Dynamic co-evolution of transposable elements and the piRNA pathway in African cichlid fishes.

East African cichlid fishes have diversified in an explosive fashion, but the (epi)genetic basis of the phenotypic diversity of these fishes remains largely unknown. Although transposable elements (TEs) have been associated with phenotypic variation in cichlids, little is known about their transcriptional activity and epigenetic silencing. Here, we describe dynamic patterns of TE expression in African cichlid gonads and during early development. Orthology inference revealed an expansion of piwil1 genes in Lake Malawi cichlids, likely driven by PiggyBac TEs. The expanded piwil1 copies have signatures of positive selection and retain amino acid residues essential for catalytic activity. Furthermore, the gonads of African cichlids express a Piwi-interacting RNA (piRNA) pathway that target TEs. We define the genomic sites of piRNA production in African cichlids and find divergence in closely related species, in line with fast evolution of piRNA-producing loci. Our findings suggest dynamic co-evolution of TEs and host silencing pathways in the African cichlid radiations. We propose that this co-evolution has contributed to cichlid genomic diversity.

rnaCrosslinkOO: An Object-Oriented R Package for the Analysis of RNA Structural Data Generated by RNA Crosslinking Experiments.

SUMMARY: RNA (Ribonucleic Acid) molecules have secondary and tertiary structures in vivo which play a crucial role in cellular processes such as the regulation of gene expression, RNA processing and localisation. The ability to investigate these structures will enhance our understanding of their function and contribute to the diagnosis and treatment of diseases caused by RNA dysregulation. However, there are no mature pipelines or packages for processing and analysing complex in vivo RNA structural data. Here, we present rnaCrosslinkOO (RNA Crosslink Object-Oriented), a novel software package for the comprehensive analysis of data derived from the COMRADES (Crosslinking of Matched RNA and Deep Sequencing) method. rnaCrosslinkOO offers a comprehensive pipeline from raw sequencing reads to the identification and comparison of RNA structural features. It includes read processing and alignment, clustering of duplexes, data exploration, folding and comparisons of RNA structures. rnaCrosslinkOO also enables comparisons between conditions, the identification of inter-RNA interactions, and the incorporation of reactivity data to improve structure prediction. AVAILABILITY AND IMPLEMENTATION: rnaCrosslinkOO is freely available to non-commercial users and implemented in R, with the source code and documentation accessible at [https://CRAN.R-project.org/package=rnaCrosslinkOO]. The software is supported on Linux, macOS, and Windows platforms.

The histone chaperone SPT2 regulates chromatin structure and function in Metazoa.

Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3-H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans ortholog of SPT2 (CeSPT-2) and show that its ability to bind histones H3-H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 null mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt-2 mutants have increased chromatin accessibility at a subset of these loci. We also show that SPT2 influences chromatin structure and controls the levels of soluble and chromatin-bound H3.3 in human cells. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa.

Canalisation and plasticity on the developmental manifold of Caenorhabditis elegans.

How do the same mechanisms that faithfully regenerate complex developmental programmes in spite of environmental and genetic perturbations also allow responsiveness to environmental signals, adaptation and genetic evolution? Using the nematode Caenorhabditis elegans as a model, we explore the phenotypic space of growth and development in various genetic and environmental contexts. Our data are growth curves and developmental parameters obtained by automated microscopy. Using these, we show that among the traits that make up the developmental space, correlations within a particular context are predictive of correlations among different contexts. Furthermore, we find that the developmental variability of this animal can be captured on a relatively low-dimensional phenotypic manifold and that on this manifold, genetic and environmental contributions to plasticity can be deconvolved independently. Our perspective offers a new way of understanding the relationship between robustness and flexibility in complex systems, suggesting that projection and concentration of dimension can naturally align these forces as complementary rather than competing.

