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The intron-exon structures of nuclear genes show striking diversity across eukaryotes. Several independent lineages have undergone convergent evolution including widespread loss of introns and transformed cis splicing signals. The causes and mechanisms of these changes remain mysterious: (i) transformation of splicing signals could reflect either selective loss of suboptimal introns or coevolution of introns and splicing machinery; and (ii) corresponding changes in the splicing machinery remain poorly characterized. A promising model to study these questions is Entamoeba. Analysis of five Entamoeba species revealed low intron densities, nearly universal atypical 5’ splice sites and 3’ intronic sequences. A flexible search for U1 snRNA genes revealed a modified 5’-AACAAAC-3’ recognition sequence, affording complete Watson-Crick basepairing potential with the atypical 5’ splice site and extended basepairing potential. A U1 candidate in the related species Mastigamoeba balumuthi revealed a separate modification complimenting a different atypical consensus splice site. Genome-wide study of intron loss and gain revealed that introns with suboptimal splicing motifs were no more likely to be lost, suggesting against genome-wide homogenization of intron splicing motifs by selective intron loss. Unexpectedly, this analysis also revealed widespread intron gain in Entamoeba invadens. In total, the current analyses: (i) provide the most direct available evidence of coevolution of spliceosomal introns and splicing machinery; (ii) illuminate the evolutionary forces responsible for concerted intron loss and splicing motif transformation; and (iii) reveal widespread intron gain in an otherwise highly reduced lineage.

Haplodiploidy and paternal genome elimination (HD/PGE) are common in invertebrates, having evolved at least two dozen times, all from male heterogamety (i.e., systems with X chromosomes). However, why X chromosomes are important for the evolution of HD/PGE remains debated. The Haploid Viability Hypothesis posits that X-linked genes promote the evolution of male haploidy by facilitating purging recessive deleterious mutations. The Intragenomic Conflict Hypothesis holds that conflict between genes drives genetic system turnover; under this model, X-linked genes could promote the evolution of male haploidy due to conflicts with autosomes over sex ratios and genetic transmission. We studied lineages where we can distinguish these hypotheses: species with germline PGE that retain an XX/X0 sex determination system (gPGE+X). Because evolving PGE in these cases involves changes in transmission without increases in male hemizygosity, a high degree of X linkage in these systems is predicted by the Intragenomic Conflict Hypothesis but not the Haploid Viability Hypothesis. To quantify the degree of X linkage, we sequenced and compared 7 gPGE+X species’ genomes with 11 related species with typical XX/XY or XX/X0 genetic systems, representing three transitions to gPGE. We find highly increased X linkage in both modern and ancestral genomes of gPGE+X species compared to non-gPGE relatives, and recover a significant positive correlation between percent X linkage and the evolution of gPGE. These are among the first empirical results suggesting a role for intragenomic conflict in the evolution of novel genetic systems like HD/PGE.Significance StatementSex determination systems such as haplodiploidy, in which males’ gene transmission is haploid, are surprisingly common, however, the evolutionary paths to these systems are poorly understood. X chromosomes may play a particularly important role, either by increasing survival of males with only maternal genomes, or due to conflicts between X-chromosomal and autosomal genes. We studied X-chromosome gene richness in three arthropod lineages in which males are diploid as adults but only transmit their maternally-inherited haploid genome. We find that species with such atypical systems have far more X chromosomal genes than related diploid species. These results suggest that conflict between genetic elements within the genome drives the evolution of unusual sex determination systems.

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.

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.

Recent work has illuminated the bizarre sex chromosomal system of the mandarin vole, Lasiopodomys mandarinus. The ancestral sex chromosomes have been replaced by 4 neo-sex chromosomes. These sex chromosomes show non-Mendelian inheritance and epistatic sex determination, as well as unaccounted-for karyotype frequencies. I suggest a model to account for the complex observed inheritance patterns. The proposed model combines putative adaptations previously observed in rodents, including feminizing X chromosomes and Y-biased spermatogenesis, with a novel proposed mechanism of genomic imprinting of X-linked genes during oogenesis in XY females. Alternative possibilities are also discussed. The proposed scenario provides a relatively simple and testable model for the function and origins of a remarkably complex mammalian sex chromosomal system.

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.

