Accurate microRNA annotation of animal genomes using trained covariance models of curated microRNA complements in MirMachine

The annotation of microRNAs depends on the availability of transcriptomics data and expert knowledge. This has led to a gap between the availability of novel genomes and high-quality microRNA complements. Using >16,000 microRNAs from the manually curated microRNA gene database MirGeneDB, we generated trained covariance models for all conserved microRNA families. These models are available in our tool MirMachine, which annotates conserved microRNAs within genomes. We successfully applied MirMachine to a range of animal species, including those with large genomes and genome duplications and extinct species, where small RNA sequencing is hard to achieve. We further describe a microRNA score of expected microRNAs that can be used to assess the completeness of genome assemblies. MirMachine closes a long-persisting gap in the microRNA field by facilitating automated genome annotation pipelines and deeper studies into the evolution of genome regulation, even in extinct organisms

Advancing the use of noncoding RNA in regulatory toxicology: Report of an ECETOC workshop

The European Centre for the Ecotoxicology and Toxicology of Chemicals (ECETOC) organised a workshop to discuss the state-of-the-art research on noncoding RNAs (ncRNAs) as biomarkers in regulatory toxicology and as analytical and therapeutic agents. There was agreement that ncRNA expression profiling data requires careful evaluation to determine the utility of specific ncRNAs as biomarkers. To advance the use of ncRNA in regulatory toxicology, the following research priorities were identified: (1) Conduct comprehensive literature reviews to identify possibly suitable ncRNAs and areas of toxicology where ncRNA expression profiling could address prevailing scientific deficiencies. (2) Develop consensus on how to conduct ncRNA expression profiling in a toxicological context. (3) Conduct experimental projects, including, e.g., rat (90-day) oral toxicity studies, to evaluate the toxicological relevance of the expression profiles of selected ncRNAs. Thereby, physiological ncRNA expression profiles should be established, including the biological variability of healthy individuals. To substantiate the relevance of key ncRNAs for cell homeostasis or pathogenesis, molecular events should be dose-dependently linked with substance-induced apical effects. Applying a holistic approach, knowledge on ncRNAs, 'omics and epigenetics technologies should be integrated into adverse outcome pathways to improve the understanding of the functional roles of ncRNAs within a regulatory context

Viroids, the simplest RNA replicons: How they manipulate their hosts for being propagated and how their hosts react for containing the infection

[EN] The discovery of viroids about 45 years ago heralded a revolution in Biology: small RNAs comprising around 350 nt were found to be able to replicate autonomously and to incite diseases in certain plants without encoding proteins, fundamental properties discriminating these infectious agents from viruses. The initial focus on the pathological effects usually accompanying infection by viroids soon shifted to their molecular features they are circular molecules that fold upon themselves adopting compact secondary conformations and then to how they manipulate their hosts to be propagated. Replication of viroids in the nucleus or chloroplasts through a rolling-circle mechanism involving polymerization, cleavage and circularization of RNA strands dealt three surprises: (i) certain RNA polymerases are redirected to accept RNA instead of their DNA templates, (ii) cleavage in chloroplastic viroids is not mediated by host enzymes but by hammerhead ribozymes, and (iii) circularization in nuclear viroids is catalyzed by a DNA ligase redirected to act upon RNA substrates. These enzymes (and ribozymes) are most probably assisted by host proteins, including transcription factors and RNA chaperones. Movement of viroids, first intracellularly and then to adjacent cells and distal plant parts, has turned out to be a tightly regulated process in which specific RNA structural motifs play a crucial role. More recently, the advent of RNA silencing has brought new views on how viroids may cause disease and on how their hosts react to contain the infection; additionally, viroid infection may be restricted by other mechanisms. Representing the lowest step on the biological size scale, viroids have also attracted considerable interest to get a tentative picture of the essential characteristics of the primitive replicons that populated the postulated RNA world. (C) 2015 Elsevier B.V. All rights reserved.Research in R.F. laboratory is currently funded by grants BFU2011-28443 and ACOMP/2014/A/103 from the Spanish Ministerio de Economia y Competitividad (MINECO) and Generalidad Valenciana, respectively. S.M., S.D. and A.L.-C. have been supported by fellowships or contracts from MINECO. Research in B.N. and F.D.S. laboratory has been funded by a dedicated grant of the Ministero dell'Economia e Finanze Italian to the CNR (CISIA, Legge n. 191/2009).Flores Pedauye, R.; Minoia, S.; Carbonell, A.; Gisel, A.; Delgado Villar, SG.; López-Carrasco, MA.; Navarro, B.... (2015). Viroids, the simplest RNA replicons: How they manipulate their hosts for being propagated and how their hosts react for containing the infection. Virus Research. 209:136-145. https://doi.org/10.1016/j.virusres.2015.02.027S13614520

