Sex chromosomes: How to make a hermaphrodite.

The existence of sex chromosomes complicates the evolution of cosexuality (hermaphroditism). Four new genomic studies from haploid-dominant plants show commonalities and differences in mechanisms of the evolution of cosexuality, raising questions about the genetics of sexual dimorphism and the fate of cosexual lineages.

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.

Genomic Assessment of the Contribution of the Wolbachia Endosymbiont of Eurosta solidaginis to Gall Induction.

We explored the genome of the Wolbachia strain, wEsol, symbiotic with the plant-gall-inducing fly Eurosta solidaginis with the goal of determining if wEsol contributes to gall induction by its insect host. Gall induction by insects has been hypothesized to involve the secretion of the phytohormones cytokinin and auxin and/or proteinaceous effectors to stimulate cell division and growth in the host plant. We sequenced the metagenome of E. solidaginis and wEsol and assembled and annotated the genome of wEsol. The wEsol genome has an assembled length of 1.66 Mbp and contains 1878 protein-coding genes. The wEsol genome is replete with proteins encoded by mobile genetic elements and shows evidence of seven different prophages. We also detected evidence of multiple small insertions of wEsol genes into the genome of the host insect. Our characterization of the genome of wEsol indicates that it is compromised in the synthesis of dimethylallyl pyrophosphate (DMAPP) and S-adenosyl L-methionine (SAM), which are precursors required for the synthesis of cytokinins and methylthiolated cytokinins. wEsol is also incapable of synthesizing tryptophan, and its genome contains no enzymes in any of the known pathways for the synthesis of indole-3-acetic acid (IAA) from tryptophan. wEsol must steal DMAPP and L-methionine from its host and therefore is unlikely to provide cytokinin and auxin to its insect host for use in gall induction. Furthermore, in spite of its large repertoire of predicted Type IV secreted effector proteins, these effectors are more likely to contribute to the acquisition of nutrients and the manipulation of the host's cellular environment to contribute to growth and reproduction of wEsol than to aid E. solidaginis in manipulating its host plant. Combined with earlier work that shows that wEsol is absent from the salivary glands of E. solidaginis, our results suggest that wEsol does not contribute to gall induction by its host.

Transposable elements drive intron gain in diverse eukaryotes.

There is massive variation in intron numbers across eukaryotic genomes, yet the major drivers of intron content during evolution remain elusive. Rapid intron loss and gain in some lineages contrast with long-term evolutionary stasis in others. Episodic intron gain could be explained by recently discovered specialized transposons called Introners, but so far Introners are only known from a handful of species. Here, we performed a systematic search across 3,325 eukaryotic genomes and identified 27,563 Introner-derived introns in 175 genomes (5.2%). Species with Introners span remarkable phylogenetic diversity, from animals to basal protists, representing lineages whose last common ancestor dates to over 1.7 billion years ago. Aquatic organisms were 6.5 times more likely to contain Introners than terrestrial organisms. Introners exhibit mechanistic diversity but most are consistent with DNA transposition, indicating that Introners have evolved convergently hundreds of times from nonautonomous transposable elements. Transposable elements and aquatic taxa are associated with high rates of horizontal gene transfer, suggesting that this combination of factors may explain the punctuated and biased diversity of species containing Introners. More generally, our data suggest that Introners may explain the episodic nature of intron gain across the eukaryotic tree of life. These results illuminate the major source of ongoing intron creation in eukaryotic genomes.

Novel RNA viruses associated with avian haemosporidian parasites.

