthologous genes from mitochondria and implications for the evolution of eukaryotes.

BackgroundMitochondria are ubiquitous membranous organelles of eukaryotic cells that evolved from an alpha-proteobacterial endosymbiont and possess a small genome that encompasses from 3 to 106 genes. Accumulation of thousands of mitochondrial genomes from diverse groups of eukaryotes provides an opportunity for a comprehensive reconstruction of the evolution of the mitochondrial gene repertoire.ResultsClusters of orthologous mitochondrial protein-coding genes (MitoCOGs) were constructed from all available mitochondrial genomes and complemented with nuclear orthologs of mitochondrial genes. With minimal exceptions, the mitochondrial gene complements of eukaryotes are subsets of the superset of 66 genes found in jakobids. Reconstruction of the evolution of mitochondrial genomes indicates that the mitochondrial gene set of the last common ancestor of the extant eukaryotes was slightly larger than that of jakobids. This superset of mitochondrial genes likely represents an intermediate stage following the loss and transfer to the nucleus of most of the endosymbiont genes early in eukaryote evolution. Subsequent evolution in different lineages involved largely parallel transfer of ancestral endosymbiont genes to the nuclear genome. The intron density in nuclear orthologs of mitochondrial genes typically is nearly the same as in the rest of the genes in the respective genomes. However, in land plants, the intron density in nuclear orthologs of mitochondrial genes is almost 1.5-fold lower than the genomic mean, suggestive of ongoing transfer of functional genes from mitochondria to the nucleus.ConclusionsThe MitoCOGs are expected to become an important resource for the study of mitochondrial evolution. The nearly complete superset of mitochondrial genes in jakobids likely represents an intermediate stage in the evolution of eukaryotes after the initial, extensive loss and transfer of the endosymbiont genes. In addition, the bacterial multi-subunit RNA polymerase that is encoded in the jakobid mitochondrial genomes was replaced by a single-subunit phage-type RNA polymerase in the rest of the eukaryotes. These results are best compatible with the rooting of the eukaryotic tree between jakobids and the rest of the eukaryotes. The land plants are the only eukaryotic branch in which the gene transfer from the mitochondrial to the nuclear genome appears to be an active, ongoing process.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-014-0237-5) contains supplementary material, which is available to authorized users.

The identification of putative RNA polymerase II C-terminal domain associated proteins in red and green algae

A tandemly repeated C-terminal domain (CTD) of the largest subunit of RNA polymerase II is functionally essential and strongly conserved in many organisms, including animal, yeast and plant models. Although present in simple, ancestral red algae, CTD tandem repeats have undergone extensive modifications and degeneration during the evolutionary transition to developmentally complex rhodophytes. In contrast, CTD repeats are conserved in both green algae and their more complex land plant relatives. Understanding the mechanistic differences that underlie these variant patterns of CTD evolution requires knowledge of CTD-associated proteins in these 2 lineages. To provide an initial baseline comparison, we bound potential phospho-CTD associated proteins (PCAPs) to artificially synthesized and phosphorylated CTD repeats from the unicellular red alga Cyanidioschyzon merolae and green alga Chlamydomonas reinhardtii. Our results indicate that red and green algae share a number of PCAPs, including kinases and proteins involved in mRNA export. There also are important taxon-specific differences, including mRNA splicing-related PCAPs recovered from Chlamydomonas but not Cyanidioschyzon, consistent with the relative intron densities in green and red algae. Our results also offer the first experimental indication that different proteins bind 2 distinct types of repeats in Cyanidioschyzon, suggesting a division of function between the proximal and distal CTD, similar to patterns identified in more developmentally complex model organisms.

Reverse transcriptase and intron number evolution.

Introns are universal in eukaryotic genomes and play important roles in transcriptional regulation, mRNA export to the cytoplasm, nonsense-mediated decay as both a regulatory and a splicing quality control mechanism, R-loop avoidance, alternative splicing, chromatin structure, and evolution by exon-shuffling. Sixteen complete fungal genomes were used 13 of which were sequenced and annotated by JGI. Ustilago maydis, Cryptococcus neoformans, and Coprinus cinereus (also named Coprinopsis cinerea) were from the Broad Institute. Gene models from JGI-annotated genomes were taken from the GeneCatalog track that contained the best representative gene models. Varying fractions of the GeneCatalog were manually curated by external users. For clarity, we used the JGI unique database identifier. The last common ancestor of eukaryotes (LECA) has an estimated 6.4 coding exons per gene (EPG) and evolved into the diverse eukaryotic life forms, which is recapitulated by the development of a stem cell. We found a parallel between the simulated reverse transcriptase (RT)-mediated intron loss and the comparative analysis of 16 fungal genomes that spanned a wide range of intron density. Although footprints of RT (RTF) were dynamic, relative intron location (RIL) to the 5'-end of mRNA faithfully traced RT-mediated intron loss and revealed 7.7 EPG for LECA. The mode of exon length distribution was conserved in simulated intron loss, which was exemplified by the shared mode of 75 nt between fungal and Chlamydomonas genomes. The dominant ancient exon length was corroborated by the average exon length of the most intron-rich genes in fungal genomes and consistent with ancient protein modules being ~25 aa. Combined with the conservation of a protein length of 400 aa, the earliest ancestor of eukaryotes could have 16 EPG. During earlier evolution, Ascomycota's ancestor had significantly more 3'-biased RT-mediated intron loss that was followed by dramatic RTF loss. There was a down trend of EPG from more conserved to less conserved genes. Moreover, species-specific genes have higher exon-densities, shorter exons, and longer introns when compared to genes conserved at the phylum level. However, intron length in species-specific genes became shorter than that of genes conserved in all species after genomes experiencing drastic intron loss. The estimated EPG from the most frequent exon length is more than double that from the RIL method. This implies significant intron loss during the very early period of eukaryotic evolution. De novo gene-birth contributes to shorter exons, longer introns, and higher exon-density in species-specific genes relative to conserved genes.