Portion Of Genome Research Articles

Glycan-reactive natural antibodies mediate blockade of endogenous retroviruses emergence

Abstract Endogenous retroviruses (ERVs), comprising a substantial portion of the vertebrate genome, are remnants of ancient genetic invaders. Although multiple layers of cell-intrinsic control are utilized to prevent retroviral reactivation, ERVs with intact coding potential are able to reactivate in immunodeficient mice. While previous studies have indicated that B cells are indispensable for preventing ERV reactivation, it is not yet clear which B cell population mediates the blockade of ERV emergence to prevent subsequent damage in the host. Here, we employed direct labeling of B cells reactive with emerged ERV particles to characterize the B cell population and clonal repertoire responsible for recognition of ERV, and to study the mechanism by which B cells provide protection against ERV emergence. We found that ERV-reactive B cells are enriched in innate-like B-1 cell compartment that predominantly reside in peritoneal and pleural cavities. B-1 cells specificity is biased toward conserved epitopes shared by bacterial- and self-antigens due to distinct developmental pathways in fetal haematopoiesis. We identified ERV-reactive antibodies in unimmunized mice, the level of which further increases upon innate sensor stimulation. B cell receptor repertoire profiling of ERV-reactive B-1 cells revealed increased usage of Igh VH genes that give rise to glycan-specific antibodies targeting glycan structures exhibited by bacterial and viral antigens. We demonstrated that these glycan-specific natural antibodies engage complement pathway to facilitate clearance of reactivated ERV particles. In conclusion, we elucidated the role of glycan-specific B-1 cells and secreted natural antibodies in mediating blockade of ERV emergence.

Comprehensive identification of somatic nucleotide variants in human brain tissue

BackgroundPost-zygotic mutations incurred during DNA replication, DNA repair, and other cellular processes lead to somatic mosaicism. Somatic mosaicism is an established cause of various diseases, including cancers. However, detecting mosaic variants in DNA from non-cancerous somatic tissues poses significant challenges, particularly if the variants only are present in a small fraction of cells.ResultsHere, the Brain Somatic Mosaicism Network conducts a coordinated, multi-institutional study to examine the ability of existing methods to detect simulated somatic single-nucleotide variants (SNVs) in DNA mixing experiments, generate multiple replicates of whole-genome sequencing data from the dorsolateral prefrontal cortex, other brain regions, dura mater, and dural fibroblasts of a single neurotypical individual, devise strategies to discover somatic SNVs, and apply various approaches to validate somatic SNVs. These efforts lead to the identification of 43 bona fide somatic SNVs that range in variant allele fractions from ~ 0.005 to ~ 0.28. Guided by these results, we devise best practices for calling mosaic SNVs from 250× whole-genome sequencing data in the accessible portion of the human genome that achieve 90% specificity and sensitivity. Finally, we demonstrate that analysis of multiple bulk DNA samples from a single individual allows the reconstruction of early developmental cell lineage trees.ConclusionsThis study provides a unified set of best practices to detect somatic SNVs in non-cancerous tissues. The data and methods are freely available to the scientific community and should serve as a guide to assess the contributions of somatic SNVs to neuropsychiatric diseases.

Druggable genome and precision medicine in cancer: current challenges.

The past decades have seen tremendous developments with respect to "specific" therapeutics that target key signaling molecules to conquer cancer. The key advancements with multiomics technologies, especially genomics, have allowed physicians and molecular oncologists to design "tailor-made" solutions to the specific oncogenes that are deregulated in individual patients, a strategy which has turned out to be successful though the patients quickly develop resistance. The swift integration of multidisciplinary approaches has led to the development of "next generation" therapeutics and, with synergistic therapeutic regimes combined with immune checkpoint inhibitors to reactivate the dampened immune response, has provided the much-needed promise for cancer patients. Despite these advances, a large portion of the druggable genome remains understudied, and the role of druggable genome in the immune system needs further attention. Establishment of patient-derived organoid models has fastened the preclinical validation of novel therapeutics for swift clinical translation. We summarized the current advances and challenges and also stress the importance of biobanking and collection of longitudinal data sets with structured clinical information, as well as the critical role these "high content data sets" will play in designing new therapeutic regimes in a tailor-made fashion.