Avian haemosporidian parasites can cause malaria-like symptoms in their hosts and have been implicated in the demise of some bird species. The newly described Matryoshka RNA viruses (MaRNAV1 and MaRNAV2) infect haemosporidian parasites that in turn infect their vertebrate hosts. MaRNAV2 was the first RNA virus discovered associated with parasites of the genus Leucocytozoon. By analyzing metatranscriptomes from the NCBI SRA database with local sequence alignment tools, we detected two novel RNA viruses; we describe them as MaRNAV3 associated with Leucocytozoon and MaRNAV4 associated with Parahaemoproteus. MaRNAV3 had ~59% amino acid identity to the RNA-dependent RNA-polymerase (RdRp) of MaRNAV1 and ~63% amino acid identity to MaRNAV2. MaRNAV4 had ~44% amino acid identity to MaRNAV1 and ~47% amino acid identity to MaRNAV2. These findings lead us to hypothesize that MaRNAV_like viruses are widespread and tightly associated with the order Haemosporida since they have been described in human Plasmodium vivax, and now two genera of avian haemosporidians.

Coupling of spliceosome complexity to intron diversity.

We determined that over 40 spliceosomal proteins are conserved between many fungal species and humans but were lost during the evolution of S. cerevisiae, an intron-poor yeast with unusually rigid splicing signals. We analyzed null mutations in a subset of these factors, most of which had not been investigated previously, in the intron-rich yeast Cryptococcus neoformans. We found they govern splicing efficiency of introns with divergent spacing between intron elements. Importantly, most of these factors also suppress usage of weak nearby cryptic/alternative splice sites. Among these, orthologs of GPATCH1 and the helicase DHX35 display correlated functional signatures and copurify with each other as well as components of catalytically active spliceosomes, identifying a conserved G patch/helicase pair that promotes splicing fidelity. We propose that a significant fraction of spliceosomal proteins in humans and most eukaryotes are involved in limiting splicing errors, potentially through kinetic proofreading mechanisms, thereby enabling greater intron diversity.

ExOrthist: a tool to infer exon orthologies at any evolutionary distance.

Several bioinformatic tools have been developed for genome-wide identification of orthologous and paralogous genes. However, no corresponding tool allows the detection of exon homology relationships. Here, we present ExOrthist, a fully reproducible Nextflow-based software enabling inference of exon homologs and orthogroups, visualization of evolution of exon-intron structures, and assessment of conservation of alternative splicing patterns. ExOrthist evaluates exon sequence conservation and considers the surrounding exon-intron context to derive genome-wide multi-species exon homologies at any evolutionary distance. We demonstrate its use in different evolutionary scenarios: whole genome duplication in frogs and convergence of Nova-regulated splicing networks ( https://github.com/biocorecrg/ExOrthist ).

Mammals with Small Populations Do Not Exhibit Larger Genomes.

Genome size in cellular organisms varies by six orders of magnitude, yet the cause of this large variation remains unexplained. The influential Drift-Barrier Hypothesis proposes that large genomes tend to evolve in small populations due to inefficient selection. However, to our knowledge no explicit tests of the Drift-Barrier Hypothesis have been reported. We performed the first explicit test, by comparing estimated census population size and genome size in mammals while incorporating potential covariates and the effect of shared evolutionary history. We found a lack of correlation between census population size and genome size among 199 species of mammals. These results suggest that population size is not the predominant factor influencing genome size and that the Drift-Barrier Hypothesis should be considered provisional.

Sex chromosome transformation and the origin of a male-specific X chromosome in the creeping vole.

The mammalian sex chromosome system (XX female/XY male) is ancient and highly conserved. The sex chromosome karyotype of the creeping vole (Microtus oregoni) represents a long-standing anomaly, with an X chromosome that is unpaired in females (X0) and exclusively maternally transmitted. We produced a highly contiguous male genome assembly, together with short-read genomes and transcriptomes for both sexes. We show that M. oregoni has lost an independently segregating Y chromosome and that the male-specific sex chromosome is a second X chromosome that is largely homologous to the maternally transmitted X. Both maternally inherited and male-specific sex chromosomes carry fragments of the ancestral Y chromosome. Consequences of this recently transformed sex chromosome system include Y-like degeneration and gene amplification on the male-specific X, expression of ancestral Y-linked genes in females, and X inactivation of the male-specific chromosome in male somatic cells. The genome of M. oregoni elucidates the processes that shape the gene content and dosage of mammalian sex chromosomes and exemplifies a rare case of plasticity in an ancient sex chromosome system.

