Systematic transcriptome wide analysis of lncRNA-miRNA interactions.

BACKGROUND: Long noncoding RNAs (lncRNAs) are a recently discovered class of non-protein coding RNAs, which have now increasingly been shown to be involved in a wide variety of biological processes as regulatory molecules. The functional role of many of the members of this class has been an enigma, except a few of them like Malat and HOTAIR. Little is known regarding the regulatory interactions between noncoding RNA classes. Recent reports have suggested that lncRNAs could potentially interact with other classes of non-coding RNAs including microRNAs (miRNAs) and modulate their regulatory role through interactions. We hypothesized that lncRNAs could participate as a layer of regulatory interactions with miRNAs. The availability of genome-scale datasets for Argonaute targets across human transcriptome has prompted us to reconstruct a genome-scale network of interactions between miRNAs and lncRNAs. RESULTS: We used well characterized experimental Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) datasets and the recent genome-wide annotations for lncRNAs in public domain to construct a comprehensive transcriptome-wide map of miRNA regulatory elements. Comparative analysis revealed that in addition to targeting protein-coding transcripts, miRNAs could also potentially target lncRNAs, thus participating in a novel layer of regulatory interactions between noncoding RNA classes. Furthermore, we have modeled one example of miRNA-lncRNA interaction using a zebrafish model. We have also found that the miRNA regulatory elements have a positional preference, clustering towards the mid regions and 3' ends of the long noncoding transcripts. We also further reconstruct a genome-wide map of miRNA interactions with lncRNAs as well as messenger RNAs. CONCLUSIONS: This analysis suggests widespread regulatory interactions between noncoding RNAs classes and suggests a novel functional role for lncRNAs. We also present the first transcriptome scale study on miRNA-lncRNA interactions and the first report of a genome-scale reconstruction of a noncoding RNA regulatory interactome involving lncRNAs

zfishbook: connecting you to a world of zebrafish revertible mutants

zfishbook is an internet-based openly accessible database of revertible protein trap gene-breaking transposon (GBT) insertional mutants in the zebrafish, Danio rerio. In these lines, a monomeric red fluorescent protein (mRFP) is encoded by an artificial 3′ exon, resulting in a translational fusion to endogenous loci. The natural transparency of the zebrafish embryo and larvae greatly facilitates the expression annotation of tagged loci using new capillary-based SCORE imaging methods. Molecular annotation of each line is facilitated by cloning methods such as 5′-Rapid Amplification of cDNA Ends (RACE) and inverse polymerase chain reaction (PCR). zfishbook (http://zfishbook.org) represents a central hub for molecular, expression and mutational information about GBT lines from the International Zebrafish Protein Trap Consortium (IZPTC) that includes researchers from around the globe. zfishbook is open to community-wide contributions including expression and functional annotation. zfishbook also represents a central location for information on how to obtain these lines from diverse members of the IZPTC and integration within other zebrafish community databases including Zebrafish Information Network (ZFIN), Ensembl and National Center for Biotechnology Information

Landscape of Pharmacogenetic Variants Associated With Non-Insulin Antidiabetic Drugs in the Indian Population

