Project description:Background: Long non-coding RNAs (lncRNAs) are increasingly implicated as gene regulators and may ultimately be more numerous than protein-coding genes in the human genome. Despite large numbers of reported lncRNAs, reference annotations are likely incomplete due to their lower and tighter tissue-specific expression compared to mRNAs. An unexplored factor potentially confounding lncRNA identification is inter-individual expression variability. Here, we characterize lncRNA natural expression variability in human primary granulocytes. Results: We annotate granulocyte lncRNAs and mRNAs in RNA-seq data from ten healthy individuals, identifying multiple lncRNAs absent from reference annotations, and use this to investigate three known features (higher tissue-specificity, lower expression, and reduced splicing efficiency) of lncRNAs relative to mRNAs. Expression variability was examined in seven individuals sampled three times at one or more than one month intervals. We show that lncRNAs display significantly more inter-individual expression variability compared to mRNAs. We confirm this finding in 2 independent human datasets by analyzing multiple tissues from the GTEx project and lymphoblastoid cell lines from the GEUVADIS project. Using the latter dataset we also show that including more human donors into the transcriptome annotation pipeline allows identification of an increasing number of lncRNAs, but minimally affects mRNA gene number. Conclusions: A comprehensive annotation of lncRNAs is known to require an approach that is sensitive to low and tight tissue-specific expression. Here we show that increased inter-individual expression variability is an additional general lncRNA feature to consider when creating a comprehensive annotation of human lncRNAs or proposing their use as prognostic or disease markers. We used PolyA+ RNA-seq data from human primary granulocytes of 10 healthy individuals to de novo annotate lncRNAs and mRNAs in this cell type and ribosomal depleted (total) RNA-seq data from seven of these individuals sampled three times to analyze lncRNA amd mRNA expression variability
Project description:The non-coding transcriptome of the hyperthermophilic archaeon Pyrococcus abyssi is investigated using the RNA-seq technology. A dedicated computational pipeline analyzes RNA-seq reads and prior genome annotation to identify small RNAs, untranslated regions of mRNAs, and cis-encoded antisense transcripts. Unlike other archaea, such as Sulfolobus and Halobacteriales, P. abyssi produces few leaderless mRNA transcripts. Antisense transcription is widespread (215 transcripts) and targets protein-coding genes that are less conserved than average genes. We identify at least three novel H/ACA-like guide RNAs among the newly characterized non-coding RNAs. Long 5' UTRs in mRNAs of ribosomal proteins and amino-acid biosynthesis genes strongly suggest the presence of cis-regulatory leaders in these mRNAs. We selected a high-interest subset of non-coding RNAs based on their strong promoters, high GC-content, phylogenetic conservation, or abundance. Some of the novel small RNAs and long 5' UTRs display high GC contents, suggesting unknown structural RNA functions. However, we were surprised to observe that most of the high-interest RNAs are AU-rich, which suggests an absence of stable secondary structure in the high-temperature environment of P. abyssi. Yet, these transcripts display other hallmarks of functionality, such as high expression or high conservation, which leads us to consider possible RNA functions that do not require extensive secondary structure. directional RNA-seq, Illumina GA-IIx
Project description:Interventions: Case series:Nil
Primary outcome(s): intestinal microecological disorders;blood non-coding RNAs and immune status
Study Design: Randomized parallel controlled trial
Project description:Deciphering the genetic architecture of human cardiac disorders is of fundamental importance but their underlying complexity is a major hurdle. We investigated the natural variation of cardiac performance in the sequenced inbred lines of the Drosophila Genetic Reference Panel (DGRP). Genome Wide Associations Studies (GWAS) identified genetic networks associated with natural variation of cardiac traits which were used to gain insights as to the molecular and cellular processes affected. Non-coding variants that we identified were used to map potential regulatory non-coding regions, which in turn were employed to predict Transcription Factors (TFs) binding sites. Cognate TFs, many of which themselves bear polymorphisms associated with variations of cardiac performance, were also validated by heart specific knockdown. Additionally, we showed that the natural variations associated with variability in cardiac performance affect a set of genes overlapping those associated with average traits but through different variants in the same genes. Furthermore, we showed that phenotypic variability was also associated with natural variation of gene regulatory networks. More importantly, we documented correlations between genes associated with cardiac phenotypes in both flies and humans, which supports a conserved genetic architecture regulating adult cardiac function from arthropods to mammals. Specifically, roles for PAX9 and EGR2 in the regulation of the cardiac rhythm were established in both models, illustrating that the characteristics of natural variations in cardiac function identified in Drosophila can accelerate discovery in humans.
Project description:Natural non-coding antisense transcripts (ncNATs) are long non-coding RNAs (lncRNA) transcribed from the opposite strand of a separate protein coding or non-coding gene and can affect the overlapped gene expression through epigenetic, transcriptional, post-transcriptional and/or translational modulations. ncNATs can influence cancerous cell proliferation, migration and therapeutic resistance. Recently, growing numbers of ncNATs were shown to be dysregulated in cancerous cells, however, actual impact of ncNATs on cancer progression remains largely unknown. We performed RNA-seq on post-surgical tumor samples from 26 glioma patients, and normal brain tissue.
Project description:Long non-coding RNAs (lncRNAs) are a heterogeneous group of transcripts that lack protein coding potential and display regulatory functions in various cellular processes. As a result of their cell- and cancer-specific expression patterns, lncRNAs have emerged as potential diagnostic and therapeutic targets. The accurate characterization of lncRNAs in bulk transcriptome data remains challenging due to their low abundance compared to protein coding genes. To tackle this issue, we describe a unique short-read custom lncRNA capture sequencing approach that relies on a comprehensive set of 565,878 capture probes for 49,372 human lncRNA genes. This custom lncRNA capture approach was evaluated on various sample types ranging from artificial high-quality RNA mixtures to more challenging formalin-fixed paraffin-embedded tissue and biofluid material. The custom enrichment approach allows the detection of a more diverse repertoire of lncRNAs, with better reproducibility and higher coverage compared to classic total RNA-sequencing.
Project description:The non-coding transcriptome of the hyperthermophilic archaeon Pyrococcus abyssi is investigated using the RNA-seq technology. A dedicated computational pipeline analyzes RNA-seq reads and prior genome annotation to identify small RNAs, untranslated regions of mRNAs, and cis-encoded antisense transcripts. Unlike other archaea, such as Sulfolobus and Halobacteriales, P. abyssi produces few leaderless mRNA transcripts. Antisense transcription is widespread (215 transcripts) and targets protein-coding genes that are less conserved than average genes. We identify at least three novel H/ACA-like guide RNAs among the newly characterized non-coding RNAs. Long 5' UTRs in mRNAs of ribosomal proteins and amino-acid biosynthesis genes strongly suggest the presence of cis-regulatory leaders in these mRNAs. We selected a high-interest subset of non-coding RNAs based on their strong promoters, high GC-content, phylogenetic conservation, or abundance. Some of the novel small RNAs and long 5' UTRs display high GC contents, suggesting unknown structural RNA functions. However, we were surprised to observe that most of the high-interest RNAs are AU-rich, which suggests an absence of stable secondary structure in the high-temperature environment of P. abyssi. Yet, these transcripts display other hallmarks of functionality, such as high expression or high conservation, which leads us to consider possible RNA functions that do not require extensive secondary structure.