The genomic pool of standing structural variation outnumbers single nucleotide polymorphism by threefold in the marine teleost Chrysophrys auratus

Recent studies have highlighted an important role of structural variation (SV) in ecological and evolutionary processes, but few have studied nonmodel species in the wild. As part of our long‐term research programme on the nonmodel teleost fish Australasian snapper (Chrysophrys auratus), we aim to build one of the first catalogues of genomic variants (SNPs and indels, and deletions, duplications and inversions) in fishes and evaluate overlap of genomic variants with regions under putative selection (Tajima's D and π), and coding sequences (genes). For this, we analysed six males and six females from three locations in New Zealand and generated a high‐resolution genomic variation catalogue. We characterized 20,385 SVs and found they intersected with almost a third of all annotated genes. Together with small indels, SVs account for three times more variation in the genome in terms of bases affected compared to SNPs. We found that a sizeable portion of detected SVs was in the upper and lower genomic regions of Tajima's D and π, indicating that some of these have an effect on the phenotype. Together, these results shed light on the often neglected genomic variation that is produced by SVs and highlights the need to go beyond the mere measure of SNPs when investigating evolutionary processes, such as species diversification and adaptation.     

Dynamic miRNA–mRNA paradigms: New faces of miRNAs

More and more evidences suggested that the flow of genetic information can be spatially and temporally regulated by non-coding RNAs (ncRNAs), such as microRNAs (miRNAs). Although biogenesis and function of miRNAs have been well detailed, elucidation of the dynamic interplays between miRNAs and mRNAs have just begun. Here, we highlighted that the miRNA–mRNA interactions which could take place in different cellular locations. During dynamic interactions, miRNA binding sites included not only 3′UTRs, but also 5′UTRs and CDSs. Under different physiological or pathological conditions, miRNAs could switch from translational inhibition to activation. Dynamic miRNA–mRNA paradigms which suggested a novel tip of the iceberg beneath the gene regulation network will provide clues for function studies of other ncRNAs.     

Classic approach revitalizes genomics: Complete characterization of a candidate gene for thermal adaptation in two coral reef fishes

Lactate dehydrogenase-B (ldh-b) encodes a metabolic enzyme (LDH-B) which plays an important role in maintaining aerobic performance and in thermal acclimation and/or adaptation of fish. As the first step in understanding the effect this enzyme has on the ability of tropical coral reef fishes to cope with thermal stress, we characterized both coding and non-coding regions of ldh-b in two congeneric perciformes, Plectropomus leopardus and Plectropomus laevis. Ldh-b was 4666 and 4539bp in length in P. leopardus and P. laevis, respectively, with coding regions comprising 1005bp in both species. We report a high level of sequence homology between the coding regions of ldh-b in these two species, with 98.1% identity of nucleotides corresponding to 100% amino acid identity between the deduced protein sequences. Comparison between non-coding (intron) regions of both species revealed the presence of several indels, despite the high level of homology observed (95.9% identity of intron nucleotides). Potential regulatory motifs and elements, including twenty-six simple sequence repeat motifs (mono-, di-, tri- and tetranucleotide) and twenty-three putative microRNA elements are identified within the introns of both species, further supporting recent demonstrations that such short motifs and elements exhibit widespread positioning throughout non-coding regions of the genome. This novel characterization of ldh-b in these two coral reef fishes allows for a wide range of future studies (e.g. analytical comparisons of ldh-b and LDH-B among different fish genera from different thermal environments and habitats).     

Novel non-coding RNAs in Dictyostelium discoideum and their expression during development

The quest for non-coding RNAs (ncRNAs) in the last few years has revealed a surprisingly large number of small RNAs belonging to previously known as well as entirely novel classes. Computational and experimental approaches have uncovered new ncRNAs in all kingdoms of life. In this work, we used a shotgun cloning approach to construct full-length cDNA libraries of small RNAs from the eukaryotic model organism Dictyostelium discoideum. Interestingly, two entirely novel classes of RNAs were identified of which one is developmentally regulated. The RNAs within each class share conserved 5'- and 3'-termini that can potentially form stem structures. RNAs of both classes show predominantly cytoplasmic localization. In addition, based on conserved structure and/or sequence motifs, several of the identified ncRNAs could be divided into classes known from other organisms, e.g. 18 small nucleolar RNA candidates (17 box C/D, of which a few are developmentally regulated, and one box H/ACA). Two ncRNAs showed a high degree of similarity to the small nuclear U2 RNA and signal recognition particle RNA (SRP RNA), respectively. Furthermore, the majority of the regions upstream of the sequences encoding the isolated RNAs share conserved motifs that may constitute new promoter elements.     