U6 snRNA m6A modification is required for accurate and efficient cis- and trans-splicing of C. elegans mRNAs.

pre-mRNA splicing is a critical feature of eukaryotic gene expression. Many eukaryotes use cis-splicing to remove intronic sequences from pre-mRNAs. In addition to cis-splicing, many organisms use trans-splicing to replace the 5' ends of mRNAs with a non-coding spliced-leader RNA. Both cis- and trans-splicing rely on accurately recognising splice site sequences by spliceosomal U snRNAs and associated proteins. Spliceosomal snRNAs carry multiple RNA modifications with the potential to affect different stages of pre-mRNA splicing. Here, we show that m6A modification of U6 snRNA A43 by the RNA methyltransferase METT-10 is required for accurate and efficient cis- and trans-splicing of C. elegans pre-mRNAs. The absence of U6 snRNA m6A modification primarily leads to alternative splicing at 5' splice sites. Furthermore, weaker 5' splice site recognition by the unmodified U6 snRNA A43 affects splicing at 3' splice sites. U6 snRNA m6A43 and the splicing factor SNRNP27K function to recognise an overlapping set of 5' splice sites with an adenosine at +4 position. Finally, we show that U6 snRNA m6A43 is required for efficient SL trans-splicing at weak 3' trans-splice sites. We conclude that the U6 snRNA m6A modification is important for accurate and efficient cis- and trans-splicing in C. elegans.

Regulation of microRNA expression by the adaptor protein GRB2.

Protein interactions with the microRNA (miRNA)-mediated gene silencing protein Argonaute 2 (AGO2) control miRNA expression. miRNA biogenesis starts with the production of precursor transcripts and culminates with the loading of mature miRNA onto AGO2 by DICER1. Here we reveal an additional component to the regulatory mechanism for miRNA biogenesis involving the adaptor protein, growth factor receptor-bound protein 2 (GRB2). The N-terminal SH3 domain of GRB2 is recruited to the PAZ domain of AGO2 forming a ternary complex containing GRB2, AGO2 and DICER1. Using small-RNA sequencing we identified two groups of miRNAs which are regulated by the binding of GRB2. First, mature and precursor transcripts of mir-17~92 and mir-221 miRNAs are enhanced. Second, mature, but not precursor, let-7 family miRNAs are diminished suggesting that GRB2 directly affects loading of these miRNAs. Notably, the resulting loss of let-7 augments expression of oncogenic targets such as RAS. Thus, a new role for GRB2 is established with implications for cancer pathogenesis through regulation of miRNA biogenesis and oncogene expression.

Genomics of cold adaptations in the Antarctic notothenioid fish radiation.

Numerous novel adaptations characterise the radiation of notothenioids, the dominant fish group in the freezing seas of the Southern Ocean. To improve understanding of the evolution of this iconic fish group, here we generate and analyse new genome assemblies for 24 species covering all major subgroups of the radiation, including five long-read assemblies. We present a new estimate for the onset of the radiation at 10.7 million years ago, based on a time-calibrated phylogeny derived from genome-wide sequence data. We identify a two-fold variation in genome size, driven by expansion of multiple transposable element families, and use the long-read data to reconstruct two evolutionarily important, highly repetitive gene family loci. First, we present the most complete reconstruction to date of the antifreeze glycoprotein gene family, whose emergence enabled survival in sub-zero temperatures, showing the expansion of the antifreeze gene locus from the ancestral to the derived state. Second, we trace the loss of haemoglobin genes in icefishes, the only vertebrates lacking functional haemoglobins, through complete reconstruction of the two haemoglobin gene clusters across notothenioid families. Both the haemoglobin and antifreeze genomic loci are characterised by multiple transposon expansions that may have driven the evolutionary history of these genes.

A highly contiguous genome assembly reveals sources of genomic novelty in the symbiotic fungus Rhizophagus irregularis.