On the Role of Transposable Elements in the Regulation of Gene Expression and Subgenomic Interactions in Crop Genomes

Transposable elements (TEs) represent a major and variable portion of plant genomes, and recent progress in genetics and genomics has highlighted the importance of different TE species as a useful genetic tool in crop breeding. TEs can cause changes in the pattern of gene expression, and regulate gene function by various means such as cis- up- or down-regulation of nearby genes through insertion at promoter, intron, exon and down-stream regions, and trans-production of short interfering RNAs (siRNAs) via two RNA-directed DNA methylation (RdDM) pathways. siRNAs generated through different RdDM pathways differ in length and have variable effects on TEs. For instance, noncoding siRNAs of 20–60 nt produced by RNA polymerase IV (dicer-independent) and 21/22 nt by Pol II (dicer-dependent) have only minor effects on TEs compared with 24 nt siRNAs produced by Pol IV (dicer-dependent pathways). Following whole-genome duplication (WGD) events after polyploidization in allopolyploids, TEs from either parent are able to induce siRNAs to regulate the complex polyploid genome. Those designated as ‘controllers’ usually reside in the dominant parent and affect the TEs of the recessive parent. Subgenome cross-talk thus appears to contribute to epigenetic regulation as well as reshuffling or restructuring of subgenomes and creation of novel patterns of genes expression/and variation in local or global copy number. In this review, we focus on recent progress in unraveling the role of TEs in gene expression regulation via TE-derived siRNAs in the context of polyploid plant evolution and environmental stress, and explore how ancient WGD and recent polyploidy affected the evolution of TE-induced epigenetic mechanisms.

Universal fluorescence in situ hybridization (FISH) protocol for mapping repetitive DNAs in insects and other arthropods.

Repetitive DNAs comprise large portion of eukaryote genomes. In genome projects, the assembly of repetitive DNAs is challenging due to the similarity between repeats, which generate ambiguities for alignment. Fluorescence in situ hybridization (FISH) is a powerful technique for the physical mapping of various sequences on chromosomes. This technique is thus very helpful in chromosome-based genome assemblies, providing information on the fine architecture of genomes and their evolution. However, various protocols are currently used for FISH mapping, most of which are relatively laborious and expensive, or work properly only with a specific type of probes or sequences, and there is a need for a universal and affordable FISH protocol. Here we tested a FISH protocol for mapping of different DNA repeats, such as multigene families (rDNAs, U snDNAs, histone genes), satellite DNAs, microsatellites, transposable elements, DOP-PCR products, and telomeric motif (TTAGG)n, on the chromosomes of various insects and other arthropods. Different cell types and stages obtained from diverse tissues were used. The FISH procedure proved high quality and reliable results in all experiments performed. We obtained data on the chromosomal distribution of DNA repeats in representatives of insects and other arthropods. Thus, our results allow us to conclude that the protocol is universal and requires only time adjustment for chromosome/DNA denaturation. The use of this FISH protocol will facilitate studies focused on understanding the evolution and role of repetitive DNA in arthropod genomes.

Filtering the Junk: Assigning Function to the Mosquito Non-Coding Genome.