Editor's choice: Functional classification of 15 million SNPs detected from diverse chicken populations

Next-generation sequencing has prompted a surge of discovery of millions of genetic variants from vertebrate genomes. Besides applications in genetic association and linkage studies, a fraction of these variants will have functional consequences. This study describes detection and characterization of 15 million SNPs from chicken genome with the goal to predict variants with potential functional implications (pfVars) from both coding and non-coding regions. The study reports: 183K amino acid-altering SNPs of which 48% predicted as evolutionary intolerant, 13K splicing variants, 51K likely to alter RNA secondary structures, 500K within most conserved elements and 3K from non-coding RNAs. Regions of local fixation within commercial broiler and layer lines were investigated as potential selective sweeps using genome-wide SNP data. Relationships with phenotypes, if any, of the pfVars were explored by overlaying the sweep regions with known QTLs. Based on this, the candidate genes and/or causal mutations for a number of important traits are discussed. Although the fixed variants within sweep regions were enriched with non-coding SNPs, some non-synonymous-intolerant mutations reached fixation, suggesting their possible adaptive advantage. The results presented in this study are expected to have important implications for future genomic research to identify candidate causal mutations and in poultry breeding.     

Non-coding RNAs in human disease

The relevance of the non-coding genome to human disease has mainly been studied in the context of the widespread disruption of microRNA (miRNA) expression and function that is seen in human cancer. However, we are only beginning to understand the nature and extent of the involvement of non-coding RNAs (ncRNAs) in disease. Other ncRNAs, such as PIWI-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), transcribed ultraconserved regions (T-UCRs) and large intergenic non-coding RNAs (lincRNAs) are emerging as key elements of cellular homeostasis. Along with microRNAs, dysregulation of these ncRNAs is being found to have relevance not only to tumorigenesis, but also to neurological, cardiovascular, developmental and other diseases. There is great interest in therapeutic strategies to counteract these perturbations of ncRNAs.     

Detection of selection signatures in Limousin cattle using whole-genome resequencing

Limousin, a renowned beef breed originating from central France, has been selectively bred over the last 100 years to improve economically important traits. We used whole-genome sequencing data from 10 unrelated Limousin bull calves to detect polymorphisms and identify regions under selection. A total of 13 943 766 variants were identified. Moreover, 311 852 bi-allelic SNPs and 92 229 indels located on autosomes were fixed for the alternative allele in all sequenced animals, including the previously reported missense deleterious F94L mutation in MSTN. We performed a whole-genome screen to discover genomic regions with excess homozygosity, using the pooled heterozygosity score and identified 171 different candidate selective sweeps. In total, 68 candidate genes were found in only 57 of these regions, indicating that a large fraction of the genome under selection might lie in non-coding regions and suggesting that a majority of adaptive mutations might be regulatory in nature. Many QTL were found within candidate selective sweep regions, including QTL associated with shear force or carcass weight. Among the putative selective sweeps, we located genes (MSTN, NCKAP5, RUNX2) that potentially contribute to important phenotypes in Limousin. Several candidate regions and genes under selection were also found in previous genome-wide selection scans performed in Limousin. In addition, we were able to pinpoint candidate causative regulatory polymorphisms in GRIK3 and RUNX2 that might have been under selection. Our results will contribute to improved understanding of the mechanisms and targets of artificial selection and will facilitate the interpretation of GWASs performed in Limousin.     

Global signatures of protein binding on structured RNAs in Saccharomyces cerevisiae

Protein binding is essential to the transport,decay and regulation of almost all RNA molecules.However,the structural preference of protein binding on RNAs and their cellular functions and dynamics upon changing environmental conditions are poorly understood.Here,we integrated various high-throughput data and introduced a computational framework to describe the global interactions between RNA binding proteins(RBPs)and structured RNAs in yeast at single-nucleotide resolution.We found that on average,in terms of percent total lengths,~15%of mRNA untranslated regions(UTRs),~37%of canonical non-coding RNAs(ncRNAs)and~11%of long ncRNAs(lncRNAs)are bound by proteins.The RBP binding sites,in general,tend to occur at single-stranded loops,with evolutionarily conserved signatures,and often facilitate a specific RNA structure conformation in vivo.We found that four nucleotide modifications of tRNA are significantly associated with RBP binding.We also identified various structural motifs bound by RBPs in the UTRs of mRNAs,associated with localization,degradation and stress responses.Moreover,we identified>200 novel lncRNAs bound by RBPs,and about half of them contain conserved secondary structures.We present the first ensemble pattern of RBP binding sites in the structured non-coding regions of a eukaryotic genome,emphasizing their structural context and cellular functions.     