The root systems of most plant species are aided by the soil-foraging capacities of symbiotic arbuscular mycorrhizal (AM) fungi of the Glomeromycotina subphylum. Despite recent advances in our knowledge of the ecology and molecular biology of this mutualistic symbiosis, our understanding of the AM fungi genome biology is just emerging. Presented here is a close to T2T genome assembly of the model AM fungus Rhizophagus irregularis DAOM197198, achieved through Nanopore long-read DNA sequencing and Hi-C data. This haploid genome assembly of R. irregularis, alongside short- and long-read RNA-Sequencing data, was used to produce a comprehensive annotation catalog of gene models, repetitive elements, small RNA loci, and DNA cytosine methylome. A phylostratigraphic gene age inference framework revealed that the birth of genes associated with nutrient transporter activity and transmembrane ion transport systems predates the emergence of Glomeromycotina. While nutrient cycling in AM fungi relies on genes that existed in ancestor lineages, a burst of Glomeromycotina-restricted genetic innovation is also detected. Analysis of the chromosomal distribution of genetic and epigenetic features highlights evolutionarily young genomic regions that produce abundant small RNAs, suggesting active RNA-based monitoring of genetic sequences surrounding recently evolved genes. This chromosome-scale view of the genome of an AM fungus genome reveals previously unexplored sources of genomic novelty in an organism evolving under an obligate symbiotic life cycle.

Loss of the E3 ubiquitin ligases UBR-5 or HECD-1 restores Caenorhabditis elegans development in the absence of SWI/SNF function.

SWItch/sucrose non-fermenting (SWI/SNF) complexes are a family of chromatin remodelers that are conserved across eukaryotes. Mutations in subunits of SWI/SNF cause a multitude of different developmental disorders in humans, most of which have no current treatment options. Here, we identify an alanine-to-valine-causing mutation in the SWI/SNF subunit snfc-5 (SMARCB1 in humans) that prevents embryonic lethality in Caenorhabditis elegans nematodes harboring a loss-of-function mutation in the SWI/SNF subunit swsn-1 (SMARCC1/2 in humans). Furthermore, we found that the combination of this specific mutation in snfc-5 and a loss-of-function mutation in either of the E3 ubiquitin ligases ubr-5 (UBR5 in humans) or hecd-1 (HECTD1 in humans) can restore development to adulthood in swsn-1 loss-of-function mutants that otherwise die as embryos. Using these mutant models, we established a set of 335 genes that are dysregulated in SWI/SNF mutants that arrest their development embryonically but exhibit near wild-type levels of expression in the presence of suppressor mutations that prevent embryonic lethality, suggesting that SWI/SNF promotes development by regulating some subset of these 335 genes. In addition, we show that SWI/SNF protein levels are reduced in swsn-1; snfc-5 double mutants and partly restored to wild-type levels in swsn-1; snfc-5; ubr-5 triple mutants, consistent with a model in which UBR-5 regulates SWI/SNF levels by tagging the complex for proteasomal degradation. Our findings establish a link between two E3 ubiquitin ligases and SWI/SNF function and suggest that UBR5 and HECTD1 could be potential therapeutic targets for the many developmental disorders caused by missense mutations in SWI/SNF subunits.

Epigenetic inheritance of gene silencing is maintained by a self-tuning mechanism based on resource competition.

Biological systems can maintain memories over long timescales, with examples including memories in the brain and immune system. It is unknown how functional properties of memory systems, such as memory persistence, can be established by biological circuits. To address this question, we focus on transgenerational epigenetic inheritance in Caenorhabditis elegans. In response to a trigger, worms silence a target gene for multiple generations, resisting strong dilution due to growth and reproduction. Silencing may also be maintained indefinitely upon selection according to silencing levels. We show that these properties imply the fine-tuning of biochemical rates in which the silencing system is positioned near the transition to bistability. We demonstrate that this behavior is consistent with a generic mechanism based on competition for synthesis resources, which leads to self-organization around a critical state with broad silencing timescales. The theory makes distinct predictions and offers insights into the design principles of long-term memory systems.

Mouse primordial germ-cell-like cells lack piRNAs.