Simple SummaryIn eukaryotes, the fraction of the genome not coding for proteins vastly outsizes the portion containing protein-coding genes. This non-coding genome, once termed “junk”, was thought for decades to be inconsequential to the biology of an organism. It is now widely acknowledged that elements within the non-coding genome serve important gene-regulatory functions impacting when, where, and to what levels genes and their protein products are expressed. Without an amino acid-like code to decipher non-coding regulatory elements within the genome, significant technology development has aided in their discovery. Currently, genome-wide identification of non-coding regulatory elements is an active area of research with significant progress made in humans, mice, and other model organisms. However, work to address the roles of these elements in mosquito disease vectors is in its infancy. In this article, we review existing methodology to generate genome-wide catalogs for three classes of non-coding elements and discuss their use in mosquito disease vectors and other insects.The portion of the mosquito genome that does not code for proteins contains regulatory elements that likely underlie variation for important phenotypes including resistance and susceptibility to infection with arboviruses and Apicomplexan parasites. Filtering the non-coding genome to uncover these functional elements is an expanding area of research, though identification of non-coding regulatory elements is challenging due to the lack of an amino acid-like code for the non-coding genome and a lack of sequence conservation across species. This review focuses on three types of non-coding regulatory elements: (1) microRNAs (miRNAs), (2) long non-coding RNAs (lncRNAs), and (3) enhancers, and summarizes current advances in technical and analytical approaches for measurement of each of these elements on a genome-wide scale. The review also summarizes and highlights novel findings following application of these techniques in mosquito-borne disease research. Looking beyond the protein-coding genome is essential for understanding the complexities that underlie differential gene expression in response to arboviral or parasite infection in mosquito disease vectors. A comprehensive understanding of the regulation of gene and protein expression will inform transgenic and other vector control methods rooted in naturally segregating genetic variation.

One-shot analysis of translated mammalian lncRNAs with AHARIBO.

A vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode peptides, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated and peptide-producing lncRNAs. Here, we present AHA-mediated RIBOsome isolation (AHARIBO), a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs, and corresponding de novo synthesized peptides. Besides identifying mRNAs under active translation and associated ribosomes, we found and distinguished lncRNAs acting as ribosome sponges or encoding micropeptides, laying the ground for a better functional understanding of hundreds of lncRNAs.

Chromosomal Mapping of Tandem Repeats Revealed Massive Chromosomal Rearrangements and Insights Into Senna tora Dysploidy.

Tandem repeats can occupy a large portion of plant genomes and can either cause or result from chromosomal rearrangements, which are important drivers of dysploidy-mediated karyotype evolution and speciation. To understand the contribution of tandem repeats in shaping the extant Senna tora dysploid karyotype, we analyzed the composition and abundance of tandem repeats in the S. tora genome and compared the chromosomal distribution of these repeats between S. tora and a closely related euploid, Senna occidentalis. Using a read clustering algorithm, we identified the major S. tora tandem repeats and visualized their chromosomal distribution by fluorescence in situ hybridization. We identified eight independent repeats covering ~85 Mb or ~12% of the S. tora genome. The unit lengths and copy numbers had ranges of 7–5,833 bp and 325–2.89 × 106, respectively. Three short duplicated sequences were found in the 45S rDNA intergenic spacer, one of which was also detected at an extra-NOR locus. The canonical plant telomeric repeat (TTTAGGG)n was also detected as very intense signals in numerous pericentromeric and interstitial loci. StoTR05_180, which showed subtelomeric distribution in Senna occidentalis, was predominantly pericentromeric in S. tora. The unusual chromosomal distribution of tandem repeats in S. tora not only enabled easy identification of individual chromosomes but also revealed the massive chromosomal rearrangements that have likely played important roles in shaping its dysploid karyotype.

The enhancer activity of long interspersed nuclear element derived microRNA 625 induced by NF-\u03baB

Transposable elements (TEs) are DNA sequences that cut or introduced into the genome, and they represent a massive portion of the human genome. TEs generate a considerable number of microRNAs (miRNAs) are derived from TEs (MDTEs). Numerous miRNAs are related to cancer, and hsa-miRNA-625 is a well-known oncomiR derived from long interspersed nuclear elements (LINEs). The relative expression of hsa-miRNA-625-5p differs in humans, chimpanzees, crab-eating monkeys, and mice, and four primers were designed against the 3′UTR of GATAD2B to analyze the different quantities of canonical binding sites and the location of miRNA binding sites. Luciferase assay was performed to score for the interaction between hsa-miRNA-625 and the 3′UTR of GATAD2B, while blocking NF-κB. In summary, the different numbers of canonical binding sites and the locations of miRNA binding sites affect gene expression, and NF-κB induces the enhancer activity of hsa-miRNA-625-5p by sharing the binding sites.

Persistence of Hepatitis A Virus RNA in Water, on Non-porous Surfaces, and on Blueberries.

