947 research outputs found

    Examples of sequence conservation analyses capture a subset of mouse long non-coding RNAs sharing homology with fish conserved genomic elements

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    Background: Long non-coding RNAs (lncRNA) are a major class of non-coding RNAs. They are involved in diverse intra-cellular mechanisms like molecular scaffolding, splicing and DNA methylation. Through these mechanisms they are reported to play a role in cellular differentiation and development. They show an enriched expression in the brain where they are implicated in maintaining cellular identity, homeostasis, stress responses and plasticity. Low sequence conservation and lack of functional annotations make it difficult to identify homologs of mammalian lncRNAs in other vertebrates. A computational evaluation of the lncRNAs through systematic conservation analyses of both sequences as well as their genomic architecture is required.Results: Our results show that a subset of mouse candidate lncRNAs could be distinguished from random sequences based on their alignment with zebrafish phastCons elements. Using ROC analyses we were able to define a measure to select significantly conserved lncRNAs. Indeed, starting from ~2,800 mouse lncRNAs we could predict that between 4 and 11% present conserved sequence fragments in fish genomes. Gene ontology (GO) enrichment analyses of protein coding genes, proximal to the region of conservation, in both organisms highlighted similar GO classes like regulation of transcription and central nervous system development. The proximal coding genes in both the species show enrichment of their expression in brain. In summary, we show that interesting genomic regions in zebrafish could be marked based on their sequence homology to a mouse lncRNA, overlap with ESTs and proximity to genes involved in nervous system development.Conclusions: Conservation at the sequence level can identify a subset of putative lncRNA orthologs. The similar protein-coding neighborhood and transcriptional information about the conserved candidates provide support to the hypothesis that they share functional homology. The pipeline herein presented represents a proof of principle showing that a portion between 4 and 11% of lncRNAs retains region of conservation between mammals and fishes. We believe this study will result useful as a reference to analyze the conservation of lncRNAs in newly sequenced genomes and transcriptomes. \uc2\ua9 2013 Basu et al.; licensee BioMed Central Ltd

    The Tetraodon nigroviridis reference transcriptome: Developmental transition, length retention and microsynteny of long non-coding RNAs in a compact vertebrate genome

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    Pufferfish such as fugu and tetraodon carry the smallest genomes among all vertebrates and are ideal for studying genome evolution. However, comparative genomics using these species is hindered by the poor annotation of their genomes. We performed RNA sequencing during key stages of maternal to zygotic transition of Tetraodon nigroviridis and report its first developmental transcriptome. We assembled 61,033 transcripts (23,837 loci) representing 80% of the annotated gene models and 3816 novel coding transcripts from 2667 loci. We demonstrate the similarities of gene expression profiles between pufferfish and zebrafish during maternal to zygotic transition and annotated 1120 long non-coding RNAs (lncRNAs) many of which differentially expressed during development. The promoters for 60% of the assembled transcripts result validated by CAGE-seq. Despite the extreme compaction of the tetraodon genome and the dramatic loss of transposons, the length of lncRNA exons remain comparable to that of other vertebrates and a small set of lncRNAs appears enriched for transposable elements suggesting a selective pressure acting on lncRNAs length and composition. Finally, a set of lncRNAs are microsyntenic between teleost and vertebrates, which indicates potential regulatory interactions between lncRNAs and their flanking coding genes. Our work provides a fundamental molecular resource for vertebrate comparative genomics and embryogenesis studies

    Bioinformatics in Italy : BITS 2012, the ninth annual meeting of the Italian Society of Bioinformatics

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    The BITS2012 meeting, held in Catania on May 2-4, 2012, brought together almost 100 Italian researchers working in the field of Bioinformatics, as well as students in the same or related disciplines. About 90 original research works were presented either as oral communication or as posters, representing a landscape of Italian current research in bioinformatics.This preface provides a brief overview of the meeting and introduces the manuscripts that were accepted for publication in this supplement, after a strict and careful peer-review by an International board of referees

    Study of the human SOX17 locus and its genetic determinants in definitive endoderm

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    Embryonic development and organogenesis depend on the precise spatiotemporal expression of specific sets of genes. Precisely controlled gene expression ensures cell state transitions, especially in the early stages of development, as gastrulation. These complex multi-layered cellular processes are orchestrated by the interfacing of the epigenome, 3-dimensional (3D) nuclear organization, cis-regulatory elements (CREs) with transcription factors (TF), and long non-coding RNAs (lncRNAs). In the gastrulating embryo, definitive endoderm is specified from the pluripotent epiblast following a series of regulatory events, including the activation of SOX17, a key TF of that particular germ layer. Although SOX17 has been extensively studied in early embryonic development, the precise control of its activation, the locus, and the epigenetic rules governing its genetic regulatory network (GRN) remains poorly investigated. In my thesis, I in-depth characterized the human SOX17 locus, exploring the relevance and regulatory impact of 3D nuclear organization, its distal CREs, and their activity. I applied a series of loss of function (LOF) and transgenic experiments to dissect the locus at a satisfactory resolution. In particular, I showed SOX17 among a subset of developmental regulators topologically isolated within CTCF-CTCF loop domains and highlighted the importance of gene control in 3D within this type of domain. I pinpointed the relevance of SOX17’s distal CREs and their definitive endoderm-specific interaction and showed this interaction to be highly dependent on CTCF-CTCF loop-formation to guarantee proper gene control. I found CRE-dependent SOX17 gene deregulation associated with poor definitive endoderm differentiation outcome and a stalled “mesendodermal-like” phenotype. Assessing the genetic identity of different CREs, I divulged the presence of a novel lncRNA within the locus, namely LNCSOX17. I fully characterized LNCSOX17 and established its identity as a bona fide lncRNA through a series of genetic perturbations. I demonstrated the importance of LNCSOX17 for forming definitive endoderm and the lack of participation in SOX17 cis-acting gene control. I associated the loss of LNCSOX17 RNA but not its active transcription at the locus with an aberrant endodermal transcriptome, a lack of epithelial-to-mesenchymal transition (EMT), and the hyperactivity of the detrimental definitive endoderm JNK/JUN/AP1 signaling pathway. I found definitive endoderm lacking LNCSOX17 to be functionally impeded in the generation of pancreatic progenitor populations. The studies within this thesis serve as valuable examples to support the functional relevance of 3D nuclear organization and its importance for developmental gene control in cis via CTCF-CTCF loop domain-mediated CRE-promoter contact facilitation. They associate developmental gene expression levels with various phenotypes, identify a so far unknown developmental lncRNA molecule, and imply its relevance for the formation of definitive endoderm. The outlined results advance our knowledge of developmental TF gene-control and its importance for the development of human definitive endoderm