Significance Some social insects exhibit split sex ratios, wherein a subset of colonies produce future queens and others produce males. This phenomenon spawned many influential theoretical studies and empirical tests, both of which have advanced our understanding of parent–offspring conflicts and the maintenance of cooperative breeding. However, previous studies assumed that split sex ratio was not under genetic control. Here, we show that split sex ratio is associated with a large genomic region in two ant species. The discovery that sex allocation can have a genetic basis provides an additional perspective on this well-studied trait of social insects., Sexually reproducing organisms usually invest equally in male and female offspring. Deviations from this pattern have led researchers to new discoveries in the study of parent–offspring conflict, genomic conflict, and cooperative breeding. Some social insect species exhibit the unusual population-level pattern of split sex ratio, wherein some colonies specialize in the production of future queens and others specialize in the production of males. Theoretical work predicted that worker control of sex ratio and variation in relatedness asymmetry among colonies would cause each colony to specialize in the production of one sex. While some empirical tests supported theoretical predictions, others deviated from them, leaving many questions about how split sex ratio emerges. One factor yet to be investigated is whether colony sex ratio may be influenced by the genotypes of queens or workers. Here, we sequence the genomes of 138 Formica glacialis workers from 34 male-producing and 34 gyne-producing colonies to determine whether split sex ratio is under genetic control. We identify a supergene spanning 5.5 Mbp that is closely associated with sex allocation in this system. Strikingly, this supergene is adjacent to another supergene spanning 5 Mbp that is associated with variation in colony queen number. We identify a similar pattern in a second related species, Formica podzolica. The discovery that split sex ratio is determined, at least in part, by a supergene in two species opens future research on the evolutionary drivers of split sex ratio.

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.

In modern biology, inquiry into proximal mechanistic and ultimate evolutionary causes are often segregated, pursued by different communities of specialists. Yet, the two are often mutually informative. As a case in point, a recent study by Long et al. on mechanisms of arabidopsis (Arabidopsisthaliana) male germline methylation promises insights into long-obscure ultimate causes.

Sex determination mechanisms vary widely across animals, but show remarkable degrees of recurrent evolution. Recurrent features of sex determination have largely been attributed to recurrent cooption of shared ancestral regulatory circuits. However, a new study on sex determination in Daphnia magna reveals both recurrent evolution of specific regulatory logic and apparently recurrent recruitment of a regulator, suggesting a role for optimization in recurrent patterns of sex determination mechanisms.

While much excitement has attended the discovery and study of circular RNAs, a new study in Cell Reports suggests that most mammalian circRNAs are not only functionless, but in fact costly. Comparison across three species is also consistent with the influential but rarely tested Drift-Barrier Hypothesis of molecular complexity. According to this hypothesis, nonessential genomic elements are slightly deleterious elements that fix by genetic drift and, thus, are generally more abundant in species with small effective population sizes. I discuss the implications of these new results for the Drift-Barrier hypothesis. In particular, I note the distinction between two classes of genomic elements, based on whether they are created by ‘standard’ small-scale mutations (basepair substitutions, indels, etc.) or larger, more idiosyncratic mutations (segmental duplications, transposable element propagation, etc.) I suggest that the Drift-Barrier Hypothesis is likely to apply to the former class, but perhaps not the latter class.

The sex-determining gene SRY has undergone rapid evolution in rodents. Curiously, a new study by Miyawaki et al. reveals that a recently evolved SRY gene sequence antagonizes SRY protein stability, necessitating splicing of a novel intron. Other data suggest that this troublesome gene region has noncoding RNA functions, possibly related to conflict between sex chromosomes.

CXCR3 is a chemokine receptor with two well-characterized isoforms that have unique, context-dependent roles: CXCR3-A and CXCR3-B, which are produced through alternative 3′ splice site selection (A3SS). RNA-seq data from The Cancer Genome Atlas (TCGA) were used to correlate CXCR3 expression with breast cancer progression. This analysis revealed significant CXCR3 expression patterns associated with survival and differential expression between the tumor and adjacent normal tissue. TCGA data were used to estimate abundance of immune cells in breast cancer, which demonstrated the association of CXCR3 with immune infiltration, particularly in the triple-negative subtype. Given the importance of A3SS in CXCR3, genome-wide analysis of A3SS events was performed to identify events that were differentially spliced between breast cancer tissue and adjacent normal tissue. A total of 481 splicing events in 424 genes were found to be differentially spliced. The parent genes of differentially spliced events were enriched in RNA processing and splicing functions, indicating an underappreciated role of A3SS in the integrated splicing network of breast cancer. These results further validated the role of CXCR3 in immune infiltration of tumors, while raising questions about the role of A3SS splicing.