Enteric viruses, such as human norovirus and hepatitis A virus (HAV), are the leading cause of transmissible foodborne illness. Fresh produce such as berries are often contaminated by infected food handlers, soiled water, or food contact surfaces. The gold-standard method for virus detection throughout the food chain is RT-qPCR, which detects portions of genomes including non-infectious viral particles and naked viral RNA. The aim of this study was to evaluate the persistence of heat-inactivated HAV in water, phosphate-buffered saline, on stainless steel and polyvinyl chloride, and on blueberries at −80°C, −20°C, 4°C, and room temperature. In water and phosphate-buffered saline, viral RNA could be detected for up to 90 days regardless of temperature when the initial load was 2.5 × 104 or 2.5 × 106 genome copies. It was detected on polyvinyl chloride and blueberries under most conditions. On stainless steel, the large initial load persisted for 90 days, while the medium-level load was detected only up to 16 days at room temperature or 60 days at 4°C. The detection of non-infectious viral RNA can confound investigations of gastroenteritis outbreaks. Pretreatments that discriminate between naked RNA, non-infectious virions and infectious virions need to be included in the RT-qPCR method in order to reduce the risk of positive results associated with non-infectious viral particles.

A chromatin-associated splicing isoform of OIP5-AS1 acts in cis to regulate the OIP5 oncogene

ABSTRACT A large portion of the human genome is transcribed into long noncoding RNAs that can range from 200 nucleotides to several kilobases in length. The number of identified lncRNAs is still growing, but only a handful of them have been functionally characterized. However, it is known that the functions of lncRNAs are closely related to their subcellular localization. Cytoplasmic lncRNAs can regulate mRNA stability, affect translation and act as miRNA sponges, while nuclear-retained long noncoding RNAs have been reported to be involved in transcriptional control, chromosome scaffolding, modulation of alternative splicing and chromatin remodelling. Through these processes, lncRNAs have diverse regulatory roles in cell biology and diseases. OIP5-AS1 (also known as Cyrano), a poorly characterized lncRNA expressed antisense to the OIP5 oncogene, is deregulated in multiple cancers. We showed that one of the OIP5-AS1 splicing forms (ENST00000501665.2) is retained in the cell nucleus where it associates with chromatin, thus narrowing down the spectrum of its possible mechanisms of action. Its knockdown with antisense LNA gapmeRs led to inhibited expression of a sense partner, OIP5, strongly suggesting a functional coupling between OIP5 and ENST00000501665.2. A subsequent bioinformatics analysis followed by RAP-MS and RNA Immunoprecipitation experiments suggested its possible mode of action; in particular, we found that ENST00000501665.2 directly binds to a number of nuclear proteins, including SMARCA4, a component of the SWI/SNF chromatin remodelling complex, whose binding motif is located in the promoter of the OIP5 oncogene.

Evolution of DNA packaging in gene transfer agents.

Gene transfer agents (GTAs) are virus-like particles encoded and produced by many bacteria and archaea. Unlike viruses, GTAs package fragments of the host genome instead of the genes that encode the components of the GTA itself. As a result of this non-specific DNA packaging, GTAs can transfer genes within bacterial and archaeal communities. GTAs clearly evolved from viruses and are thought to have been maintained in prokaryotic genomes due to the advantages associated with their DNA transfer capacity. The most-studied GTA is produced by the alphaproteobacterium Rhodobacter capsulatus (RcGTA), which packages random portions of the host genome at a lower DNA density than usually observed in tailed bacterial viruses. How the DNA packaging properties of RcGTA evolved from those of the ancestral virus remains unknown. To address this question, we reconstructed the evolutionary history of the large subunit of the terminase (TerL), a highly conserved enzyme used by viruses and GTAs to package DNA. We found that RcGTA-like TerLs grouped within viruses that employ the headful packaging strategy. Because distinct mechanisms of viral DNA packaging correspond to differences in the TerL amino acid sequence, our finding suggests that RcGTA evolved from a headful packaging virus. Headful packaging is the least sequence-specific mode of DNA packaging, which would facilitate the switch from packaging of the viral genome to packaging random pieces of the host genome during GTA evolution.