PIWI-interacting RNAs (piRNAs) are small RNAs bound by PIWI-clade Argonaute proteins that function to silence transposable elements (TEs). Following mouse primordial germ cell (mPGC) specification around E6.25, fetal piRNAs emerge in male gonocytes from E13.5 onward. The in vitro differentiation of mPGC-like cells (mPGCLCs) has raised the possibility of studying the fetal piRNA pathway in greater depth. However, using single-cell RNA-seq and RT-qPCR along mPGCLC differentiation, we find that piRNA pathway factors are not fully expressed in Day 6 mPGCLCs. Moreover, we do not detect piRNAs across a panel of Day 6 mPGCLC lines using small RNA-seq. Our combined efforts highlight that in vitro differentiated Day 6 mPGCLCs do not yet resemble E13.5 or later mouse gonocytes where the piRNA pathway is active. This Matters Arising paper is in response to von Meyenn et al. (2016), published in Developmental Cell. See also the correction by von Meyenn et al. published in this issue.

ER-associated RNA silencing promotes ER quality control.

The endoplasmic reticulum (ER) coordinates mRNA translation and processing of secreted and endomembrane proteins. ER-associated degradation (ERAD) prevents the accumulation of misfolded proteins in the ER, but the physiological regulation of this process remains poorly characterized. Here, in a genetic screen using an ERAD model substrate in Caenorhabditis elegans, we identified an anti-viral RNA interference pathway, referred to as ER-associated RNA silencing (ERAS), which acts together with ERAD to preserve ER homeostasis and function. Induced by ER stress, ERAS is mediated by the Argonaute protein RDE-1/AGO2, is conserved in mammals and promotes ER-associated RNA turnover. ERAS and ERAD are complementary, as simultaneous inactivation of both quality-control pathways leads to increased ER stress, reduced protein quality control and impaired intestinal integrity. Collectively, our findings indicate that ER homeostasis and organismal health are protected by synergistic functions of ERAS and ERAD.

Ecological Speciation Promoted by Divergent Regulation of Functional Genes Within African Cichlid Fishes.

Rapid ecological speciation along depth gradients has taken place repeatedly in freshwater fishes, yet molecular mechanisms facilitating such diversification are typically unclear. In Lake Masoko, an African crater lake, the cichlid Astatotilapia calliptera has diverged into shallow-littoral and deep-benthic ecomorphs with strikingly different jaw structures within the last 1,000 years. Using genome-wide transcriptome data, we explore two major regulatory transcriptional mechanisms, expression and splicing-QTL variants, and examine their contributions to differential gene expression underpinning functional phenotypes. We identified 7,550 genes with significant differential expression between ecomorphs, of which 5.4% were regulated by cis-regulatory expression QTLs, and 9.2% were regulated by cis-regulatory splicing QTLs. We also found strong signals of divergent selection on differentially expressed genes associated with craniofacial development. These results suggest that large-scale transcriptome modification plays an important role during early-stage speciation. We conclude that regulatory variants are important targets of selection driving ecologically relevant divergence in gene expression during adaptive diversification.

Epigenetic divergence during early stages of speciation in an African crater lake cichlid fish.

Epigenetic variation can alter transcription and promote phenotypic divergence between populations facing different environmental challenges. Here, we assess the epigenetic basis of diversification during the early stages of speciation. Specifically, we focus on the extent and functional relevance of DNA methylome divergence in the very young radiation of Astatotilapia calliptera in crater Lake Masoko, southern Tanzania. Our study focuses on two lake ecomorphs that diverged approximately 1,000 years ago and a population in the nearby river from which they separated approximately 10,000 years ago. The two lake ecomorphs show no fixed genetic differentiation, yet are characterized by different morphologies, depth preferences and diets. We report extensive genome-wide methylome divergence between the two lake ecomorphs, and between the lake and river populations, linked to key biological processes and associated with altered transcriptional activity of ecologically relevant genes. Such genes differing between lake ecomorphs include those involved in steroid metabolism, hemoglobin composition and erythropoiesis, consistent with their divergent habitat occupancy. Using a common-garden experiment, we found that global methylation profiles are often rapidly remodeled across generations but ecomorph-specific differences can be inherited. Collectively, our study suggests an epigenetic contribution to the early stages of vertebrate speciation.