High Variation in Single Nucleotide Polymorphisms (SNPs) and Insertions/Deletions (Indels) in the Highly Invasive Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) Middle East-Asia Minor 1 (MEAM1)

Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) Middle East-Asia Minor 1 (MEAM1) is invasive and adaptive to varied environments throughout the world. The adaptability is closely related to genomic variation such as single nucleotide polymorphisms (SNPs) and insertions/deletions (indels). In order to elucidate the feature of SNPs and indels in MEAM1, and reveal the association between SNPs/indels and adaptive capacity to various environments, a computational approach with QualitySNP was used to identify reliable SNPs and indels on the basis of 9110-expressed sequence tags of MEAM1 present in the NCBI database. There were 575 SNPs detected with a density of 10.1 SNPs/kb and 6.4 SNPs/contig. Also, 237 transitions (39.3%) and 366 transversions (60.7%) were obtained, where the ratio of transitions to transversions was 0.65:1. In addition, 581 indels with a density of 14.1 indels/kb and 9.2 indels/contig were detected. Collectively, it showed that invasive MEAM1 has high SNPs density, and higher SNPs percentage than non-invasive B. tabaci species. A high SNPs density/percentage in MEAM1 yielded a high genomic variation that might have allowed it to adapt to varied environments, which provides some support to understand the invasive nature of MEAM1 at the genomic level. High levels of genomic variation are implicated in the level of adaptive capacity and invasive species are thought to exhibit higher levels of adaptive capacity than non-invasive species.     

Characterizing linkage disequilibrium and evaluating imputation power of human genomic insertion-deletion polymorphisms

Background

Indels are an important cause of human variation and central to the study of human disease. The 1000 Genomes Project Low-Coverage Pilot identified over 1.3 million indels shorter than 50 bp, of which over 890 were identified as potentially disruptive variants. Yet, despite their ubiquity, the local genomic characteristics of indels remain unexplored.

Results

Herein we describe population- and minor allele frequency-based differences in linkage disequilibrium and imputation characteristics for indels included in the 1000 Genomes Project Low-Coverage Pilot for the CEU, YRI and CHB+JPT populations. Common indels were well tagged by nearby SNPs in all studied populations, and were also tagged at a similar rate to common SNPs. Both neutral and functionally deleterious common indels were imputed with greater than 95% concordance from HapMap Phase 3 and OMNI SNP sites. Further, 38 to 56% of low frequency indels were tagged by low frequency SNPs. We were able to impute heterozygous low frequency indels with over 50% concordance. Lastly, our analysis also revealed evidence of ascertainment bias. This bias prevents us from extending the applicability of our results to highly polymorphic indels that could not be identified in the Low-Coverage Pilot.

Conclusions

Although further scope exists to improve the imputation of low frequency indels, our study demonstrates that there are already ample opportunities to retrospectively impute indels for prior genome-wide association studies and to incorporate indel imputation into future case/control studies.     

Exonic remnants of whole-genome duplication reveal cis-regulatory function of coding exons

Using a comparative genomics approach to reconstruct the fate of genomic regulatory blocks (GRBs) and identify exonic remnants that have survived the disappearance of their host genes after whole-genome duplication (WGD) in teleosts, we discover a set of 38 candidate cis-regulatory coding exons (RCEs) with predicted target genes. These elements demonstrate evolutionary separation of overlapping protein-coding and regulatory information after WGD in teleosts. We present evidence that the corresponding mammalian exons are still under both coding and non-coding selection pressure, are more conserved than other protein coding exons in the host gene and several control sets, and share key characteristics with highly conserved non-coding elements in the same regions. Their dual function is corroborated by existing experimental data. Additionally, we show examples of human exon remnants stemming from the vertebrate 2R WGD. Our findings suggest that long-range cis-regulatory inputs for developmental genes are not limited to non-coding regions, but can also overlap the coding sequence of unrelated genes. Thus, exonic regulatory elements in GRBs might be functionally equivalent to those in non-coding regions, calling for a re-evaluation of the sequence space in which to look for long-range regulatory elements and experimentally test their activity.     

Genomic rearrangements at the FRA2H common fragile site frequently involve non-homologous recombination events across LTR and L1(LINE) repeats

Common fragile sites (cFSs) are non-random chromosomal regions that are prone to breakage under conditions of replication stress. DNA damage and chromosomal alterations at cFSs appear to be critical events in the development of various human diseases, especially carcinogenesis. Despite the growing interest in understanding the nature of cFS instability, only a few cFSs have been molecularly characterised. In this study, we fine-mapped the location of FRA2H using six-colour fluorescence in situ hybridisation and showed that it is one of the most active cFSs in the human genome. FRA2H encompasses approximately 530 kb of a gene-poor region containing a novel large intergenic non-coding RNA gene (AC097500.2). Using custom-designed array comparative genomic hybridisation, we detected gross and submicroscopic chromosomal rearrangements involving FRA2H in a panel of 54 neuroblastoma, colon and breast cancer cell lines. The genomic alterations frequently involved different classes of long terminal repeats and long interspersed nuclear elements. An analysis of breakpoint junction sequence motifs predominantly revealed signatures of microhomology-mediated non-homologous recombination events. Our data provide insight into the molecular structure of cFSs and sequence motifs affected by their activation in cancer. Identifying cFS sequences will accelerate the search for DNA biomarkers and targets for individualised therapies.