Ancient Genomes Reveal Hybridisation between Extinct Short-Faced Bears and the Extant Spectacled Bear ( <i>Tremarctos Ornatus</i>)

Two genera and multiple species of short-faced bear from the Americas went extinct during or toward the end of the Pleistocene, and all belonged to the endemic New World subfamily Tremarctinae. Two of these species were giants, growing in excess of 1,000 kg, but it remains uncertain how these extinct bears were related to the sole surviving short-faced bear: the spectacled bear (Tremarctos ornatus). Ancient mitochondrial DNA has recently suggested phylogenetic relationships among these lineages that conflict with interpretations based on morphology. However, widespread hybridisation and incomplete lineage sorting among extant bears mean that the mitochondrial phylogeny frequently does not reflect the true species tree. Here we present ancient nuclear genome sequences from representatives of the two extinct short-faced bear genera, Arctotherium and Arctodus. Our new data support a third hypothesis for the relationships among short-faced bears, which conflicts with existing mitochondrial and morphological data. Based on genome-wide D-statistics, we suggest that the extant spectacled bear derives substantial ancestry from Pleistocene hybridisation with an extinct short-faced bear lineage, resulting in a discordant phylogenetic signal between the mitochondrion and portions of the nuclear genome.

Probably Correct: Rescuing Repeats with Short and Long Reads.

Ever since the introduction of high-throughput sequencing following the human genome project, assembling short reads into a reference of sufficient quality posed a significant problem as a large portion of the human genome—estimated 50–69%—is repetitive. As a result, a sizable proportion of sequencing reads is multi-mapping, i.e., without a unique placement in the genome. The two key parameters for whether or not a read is multi-mapping are the read length and genome complexity. Long reads are now able to span difficult, heterochromatic regions, including full centromeres, and characterize chromosomes from “telomere to telomere”. Moreover, identical reads or repeat arrays can be differentiated based on their epigenetic marks, such as methylation patterns, aiding in the assembly process. This is despite the fact that long reads still contain a modest percentage of sequencing errors, disorienting the aligners and assemblers both in accuracy and speed. Here, I review the proposed and implemented solutions to the repeat resolution and the multi-mapping read problem, as well as the downstream consequences of reference choice, repeat masking, and proper representation of sex chromosomes. I also consider the forthcoming challenges and solutions with regards to long reads, where we expect the shift from the problem of repeat localization within a single individual to the problem of repeat positioning within pangenomes.

Dark Matter of Primate Genomes: Satellite DNA Repeats and Their Evolutionary Dynamics.

A substantial portion of the primate genome is composed of non-coding regions, so-called “dark matter”, which includes an abundance of tandemly repeated sequences called satellite DNA. Collectively known as the satellitome, this genomic component offers exciting evolutionary insights into aspects of primate genome biology that raise new questions and challenge existing paradigms. A complete human reference genome was recently reported with telomere-to-telomere human X chromosome assembly that resolved hundreds of dark regions, encompassing a 3.1 Mb centromeric satellite array that had not been identified previously. With the recent exponential increase in the availability of primate genomes, and the development of modern genomic and bioinformatics tools, extensive growth in our knowledge concerning the structure, function, and evolution of satellite elements is expected. The current state of knowledge on this topic is summarized, highlighting various types of primate-specific satellite repeats to compare their proportions across diverse lineages. Inter- and intraspecific variation of satellite repeats in the primate genome are reviewed. The functional significance of these sequences is discussed by describing how the transcriptional activity of satellite repeats can affect gene expression during different cellular processes. Sex-linked satellites are outlined, together with their respective genomic organization. Mechanisms are proposed whereby satellite repeats might have emerged as novel sequences during different evolutionary phases. Finally, the main challenges that hinder the detection of satellite DNA are outlined and an overview of the latest methodologies to address technological limitations is presented.

Emergence of artemisinin-resistant Plasmodium falciparum with kelch13 C580Y mutations on the island of New Guinea.