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.

Reproductive proteins, including those expressed in the testes, are among the fastest evolving proteins across the tree of life. Sexual selection on traits involved in sperm competition is thought to be a primary driver of testes gene evolution and is expected to differ between promiscuous and monogamous species due to intense competition between males to fertilize females in promiscuous lineages and lack thereof in monogamous ones. Here, we employ the rodent genus Peromyscus as a model to explore differences in evolutionary rates between testes-expressed genes in monogamous and promiscuous species. We find candidate genes that may be associated with increased sperm production in promiscuous species and gene ontology categories that show patterns of molecular convergence associated with phenotypic convergence in independently evolved monogamous species. Overall, our results highlight the possible molecular consequences of differences in mating system, likely due to differences in selective pressures.

Haldane's rule, which states that the heterogametic sex (XY or ZW females) fares more poorly in interspecific hybrids, is generally attributed to absence of one of the two species' X/Z chromosomes. However, Haldane's rule is also observed in mouse placentas despite paternal X silencing. This pattern could reflect Y chromosomes having evolved to promote growth due to maternal-paternal conflict. If so, balanced sex investment arises from a complex intra- and intergenomic duel.

A recent paper (Li et al.) reports a novel RNA-based Cas-dependent toxin-antitoxin system with the effect of 'addicting' cells to the cassette. Broadly-defined addiction systems could stabilize diverse genomic features, raising the question of the role of selfish elements and intragenomic conflict in the evolution of biological complexity.

Multicellular eukaryotes exhibit a remarkable diversity of sexual systems; however, trioecy, the coexistence of male, female, and cosexual or hermaphrodite individuals in a single species, is remarkably rare. Takahashi et al. (2021) report the first known instance of trioecy in a haploid organism. In contrast to other known cases of trioecy, the authors report evidence for genetic control of all three sexes by two loci. These results complicate models for sexual system turnover and expand the known diversity of trioecy species in several ways.

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). The research has been funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (ERC-StG-LS2-637591 to MI), the Spanish Ministerio de Ciencia (BFU2017-89201-P to MI, including and FPI PhD fellowship to FM), the “Centro de Excelencia Severo Ochoa 2013-2017” (SEV-2012-0208), EMBO Long Term postdoctoral fellowship (ALTF 1505-2015 to YM), and Marie Skłodowska-Curie actions (MSCA) grant (705938 to YM). We acknowledge the support of the CERCA Programme/Generalitat de Catalunya and of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership

New work suggests ‘subgenome dominance' in polyploids may only occur in angiosperms. Subgenome dominance could explain angiosperm-specific genome reduction, with potential implications for angiosperms' global dominance. I suggest that evolution of the endosperm could have selected for the evolution of subgenome dominance, due to increased hybrid/polyploid incompatibilities and/or through direct reciprocal suppression of maternally- and paternally-inherited genomes.

SummarySpliceosomal introns, which interrupt nuclear genes and are removed from RNA transcripts by machinery termed spliceosomes, are ubiquitous features of eukaryotic nuclear genes [1]. Patterns of spliceosomal intron evolution are complex, with some lineages exhibiting virtually no intron creation while others experience thousands of intron gains [2–5]. One possibility is that this punctate phylogenetic distribution is explained by intron creation by Introner-Like Elements (ILEs), transposable elements capable of creating introns, with only those lineages harboring ILEs undergoing massive intron gain [6–10]. However, ILEs have been reported in only four lineages. Here we study intron evolution in dinoflagellates. The remarkable fragmentation of nuclear genes by spliceosomal introns reaches its apex in dinoflagellates, which have some twenty introns per gene [11,12]. Despite this, almost nothing is known about the molecular and evolutionary mechanisms governing dinoflagellate intron evolution. We reconstructed intron evolution in five dinoflagellate genomes, revealing a dynamic history of intron loss and gain. ILEs are found in 4/5 studied species. In one species, Polarella glacialis, we find an unprecedented diversity of ILEs, with ILE insertion leading to creation of some 12,253 introns, and with 15 separate families of ILEs accounting for at least 100 introns each. These ILE families range in mobilization mechanism, mechanism of intron creation, and flexibility of mechanism of intron creation. Comparison within and between ILE families provides evidence that biases in so-called intron phase, the distribution of introns relative to codon periodicity, are driven by ILE insertion site requirements [9,13,14]. Finally, we find evidence for multiple additional transformations of the spliceosomal system in dinoflagellates, including widespread loss of ancestral introns, and alterations in required, tolerated and favored splice motifs. These results reveal unappreciated intron creating elements diversity and spliceosomal evolutionary capacity, and suggest complex evolutionary dependencies shaping genome structures.