    Elucidating the Molecular Mechanism of Cis-Regulation by the Long Noncoding RNA LincRNA-p21

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    Pervasive transcription is a hallmark of mammalian genomes. Although protein-coding genes span only a small fraction of the genome, more than two-thirds is transcribed, yielding thousands of noncoding transcripts whose expression exhibits a tight correlation with cell type, disease state, and other biological phenomena. A subset of these transcripts, termed long noncoding RNAs (lncRNAs) on account of their length (\u3e200 nucleotides) and lack of apparent coding potential, have been shown to play functional roles in processes ranging from immune signaling to organogenesis. In contrast to trans-acting lncRNAs, which may operate in either the nucleus or cytoplasm, cis-acting lncRNAs remain at their site of transcription and regulate the expression of nearby protein-coding genes. These lncRNAs have been proposed to act through three main mechanisms: (1) the RNA molecule may interact with protein factors to enact transcriptional activation or repression; (2) the act of lncRNA transcription may increase the local concentration of RNA polymerase II or chromatin-modifying factors; or (3) DNA elements within a lncRNA locus may directly regulate the expression of both the lncRNA and its neighboring gene. Deconvolving these interlinked mechanisms has proven challenging and necessitates the development and implementation of new experimental techniques. In this work, we used a suite of independent molecular and genetic approaches, including a novel ribozyme-based tool for targeted transcript degradation, to expand our understanding of the molecular “logic” through which cis-acting lncRNAs enact gene regulation. We focused on the p53-inducible lncRNA LincRNA-p21, which acts in cis to reinforce the expression of the nearby protein-coding gene and key p53 target p21/Cdkn1a. To identify the functional element of cis-regulation at this locus, we generated four mouse strains harboring complementary LincRNA-p21 loss-of-function mutations that allowed us to separately examine the importance of the LincRNA-p21 transcriptional process and the underlying DNA sequence. This parallel genetic approach demonstrated that full-length LincRNA-p21 transcription, processing, and accumulation are dispensable for cis-regulation and revealed a requirement for a conserved sequence element within exon 1. Further experiments with molecular tools suggested that active transcription through this conserved region promotes p21 expression, implicating a dual role for the transcriptional process and sequence elements within the locus. This comprehensive functional dissection of a single lncRNA locus attests to the regulatory potential of lncRNA loci and further reveals the biological significance of pervasive genomic transcription

    Conservation of different mechanisms of Hox cluster regulation within chordates

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    [eng] In this thesis we have covered the importance of finding underlying conservation events to better understand the regulatory mechanisms of important development orchestrators like the Hox cluster. As an example of these non-evident conservation, we have shown two cases, as described below. The first case studied, after developing a software able to detect homologous long noncoding RNAs by means of microsynteny analyses, is the conservation of Hotairm1 in Chordata. For assessing the homology of this lncRNA, first we had to identify the lncRNA fraction within the B. lanceolatum transcriptome. With a reliable lincRNA dataset, we used our pipeline, LincOFinder, to identify orthologs between human and amphioxus through microsynteny. After the identification of Hotairm1 as one of the lincRNAs with conserved microsynteny, we used Xenopus as a proxy to analyse the homologies in the expression and the function. We had to proceed this way due to the difficulties associated with the inhibition of genes in B. lanceolatum, and the unavailability of expression patterns for Hotairm1 in the bibliography. After we successfully characterised Hotairm1 expression in amphioxus and Xenopus, we injected morpholino oligonucleotides to target and inhibit the splicing of Hotairm1 to promote an isoform imbalance. Through the phenotype obtained and the performing of qPCRs, we were able to deduct the mechanism of Hotairm1 and successfully relate this mechanism with the one described in human cells. With all the data obtained we were able to strongly suggest that the amphioxus Hotairm1 is homologous to the Xenopus and human Hotairm1, thus being conserved in most of the lineages within chordates. The second case studied was the conservation of the regulation of the Hox cluster mediated by Cdx. When analysing the B. floridae knockouts of Cdx and Pdx obtained using the TALEN technique, we found a severe phenotype of the developing larvae in Cdx-/- and a mild phenotype in Pdx-/-. The Cdx-/- phenotype consisted in the disruption of posterior gut development, as well as an underdevelopment of the postanal tail, coupled with a non-opening anus. When looking at changes in the expression of the Hox cluster in this Cdx-/- embryos, we found collinear misregulation of the expressed Hox genes, with the most anterior Hox cluster genes upregulated, and the most posterior ones downregulated. This is very similar to findings seen in triple morpholino knockdowns of the Cdx genes in Xenopus, indicating that in both, Xenopus and amphioxus, Cdx is regulating the Hox cluster through a homologous mechanism
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