Alternative splicing modulation by G-quadruplexes.

Alternative splicing is central to metazoan gene regulation, but the regulatory mechanisms are incompletely understood. Here, we show that G-quadruplex (G4) motifs are enriched ~3-fold near splice junctions. The importance of G4s in RNA is emphasised by a higher enrichment for the non-template strand. RNA-seq data from mouse and human neurons reveals an enrichment of G4s at exons that were skipped following depolarisation induced by potassium chloride. We validate the formation of stable RNA G4s for three candidate splice sites by circular dichroism spectroscopy, UV-melting and fluorescence measurements. Moreover, we find that sQTLs are enriched at G4s, and a minigene experiment provides further support for their role in promoting exon inclusion. Analysis of >1,800 high-throughput experiments reveals multiple RNA binding proteins associated with G4s. Finally, exploration of G4 motifs across eleven species shows strong enrichment at splice sites in mammals and birds, suggesting an evolutionary conserved splice regulatory mechanism.

Oca2 targeting using CRISPR/Cas9 in the Malawi cichlid Astatotilapia calliptera.

Identifying genetic loci underlying trait variation provides insights into the mechanisms of diversification, but demonstrating causality and characterizing the role of genetic loci requires testing candidate gene function, often in non-model species. Here we establish CRISPR/Cas9 editing in Astatotilapia calliptera, a generalist cichlid of the remarkably diverse Lake Malawi radiation. By targeting the gene oca2 required for melanin synthesis in other vertebrate species, we show efficient editing and germline transmission. Gene edits include indels in the coding region, probably a result of non-homologous end joining, and a large deletion in the 3' untranslated region due to homology-directed repair. We find that oca2 knock-out A. calliptera lack melanin, which may be useful for developmental imaging in embryos and studying colour pattern formation in adults. As A. calliptera resembles the presumed generalist ancestor of the Lake Malawi cichlids radiation, establishing genome editing in this species will facilitate investigating speciation, adaptation and trait diversification in this textbook radiation.

Taming transposable elements in vertebrates: from epigenetic silencing to domestication.

Transposable element (TE)-derived sequences are ubiquitous in most eukaryotic genomes known to date. Because their expression and mobility can lead to genomic instability, several pathways have evolved to control TEs. Nevertheless, TEs represent an important source of genomic novelty and are often co-opted for novel functions that are relevant for phenotypic divergence and adaptation. Here, we review how animals, in particular vertebrates, mitigate TE mobility and expression, alongside known examples of TE domestication. We argue that the next frontier is to understand the determinants and dynamics of TE domestication: how they shift from 'non-self' targets of epigenetic silencing to 'self' genetic elements. New technologies enable avenues of research that may close the gap between epigenetic silencing and domestication of TEs.

TransposonUltimate: software for transposon classification, annotation and detection.

Most genomes harbor a large number of transposons, and they play an important role in evolution and gene regulation. They are also of interest to clinicians as they are involved in several diseases, including cancer and neurodegeneration. Although several methods for transposon identification are available, they are often highly specialised towards specific tasks or classes of transposons, and they lack common standards such as a unified taxonomy scheme and output file format. We present TransposonUltimate, a powerful bundle of three modules for transposon classification, annotation, and detection of transposition events. TransposonUltimate comes as a Conda package under the GPL-3.0 licence, is well documented and it is easy to install through https://github.com/DerKevinRiehl/TransposonUltimate. We benchmark the classification module on the large TransposonDB covering 891,051 sequences to demonstrate that it outperforms the currently best existing solutions. The annotation and detection modules combine sixteen existing softwares, and we illustrate its use by annotating Caenorhabditis elegans, Rhizophagus irregularis and Oryza sativa subs. japonica genomes. Finally, we use the detection module to discover 29 554 transposition events in the genomes of 20 wild type strains of C. elegans. Databases, assemblies, annotations and further findings can be downloaded from (https://doi.org/10.5281/zenodo.5518085).