The rapid and aggressive spread of artemisinin-resistant Plasmodium falciparum carrying the C580Y mutation in the kelch13 gene is a growing threat to malaria elimination in Southeast Asia, but there is no evidence of their spread to other regions. We conducted cross-sectional surveys in 2016 and 2017 at two clinics in Wewak, Papua New Guinea (PNG) where we identified three infections caused by C580Y mutants among 239 genotyped clinical samples. One of these mutants exhibited the highest survival rate (6.8%) among all parasites surveyed in ring-stage survival assays (RSA) for artemisinin. Analyses of kelch13 flanking regions, and comparisons of deep sequencing data from 389 clinical samples from PNG, Indonesian Papua and Western Cambodia, suggested an independent origin of the Wewak C580Y mutation, showing that the mutants possess several distinctive genetic features. Identity by descent (IBD) showed that multiple portions of the mutants' genomes share a common origin with parasites found in Indonesian Papua, comprising several mutations within genes previously associated with drug resistance, such as mdr1, ferredoxin, atg18 and pnp. These findings suggest that a P. falciparum lineage circulating on the island of New Guinea has gradually acquired a complex ensemble of variants, including kelch13 C580Y, which have affected the parasites' drug sensitivity. This worrying development reinforces the need for increased surveillance of the evolving parasite populations on the island, to contain the spread of resistance.

Refinement of Draft Genome Assemblies of Pigeonpea (Cajanus cajan).

Genome assembly of short reads from large plant genomes remains a challenge in computational biology despite major developments in next generation sequencing. Of late several draft assemblies have been reported in sequenced plant genomes. The reported draft genome assemblies of Cajanus cajan have different levels of genome completeness, a large number of repeats, gaps, and segmental duplications. Draft assemblies with portions of genome missing are shorter than the referenced original genome. These assemblies come with low map accuracy affecting further functional annotation and the prediction of gene components as desired by crop researchers. Genome coverage, i.e., the number of sequenced raw reads mapped onto a certain location of the genome is an important quality indicator of completeness and assembly quality in draft assemblies. The present work aimed to improve the coverage in reported de novo sequenced draft genomes (GCA_000340665.1 and GCA_000230855.2) of pigeonpea, a legume widely cultivated in India. The two recently sequenced assemblies, A1 and A2 comprised 72% and 75% of the estimated coverage of the genome, respectively. We employed an assembly reconciliation approach to compare the draft assemblies and merge them, filling the gaps by employing an algorithm size sorting mate-pair library to generate a high quality and near complete assembly with enhanced contiguity. The majority of gaps present within scaffolds were filled with right-sized mate-pair reads. The improved assembly reduced the number of gaps than those reported in draft assemblies resulting in an improved genome coverage of 82.4%. Map accuracy of the improved assembly was evaluated using various quality metrics and for the presence of specific trait-related functional genes. Employed pair-end and mate-pair local libraries helped us to reduce gaps, repeats, and other sequence errors resulting in lengthier scaffolds compared to the two draft assemblies. We reported the prediction of putative host resistance genes against Fusarium wilt disease by their performance and evaluated them both in wet laboratory and field phenotypic conditions.

Runs of Homozygosity in Modern Chicken Revealed by Sequence Data.

Runs of homozygosity (ROH) are chromosomal stretches that in a diploid genome appear in a homozygous state and display identical alleles at multiple contiguous loci. This study aimed to systematically compare the genomic distribution of the ROH islands among five populations of wild vs. commercial chickens of both layer and broiler type. To this end, we analyzed whole genome sequences of 115 birds including white layer (WL, n = 25), brown layer (BL, n = 25), broiler line A (BRA, n = 20), broiler line B (BRB, n = 20) and Red Junglefowl (RJF, n = 25). The ROH segments varied in size markedly among populations, ranging from 0.3 to 21.83 Mb reflecting their past genealogy. White layers contained the largest portion of the genome in homozygous state with an average ROH length of 432.1 Mb (±18.7) per bird, despite carrying it in short segments (0.3-1 Mb). Population-wise inbreeding measures based on Wright’s (Fis) and genomic (FROH) metrics revealed highly inbred genome of layer lines relative to the broilers and Red Junglefowl. We further revealed the ROH islands, among commercial lines overlapped with QTL related to limb development (GREM1, MEOX2), body weight (Meis2a.1, uc_338), eggshell color (GLCCI1, ICA1, UMAD1), antibody response to Newcastle virus (ROBO2), and feather pecking. Comparison of ROH landscape in sequencing resolution demonstrated that a sizable portion of genome of commercial lines segregates in homozygote state, reflecting many generations of assortative mating and intensive selection in their recent history. In contrary, wild birds carry shorter ROH segments, likely suggestive of older evolutionary events.