Spliceosomal introns, which interrupt nuclear genes, are ubiquitous features of eukaryotic nuclear genes.

Summary 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.

Current evolutionary reconstructions predict that early eukaryotic ancestors including both the last common ancestor of eukaryotes and of all fungi had intron-rich genomes. However, some extant eukaryotes have few introns, raising the question as to why these few introns are retained. Here we have used recently available fungal genomes to address this question. Evolutionary reconstruction of intron presence and absence using 263 diverse fungal species support the idea that massive intron loss has occurred in multiple clades. The intron densities estimated in the fungal ancestral states differ from zero to 8.28 introns per one kbp of protein-coding gene. Massive intron loss has occurred not only in microsporidian parasites and saccharomycetous yeasts (0.01 and 0.05 introns/kbp on average, respectively), but also in diverse smuts and allies (e.g.Ustilago maydis, Meira miltonrushiiandMalassezia globosahave 0.06, 0.10 and 0.20 introns/kbp, respectively). To investigate the roles of introns, we searched for their special characteristics using 1302 orthologous genes from eight intron-poor fungi. Notably, most of these introns are found close to the translation initiation codons. Our transcriptome and translatome data analyses showed that these introns are from genes with both higher mRNA expression and translation efficiency. Furthermore, these introns are common in specific classes of genes (e.g. genes involved in translation and Golgi vesicle transport), and rare in others (e.g. base-excision repair genes). Our study shows that fungal introns have a complex evolutionary history and underappreciated roles in gene expression.

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.

In human cells, the U12 spliceosome, also known as the minor spliceosome, is responsible for the splicing of 0.5% of introns, while the major U2 spliceosome is responsible for the other 99.5%. While many studies have been done to characterize and understand splicing dysregulation in cancer, almost all of them have focused on U2 splicing and ignored U12 splicing, despite evidence suggesting minor splicing is involved in cell cycle regulation. In this study, we analyzed RNA-seq data from The Cancer Genome Atlas for 14 different cohorts to determine differential splicing of minor introns in tumor and adjacent normal tissue. We found that in some cohorts, such as breast cancer, there was a strong skew towards minor introns showing increased splicing in the tumor; in others, such as the renal chromophobe cell carcinoma cohort, the opposite pattern was found, with minor introns being much more likely to have decreased splicing in the tumor. Further analysis of gene expression did not reveal any candidate regulatory mechanisms that could cause these different minor splicing phenotypes between cohorts. Our data suggest context-dependent roles of the minor spliceosome in tumorigenesis and provides a foundation for further investigation of minor splicing in cancer, which could then serve as a basis for novel therapeutic strategies.

The Aco2 gene of Schizosaccharomyces pombe was formed by gene fusion between curious partners, namely genes encoding the enzyme aconitase and a mitochondrial ribosomal protein. In addition to a full-length transcript, a truncated mRNA encoding only the N-terminal aconitase domain is produced by polyadenylation at an internal site. Protein products of the gene are found in the nucleus, mitochondria and cytoplasm, consistent with the presence of multiple subcellular targeting signals. To reconstruct the evolution of this complex gene, we studied homologous genes from a range of related species. We find evidence for a dynamic history within Taphrinomycotina, including: early evolution of a nuclear localization signal; creation of a 3’ intron that could be involved in regulating subcellular targeting; evolution of multiple peroxisomal targeting signals in different lineages; and recurrent gene loss. We present a likely stepwise model for the evolution of this remarkable gene and discuss alternative scenarios.

SUMMARYGlial cells regulate synaptic connectivity during development, but whether they selectively instruct the formation of specific synaptic circuits is not known. Here we show that the major perisynaptic glia of the retina, the Muller glia (MG), control the proper establishment of the direction-selective (DS) circuit by a synaptogenic protein, Thrombospondin 1 (TSP1). We found that TSP1 promotes excitatory synapse formation specifically in on-off Direction-Selective retinal Ganglion Cells (ooDSGCs). Lack of TSP1 leads to reduced synapse formation within the inner plexiform sublayers containing DS-circuit, resulting in deficits of ooDSGC function. Even though pan-TSP receptor, α2δ-1, interaction is required for TSP1-induced synapse formation, the ooDSGC-subtype specificity of TSP1 is conferred by a second neuronal TSP1 receptor, β1-Integrin. Furthermore, conditional deletion of β1-Integrin in ooDSGCs results in diminished excitatory synapse formation without disturbing laminar organization showing that MG-secreted TSP1 controls circuit-specific synapse formation via β1-Integrin.