Expansion and transformation of the minor spliceosomal system in the slime mold Physarum polycephalum.

Spliceosomal introns interrupt nuclear genes and are removed from RNA transcripts ("spliced") by machinery called spliceosomes. Although the vast majority of spliceosomal introns are removed by the so-called major (or "U2") spliceosome, diverse eukaryotes also contain a rare second form, the minor ("U12") spliceosome, and associated ("U12-type") introns.1-3 In all characterized species, U12-type introns are distinguished by several features, including being rare in the genome (∼0.5% of all introns),4-6 containing extended evolutionarily conserved splicing motifs,4,5,7,8 being generally ancient,9,10 and being inefficiently spliced.11-13 Here, we report a remarkable exception in the slime mold Physarum polycephalum. The P. polycephalum genome contains >20,000 U12-type introns-25 times more than any other species-enriched in a diversity of non-canonical splice boundaries as well as transformed splicing signals that appear to have co-evolved with the spliceosome due to massive gain of efficiently spliced U12-type introns. These results reveal an unappreciated dynamism of minor spliceosomal introns and spliceosomal introns in general.

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.

Author Correction: Elephant shark genome provides unique insights into gnathostome evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Comprehensive database and evolutionary dynamics of U12-type introns.

During nuclear maturation of most eukaryotic pre-messenger RNAs and long non-coding RNAs, introns are removed through the process of RNA splicing. Different classes of introns are excised by the U2-type or the U12-type spliceosomes, large complexes of small nuclear ribonucleoprotein particles and associated proteins. We created intronIC, a program for assigning intron class to all introns in a given genome, and used it on 24 eukaryotic genomes to create the Intron Annotation and Orthology Database (IAOD). We then used the data in the IAOD to revisit several hypotheses concerning the evolution of the two classes of spliceosomal introns, finding support for the class conversion model explaining the low abundance of U12-type introns in modern genomes.

Molecular Evolution: RNA Splicing Machinery Moonlights in Junk Removal.

A close relative of vertebrates solves the problem of gene-disrupting transposable element insertions by splicing them out at the RNA level. Why is such an elegant solution so rare across eukaryotes?

On the Function of Trans-Splicing: No Evidence for Widespread Proteome Diversification in Trypanosomes.

A long-standing mystery of genomic/transcriptomic structure involves spliced leader trans-splicing (SLTS), in which short RNA "tags" transcribed from a distinct genomic locus is added near the 5' end of RNA transcripts by the spliceosome. SLTS has been observed in diverse eukaryotes in a phylogenetic pattern implying recurrent independent evolution. This striking convergence suggests important functions for SLTS, however no general novel function is known. Recent findings of frequent alternative SLTS (ALT-TS) suggest that ALT-TS could impart widespread functionality. Here, we tested the hypothesis that ALT-TS diversifies proteomes by comparing splicing patterns in orthologous genes between two deeply diverged trypanosome parasites. We also tested proteome diversification functions of ALT-TS by utilizing ribosome profiling sequence data. Finally, we investigated ALT-TS as a mechanism to regulate the expression of unproductive transcripts. Although our results indicate the functional importance of some cases of trans-splicing, we find no evidence for the hypothesis that proteome diversification is a general function of trans-splicing.

Patterns of conservation of spliceosomal intron structures and spliceosome divergence in representatives of the diplomonad and parabasalid lineages.