Using SHAPE-MaP To Model RNA Secondary Structure and Identify 3'UTR Variation in Chikungunya Virus.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus associated with debilitating arthralgia in humans. RNA secondary structure in the viral genome plays an important role in the lifecycle of alphaviruses; however, the specific role of RNA structure in regulating CHIKV replication is poorly understood. Our previous studies found little conservation in RNA secondary structure between alphaviruses, and this structural divergence creates unique functional structures in specific alphavirus genomes. Therefore, to understand the impact of RNA structure on CHIKV biology, we used SHAPE-MaP to inform the modeling of RNA secondary structure throughout the genome of a CHIKV isolate from the 2013 Caribbean outbreak. We then analyzed regions of the genome with high levels of structural specificity to identify potentially functional RNA secondary structures and identified 23 regions within the CHIKV genome with higher than average structural stability, including four previously identified, functionally important CHIKV RNA structures. We also analyzed the RNA flexibility and secondary structures of multiple 3'UTR variants of CHIKV that are known to affect virus replication in mosquito cells. This analysis found several novel RNA structures within these 3'UTR variants. A duplication in the 3'UTR that enhances viral replication in mosquito cells led to an overall increase in the amount of unstructured RNA in the 3'UTR. This analysis demonstrates that the CHIKV genome contains a number of unique, specific RNA secondary structures and provides a strategy for testing these secondary structures for functional importance in CHIKV replication and pathogenesis.IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne RNA virus that causes febrile illness and debilitating arthralgia in humans. CHIKV causes explosive outbreaks but there are no approved therapies to treat or prevent CHIKV infection. The CHIKV genome contains functional RNA secondary structures that are essential for proper virus replication. Since RNA secondary structures have only been defined for a small portion of the CHIKV genome, we used a chemical probing method to define the RNA secondary structures of CHIKV genomic RNA. We identified 23 highly specific structured regions of the genome, and confirmed the functional importance of one structure using mutagenesis. Furthermore, we defined the RNA secondary structure of three CHIKV 3'UTR variants that differ in their ability to replicate in mosquito cells. Our study highlights the complexity of the CHIKV genome and describes new systems for designing compensatory mutations to test the functional relevance of viral RNA secondary structures.

Epigenetic conflict on a degenerating Y chromosome increases mutational burden in Drosophila males

Large portions of eukaryotic genomes consist of transposable elements (TEs), and the establishment of transcription-repressing heterochromatin during early development safeguards genome integrity in Drosophila. Repeat-rich Y chromosomes can act as reservoirs for TEs (‘toxic’ Y effect), and incomplete epigenomic defenses during early development can lead to deleterious TE mobilization. Here, we contrast the dynamics of early TE activation in two Drosophila species with vastly different Y chromosomes of different ages. Zygotic TE expression is elevated in male embryos relative to females in both species, mostly due to expression of Y-linked TEs. Interestingly, male-biased TE expression diminishes across development in D. pseudoobscura, but remains elevated in D. miranda, the species with the younger and larger Y chromosome. The repeat-rich Y of D. miranda still contains many actively transcribed genes, which compromise the formation of silencing heterochromatin. Elevated TE expression results in more de novo insertions of repeats in males compared to females. This lends support to the idea that the ‘toxic’ Y chromosome can create a mutational burden in males when genome-wide defense mechanisms are compromised, and suggests a previously unappreciated epigenetic conflict on evolving Y chromosomes between transcription of essential genes and silencing of selfish DNA.