Identification of putative reader proteins of 5-methylcytosine and its derivatives in Caenorhabditis elegans RNA.

Background: Methylation of carbon-5 of cytosines (m 5C) is a conserved post-transcriptional nucleotide modification of RNA with widespread distribution across organisms. It can be further modified to yield 5-hydroxymethylcytidine (hm 5C), 5-formylcytidine (f 5C), 2´-O-methyl-5-hydroxymethylcytidine (hm 5Cm) and 2´-O-methyl-5-formylcytidine (f 5Cm). How m 5C, and specially its derivates, contribute to biology mechanistically is poorly understood. We recently showed that m 5C is required for Caenorhabditis elegans development and fertility under heat stress. m 5C has been shown to participate in mRNA transport and maintain mRNA stability through its recognition by the reader proteins ALYREF and YBX1, respectively. Hence, identifying readers for RNA modifications can enhance our understanding in the biological roles of these modifications. Methods: To contribute to the understanding of how m 5C and its oxidative derivatives mediate their functions, we developed RNA baits bearing modified cytosines in diverse structural contexts to pulldown potential readers in C. elegans. Potential readers were identified using mass spectrometry. The interaction of two of the putative readers with m 5C was validated using immunoblotting. Results: Our mass spectrometry analyses revealed unique binding proteins for each of the modifications. In silico analysis for phenotype enrichments suggested that hm 5Cm unique readers are enriched in proteins involved in RNA processing, while readers for m 5C, hm 5C and f 5C are involved in germline processes. We validated our dataset by demonstrating that the nematode ALYREF homologues ALY-1 and ALY-2 preferentially bind m 5C in vitro. Finally, sequence alignment analysis showed that several of the putative m 5C readers contain the conserved RNA recognition motif (RRM), including ALY-1 and ALY-2. Conclusions: The dataset presented here serves as an important scientific resource that will support the discovery of new functions of m 5C and its derivatives. Furthermore, we demonstrate that ALY-1 and ALY-2 bind to m 5C in C. elegans.

RNA uridyl transferases TUT4/7 differentially regulate miRNA variants depending on the cancer cell type.

The human terminal uridyl transferases TUT4 and TUT7 (TUT4/7) catalyze the additions of uridines at the 3' end of RNAs, including the precursors of the tumor suppressor miRNA let-7 upon recruitment by the oncoprotein LIN28A. As a consequence, let-7 family miRNAs are down-regulated. Disruption of this TUT4/7 activity inhibits tumorigenesis. Hence, targeting TUT4/7 could be a potential anticancer therapy. In this study, we investigate TUT4/7-mediated RNA regulation in two cancer cell lines by establishing catalytic knockout models. Upon TUT4/7 mutation, we observe a significant reduction in miRNA uridylation, which results in defects in cancer cell properties such as cell proliferation and migration. With the loss of TUT4/7-mediated miRNA uridylation, the uridylated miRNA variants are replaced by adenylated isomiRs. Changes in miRNA modification profiles are accompanied by deregulation of expression levels in specific cases. Unlike let-7s, most miRNAs do not depend on LIN28A for TUT4/7-mediated regulation. Additionally, we identify TUT4/7-regulated cell-type-specific miRNA clusters and deregulation in their corresponding mRNA targets. Expression levels of miR-200c-3p and miR-141-3p are regulated by TUT4/7 in a cancer cell-type-specific manner. Subsequently, BCL2, which is a well-established target of miR-200c is up-regulated. Therefore, TUT4/7 loss causes deregulation of miRNA-mRNA networks in a cell-type-specific manner. Understanding of the underlying biology of such cell-type-specific deregulation will be an important aspect of targeting TUT4/7 for potential cancer therapies.