INTRODUCTION: Genetic variants contribute to differential responses to non-insulin antidiabetic drugs (NIADs), and consequently to variable plasma glucose control. Optimal control of plasma glucose is paramount to minimizing type 2 diabetes-related long-term complications. India\u27s distinct genetic architecture and its exploding burden of type 2 diabetes warrants a population-specific survey of NIAD-associated pharmacogenetic (PGx) variants. The recent availability of large-scale whole genomes from the Indian population provides a unique opportunity to generate a population-specific map of NIAD-associated PGx variants. RESEARCH DESIGN AND METHODS: We mined 1029 Indian whole genomes for PGx variants, drug-drug interaction (DDI) and drug-drug-gene interactions (DDGI) associated with 44 NIADs. Population-wise allele frequencies were estimated and compared using Fisher\u27s exact test. RESULTS: Overall, we found 76 known and 52 predicted deleterious common PGx variants associated with response to type 2 diabetes therapy among Indians. We report remarkable interethnic differences in the relative cumulative counts of decreased and increased response-associated alleles across NIAD classes. Indians and South Asians showed a significant excess of decreased metformin response-associated alleles compared with other global populations. Network analysis of shared PGx genes predicts high DDI risk during coadministration of NIADs with other metabolic disease drugs. We also predict an increased CYP2C19-mediated DDGI risk for CYP3A4/3A5-metabolized NIADs, saxagliptin, linagliptin and glyburide when coadministered with proton-pump inhibitors (PPIs). CONCLUSIONS: Indians and South Asians have a distinct PGx profile for antidiabetes drugs, marked by an excess of poor treatment response-associated alleles for various NIAD classes. This suggests the possibility of a population-specific reduced drug response in atleast some NIADs. In addition, our findings provide an actionable resource for accelerating future diabetes PGx studies in Indians and South Asians and reconsidering NIAD dosing guidelines to ensure maximum efficacy and safety in the population

Chromosomal-level assembly of the Asian Seabass genome using long sequence reads and multi-layered scaffolding

We report here the ~670 Mb genome assembly of the Asian seabass (Lates calcarifer), a tropical marine teleost. We used long-read sequencing augmented by transcriptomics, optical and genetic mapping along with shared synteny from closely related fish species to derive a chromosome-level assembly with a contig N50 size over 1 Mb and scaffold N50 size over 25 Mb that span ~90% of the genome. The population structure of L. calcarifer species complex was analyzed by re-sequencing 61 individuals representing various regions across the species' native range. SNP analyses identified high levels of genetic diversity and confirmed earlier indications of a population stratification comprising three clades with signs of admixture apparent in the South-East Asian population. The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics

Identification of factors required for meristem function in Arabidopsis using a novel next generation sequencing fast forward genetics approach

<p>Abstract</p> <p>Background</p> <p>Phenotype-driven forward genetic experiments are powerful approaches for linking phenotypes to genomic elements but they still involve a laborious positional cloning process. Although sequencing of complete genomes now becomes available, discriminating causal mutations from the enormous amounts of background variation remains a major challenge.</p> <p>Method</p> <p>To improve this, we developed a universal two-step approach, named 'fast forward genetics', which combines traditional bulk segregant techniques with targeted genomic enrichment and next-generation sequencing technology</p> <p>Results</p> <p>As a proof of principle we successfully applied this approach to two Arabidopsis mutants and identified a novel factor required for stem cell activity.</p> <p>Conclusion</p> <p>We demonstrated that the 'fast forward genetics' procedure efficiently identifies a small number of testable candidate mutations. As the approach is independent of genome size, it can be applied to any model system of interest. Furthermore, we show that experiments can be multiplexed and easily scaled for the identification of multiple individual mutants in a single sequencing run.</p

High Resolution Methylome Map of Rat Indicates Role of Intragenic DNA Methylation in Identification of Coding Region

DNA methylation is crucial for gene regulation and maintenance of genomic stability. Rat has been a key model system in understanding mammalian systemic physiology, however detailed rat methylome remains uncharacterized till date. Here, we present the first high resolution methylome of rat liver generated using Methylated DNA immunoprecipitation and high throughput sequencing (MeDIP-Seq) approach. We observed that within the DNA/RNA repeat elements, simple repeats harbor the highest degree of methylation. Promoter hypomethylation and exon hypermethylation were common features in both RefSeq genes and expressed genes (as evaluated by proteomic approach). We also found that although CpG islands were generally hypomethylated, about 6% of them were methylated and a large proportion (37%) of methylated islands fell within the exons. Notably, we obeserved significant differences in methylation of terminal exons (UTRs); methylation being more pronounced in coding/partially coding exons compared to the non-coding exons. Further, events like alternate exon splicing (cassette exon) and intron retentions were marked by DNA methylation and these regions are retained in the final transcript. Thus, we suggest that DNA methylation could play a crucial role in marking coding regions thereby regulating alternative splicing. Apart from generating the first high resolution methylome map of rat liver tissue, the present study provides several critical insights into methylome organization and extends our understanding of interplay between epigenome, gene expression and genome stability