BACKGROUND: Two spliceosomal intron types co-exist in eukaryotic precursor mRNAs and are excised by distinct U2-dependent and U12-dependent spliceosomes. In the diplomonad Giardia lamblia, small nuclear (sn) RNAs show hybrid characteristics of U2- and U12-dependent spliceosomal snRNAs and 5 of 11 identified remaining spliceosomal introns are trans-spliced. It is unknown whether unusual intron and spliceosome features are conserved in other diplomonads. RESULTS: We have identified spliceosomal introns, snRNAs and proteins from two additional diplomonads for which genome information is currently available, Spironucleus vortens and Spironucleus salmonicida, as well as relatives, including 6 verified cis-spliceosomal introns in S. vortens. Intron splicing signals are mostly conserved between the Spironucleus species and G. lamblia. Similar to 'long' G. lamblia introns, RNA secondary structural potential is evident for 'long' (> 50 nt) Spironucleus introns as well as introns identified in the parabasalid Trichomonas vaginalis. Base pairing within these introns is predicted to constrain spatial distances between splice junctions to similar distances seen in the shorter and uniformly-sized introns in these organisms. We find that several remaining Spironucleus spliceosomal introns are ancient. We identified a candidate U2 snRNA from S. vortens, and U2 and U5 snRNAs in S. salmonicida; cumulatively, illustrating significant snRNA differences within some diplomonads. Finally, we studied spliceosomal protein complements and find protein sets in Giardia, Spironucleus and Trepomonas sp. PC1 highly- reduced but well conserved across the clade, with between 44 and 62 out of 174 studied spliceosomal proteins detectable. Comparison with more distant relatives revealed a highly nested pattern, with the more intron-rich fornicate Kipferlia bialata retaining 87 total proteins including nearly all those observed in the diplomonad representatives, and the oxymonad Monocercomonoides retaining 115 total proteins including nearly all those observed in K. bialata. CONCLUSIONS: Comparisons in diplomonad representatives and species of other closely-related metamonad groups indicates similar patterns of intron structural conservation and spliceosomal protein composition but significant divergence of snRNA structure in genomically-reduced species. Relative to other eukaryotes, loss of evolutionarily-conserved snRNA domains and common sets of spliceosomal proteins point to a more streamlined splicing mechanism, where intron sequences and structures may be functionally compensating for the minimalization of spliceosome components.

Intragenomic Conflict and Immune Tolerance: Do Selfish X-Linked Alleles Drive Skewed X Chromosome Inactivation?

In mammalian females, diploid somatic cells contain two X chromosomes, one of which is transcriptionally silenced, in a process termed X chromosome inactivation (XCI). Whereas XCI is largely random in placental females, many women exhibit skewed XCI (SXCI), in which the vast majority cells have the same X chromosome inactivated. SXCI has serious health consequences, associated with conditions ranging from Alzheimer's to various autoimmune disorders. SXCI is also associated with outcomes of pregnancies, with higher rates of recurrent spontaneous abortion in women with SXCI. Here, I suggest that SXCI could be driven by selfish X-linked alleles. Consistent with the association of SXCI with autoimmunity, I first note the possibility that recurrent spontaneous abortion could reflect immune rejection of fetuses inheriting alleles from the largely silenced maternal X chromosome. Preferential abortion of fetuses carrying silenced X-linked alleles implies a transmission advantage for X-linked alleles on the largely expressed chromosome, which could drive the emergence of X-linked alleles that make the chromosome resistant to XCI. I discuss the evolutionary dynamics, fitness tradeoffs and implications of this hypothesis, and suggest future directions.

The Macronuclear Genome of Stentor coeruleus Reveals Tiny Introns in a Giant Cell.