SUMMARYWe 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.

Despite its importance, the selective and mechanistic forces governing recombination remain obscure. A recent study of facultatively asexual honeybees suggests a clear case of adaptive adjustment of recombination rate. That these bees' atypical genetics were central to the experiment underscores the utility of genetic oddities as model organisms for studying fundamental questions.

Spliced leader trans-splicing (SLTS) is a poorly understood mechanism that is found in a diversity of eukaryotic lineages. In SLTS, a short RNA sequence is added near the 5′ ends of the transcripts of protein-coding genes by a modified spliceosomal reaction. Available data suggest that SLTS has evolved many times, and might be more likely to evolve in animals. That SLTS might be more likely to evolve in the context of the generally complex transcriptomes characteristic of animals suggests the possibility that SLTS functions in gene regulation or transcriptome diversification, however no general novel function for SLTS is known. Here, I report SLTS in a lineage of cellularly complex unicellular eukaryotes. Cryptomonads are a group of eukaryotic algae that acquired photosynthetic capacity by secondary endosymbiosis of a red alga, and that retain a reduced copy of the nucleus of the engulfed alga. I estimate that at least one-fifth of genes in the model cryptomonad Guillardia theta and its relative Hanusia phi undergo SLTS. I show that hundreds of genes in G. theta generate alternative transcripts by SLTS at alternative sites, however I find little evidence for alternative protein production by alternative SLTS splicing. Interestingly, I find no evidence for substantial operon structure in the G. theta genome, in contrast to previous findings in other lineages with SLTS. These results extend SLTS to another major group of eukaryotes, and heighten the mystery of the evolution of SLTS and its association with cellular and transcriptomic complexity.

In the original article, the co-author name "Jennifer Kim" has been inadvertently missed during the publication process. The complete author group is given in this correction.

Summary 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?

Mutations can occur throughout the virus genome and may be beneficial, neutral or deleterious. We are interested in mutations that yield a C next to a G, producing CpG sites. CpG sites are rare in eukaryotic and viral genomes. For the eukaryotes, it is thought that CpG sites are rare because they are prone to mutation when methylated. In viruses, we know less about why CpG sites are rare. A previous study in HIV suggested that CpG-creating transition mutations are more costly than similar non-CpG-creating mutations. To determine if this is the case in other viruses, we analyzed the allele frequencies of CpG-creating and non-CpG-creating mutations across various strains, subtypes, and genes of viruses using existing data obtained from Genbank, HIV Databases, and Virus Pathogen Resource. Our results suggest that CpG sites are indeed costly for most viruses. By understanding the cost of CpG sites, we can obtain further insights into the evolution and adaptation of viruses., Evolutionary Ecology, 34, ISSN:0269-7653, ISSN:1573-8477

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.

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. Electronic supplementary material The online version of this article (10.1186/s12862-019-1488-y) contains supplementary material, which is available to authorized users.

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.

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.

The evolutionary history of the spliceosomal introns that interrupt nuclear genes in eukaryotes has been debated for four decades. Positions of introns show a high degree of coincidence between various eukaryotes, implying either than many modern introns are very old and/or that intron creation is highly biased toward certain sites, leading to rampant parallel intron gain. A series of articles in this and other journals reported evidence for a strikingly high degree of parallel insertion of introns in different alleles of the water flea Daphnia pulex Here, I report several new analyses of these data. Among the 23 loci reported to be undergoing parallel intron gain, I find that in five cases introns reported to be unrelated show extended sequence similarity strongly suggesting that the introns are in fact homologous. Five additional cases show extended conserved motifs between allegedly unrelated introns. For nearly all loci including the 13 remaining loci, at least one intron shows hallmarks of rapid sequence evolution, thwarting confident inference about homology. In addition, I reanalyze gene trees reconstructed from flanking exonic sequences, claimed by the original authors as additional evidence for parallel gain. I show that these phylogenetic trees frequently fail to recover expected relationships, and in any case show relationships not consistent with parallel intron gains. In total, I conclude that the data do not support widespread parallel intron gain in D. pulex These findings strengthen the notion that shared intron positions generally reflect ancestral introns, and thus the notion of complex genes in early eukaryotes.