Somatic Mutagenesis with a Sleeping Beauty Transposon System Leads to Solid Tumor Formation in Zebrafish

Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB) T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700–6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers

SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion

The B.1.617.2 (Delta) variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in the state of Maharashtra in late 2020 and spread throughout India, outcompeting pre-existing lineages including B.1.617.1 (Kappa) and B.1.1.7 (Alpha)1. In vitro, B.1.617.2 is sixfold less sensitive to serum neutralizing antibodies from recovered individuals, and eightfold less sensitive to vaccine-elicited antibodies, compared with wild-type Wuhan-1 bearing D614G. Serum neutralizing titres against B.1.617.2 were lower in ChAdOx1 vaccinees than in BNT162b2 vaccinees. B.1.617.2 spike pseudotyped viruses exhibited compromised sensitivity to monoclonal antibodies to the receptor-binding domain and the amino-terminal domain. B.1.617.2 demonstrated higher replication efficiency than B.1.1.7 in both airway organoid and human airway epithelial systems, associated with B.1.617.2 spike being in a predominantly cleaved state compared with B.1.1.7 spike. The B.1.617.2 spike protein was able to mediate highly efficient syncytium formation that was less sensitive to inhibition by neutralizing antibody, compared with that of wild-type spike. We also observed that B.1.617.2 had higher replication and spike-mediated entry than B.1.617.1, potentially explaining the B.1.617.2 dominance. In an analysis of more than 130 SARS-CoV-2-infected health care workers across three centres in India during a period of mixed lineage circulation, we observed reduced ChAdOx1 vaccine effectiveness against B.1.617.2 relative to non-B.1.617.2, with the caveat of possible residual confounding. Compromised vaccine efficacy against the highly fit and immune-evasive B.1.617.2 Delta variant warrants continued infection control measures in the post-vaccination era

The Genomic Distribution and Function of Histone Variant HTZ-1 during C. elegans Embryogenesis

In all eukaryotes, histone variants are incorporated into a subset of nucleosomes to create functionally specialized regions of chromatin. One such variant, H2A.Z, replaces histone H2A and is required for development and viability in all animals tested to date. However, the function of H2A.Z in development remains unclear. Here, we use ChIP-chip, genetic mutation, RNAi, and immunofluorescence microscopy to interrogate the function of H2A.Z (HTZ-1) during embryogenesis in Caenorhabditis elegans, a key model of metazoan development. We find that HTZ-1 is expressed in every cell of the developing embryo and is essential for normal development. The sites of HTZ-1 incorporation during embryogenesis reveal a genome wrought by developmental processes. HTZ-1 is incorporated upstream of 23% of C. elegans genes. While these genes tend to be required for development and occupied by RNA polymerase II, HTZ-1 incorporation does not specify a stereotypic transcription program. The data also provide evidence for unexpectedly widespread independent regulation of genes within operons during development; in 37% of operons, HTZ-1 is incorporated upstream of internally encoded genes. Fewer sites of HTZ-1 incorporation occur on the X chromosome relative to autosomes, which our data suggest is due to a paucity of developmentally important genes on X, rather than a direct function for HTZ-1 in dosage compensation. Our experiments indicate that HTZ-1 functions in establishing or maintaining an essential chromatin state at promoters regulated dynamically during C. elegans embryogenesis

Zebrafish as a model for kidney function and disease

Kidney disease is a global problem with around three million people diagnosed in the UK alone and the incidence is rising. Research is critical to develop better treatments. Animal models can help to better understand the pathophysiology behind the various kidney diseases and to screen for therapeutic compounds, but the use especially of mammalian models should be minimised in the interest of animal welfare. Zebrafish are increasingly used, as they are genetically tractable and have a basic renal anatomy comparable to mammalian kidneys with glomerular filtration and tubular filtration processing. Here, we discuss how zebrafish have advanced the study of nephrology and the mechanisms underlying kidney disease