The giant, single-celled organism Stentor coeruleus has a long history as a model system for studying pattern formation and regeneration in single cells. Stentor [1, 2] is a heterotrichous ciliate distantly related to familiar ciliate models, such as Tetrahymena or Paramecium. The primary distinguishing feature of Stentor is its incredible size: a single cell is 1 mm long. Early developmental biologists, including T.H. Morgan [3], were attracted to the system because of its regenerative abilities-if large portions of a cell are surgically removed, the remnant reorganizes into a normal-looking but smaller cell with correct proportionality [2, 3]. These biologists were also drawn to Stentor because it exhibits a rich repertoire of behaviors, including light avoidance, mechanosensitive contraction, food selection, and even the ability to habituate to touch, a simple form of learning usually seen in higher organisms [4]. While early microsurgical approaches demonstrated a startling array of regenerative and morphogenetic processes in this single-celled organism, Stentor was never developed as a molecular model system. We report the sequencing of the Stentor coeruleus macronuclear genome and reveal key features of the genome. First, we find that Stentor uses the standard genetic code, suggesting that ciliate-specific genetic codes arose after Stentor branched from other ciliates. We also discover that ploidy correlates with Stentor's cell size. Finally, in the Stentor genome, we discover the smallest spliceosomal introns reported for any species. The sequenced genome opens the door to molecular analysis of single-cell regeneration in Stentor.

Mechanism for DNA transposons to generate introns on genomic scales.

The discovery of introns four decades ago was one of the most unexpected findings in molecular biology. Introns are sequences interrupting genes that must be removed as part of messenger RNA production. Genome sequencing projects have shown that most eukaryotic genes contain at least one intron, and frequently many. Comparison of these genomes reveals a history of long evolutionary periods during which few introns were gained, punctuated by episodes of rapid, extensive gain. However, although several detailed mechanisms for such episodic intron generation have been proposed, none has been empirically supported on a genomic scale. Here we show how short, non-autonomous DNA transposons independently generated hundreds to thousands of introns in the prasinophyte Micromonas pusilla and the pelagophyte Aureococcus anophagefferens. Each transposon carries one splice site. The other splice site is co-opted from the gene sequence that is duplicated upon transposon insertion, allowing perfect splicing out of the RNA. The distributions of sequences that can be co-opted are biased with respect to codons, and phasing of transposon-generated introns is similarly biased. These transposons insert between pre-existing nucleosomes, so that multiple nearby insertions generate nucleosome-sized intervening segments. Thus, transposon insertion and sequence co-option may explain the intron phase biases and prevalence of nucleosome-sized exons observed in eukaryotes. Overall, the two independent examples of proliferating elements illustrate a general DNA transposon mechanism that can plausibly account for episodes of rapid, extensive intron gain during eukaryotic evolution.

Is Genome Complexity a Consequence of Inefficient Selection? Evidence from Intron Creation in Nonrecombining Regions.

Genomes show remarkable variation in architecture and complexity across organisms, with large differences in genome size and in numbers of genes, gene duplicates, introns and transposable elements. These differences have important implications for transcriptome and regulatory complexity and ultimately for organismal complexity. Numbers of spliceosomal introns show particularly striking differences, ranging across organisms from zero to hundreds of thousands of introns per genome. The causes of these differences remain poorly understood. According to one influential perspective, differences across species reflect the differential ability of selection in different populations to eliminate allegedly deleterious intron-containing alleles. Direct tests of this theory have been elusive. Here, I study evolution of intron-exon structures in genomic regions of recombination suppression (RRSs), which experience drastically reduced selective efficiency due to hitchhiking and background selection. I studied intron creation in eight independently evolved RRSs, spanning substantial diversity phylogenetically (plants, animals, fungi and brown algae) and biologically (sex chromosomes, mating type chromosomes, genomic regions flanking self-incompatibility loci, and the Drosophila "dot" chromosome). To identify newly created introns in RRSs, I compared intron positions in RRS genes with those in homologous genes. I found very few intron gains: no intron gains were observed in 7/8 studied data sets, and only three intron gains were observed overall (on the Drosophila dot chromosome). These results suggest that efficiency of selection may not be a major cause of differences in intron-exon structures across organisms. Instead, rates of spontaneous intron-creating and intron-deleting mutations may play the central role in shaping intron-exon structures.