Coupling and Coordination in Gene Expression Processes with Pre-mRNA Splicing

A processing is a tightly regulated and highly complex pathway which includes transcription, splicing, editing, transportation, translation and degradation. It has been well-documented that splicing of RNA polymerase II medicated nascent transcripts occurs co-transcriptionally and is functionally coupled to other RNA processing. Recently, increasing experimental evidence indicated that pre-mRNA splicing influences RNA degradation and vice versa. In this review, we summarized the recent findings demonstrating the coupling of these two processes. In addition, we highlighted the importance of splicing in the production of intronic miRNA and circular RNAs, and hence the discovery of the novel mechanisms in the regulation of gene expression.published_or_final_versio

Transcriptome Analysis of Non‐Coding RNAs in Livestock Species: Elucidating the Ambiguity

The recent remarkable development of transcriptomics technologies, especially next generation sequencing technologies, allows deeper exploration of the hidden landscapes of complex traits and creates great opportunities to improve livestock productivity and welfare. Non-coding RNAs (ncRNAs), RNA molecules that are not translated into proteins, are key transcriptional regulators of health and production traits, thus, transcriptomics analyses of ncRNAs are important for a better understanding of the regulatory architecture of livestock phenotypes. In this chapter, we present an overview of common frameworks for generating and processing RNA sequence data to obtain ncRNA transcripts. Then, we review common approaches for analyzing ncRNA transcriptome data and present current state of the art methods for identification of ncRNAs and functional inference of identified ncRNAs, with emphasis on tools for livestock species. We also discuss future challenges and perspectives for ncRNA transcriptome data analysis in livestock species

Nuclear RNA purification by flow cytometry to study nuclear processes in plants

The nature of plant tissues has continuously hampered understanding of the spatio-temporal and subcellular distribution of RNA-guided processes. Here, we describe a universal protocol based on Arabidopsis to investigate subcellular RNA distribution from virtually any plant species using flow cytometry sorting. This protocol includes all necessary control steps to assess the quality of the nuclear RNA purification. Moreover, it can be easily applied to different plant developmental stages, tissues, cell cycle phases, experimental growth conditions, and specific cell type(s). For complete information on the use and execution of this protocol, please refer to and . The nature of plant tissues has continuously hampered understanding of the spatio-temporal and subcellular distribution of RNA-guided processes. Here, we describe a universal protocol based on Arabidopsis to investigate subcellular RNA distribution from virtually any plant species using flow cytometry sorting. This protocol includes all necessary control steps to assess the quality of the nuclear RNA purification. Moreover, it can be easily applied to different plant developmental stages, tissues, cell cycle phases, experimental growth conditions, and specific cell type(s)

Computational frameworks for microRNA functional analysis of inter-kingdom and indirect targeting.

Genes are DNA sequences that encode the information needed to synthesize molecules necessary for the function of the cell. Some genes are called protein-coding genes because they have the code required to manufacture proteins. The expression of a certain gene means its product (protein) is produced. Although some genes are not protein-coding, they regulate the gene expression of other protein-coding genes. Of these, microRNAs (miRNAs) are small RNA molecules that inhibit the expression of other genes by binding to their mRNA transcripts. miRNAs have been shown to be linked to several biological processes like development and diseases like cancer. Recently, researchers have hypothesized that miRNAs are involved in the regulation of the expression of genes from other species. Although tools to predict miRNA target genes are available in the case when miRNAs and target genes belong to the same species, to our knowledge there are no available tools to predict inter-kingdom miRNA target genes (miRNA and target genes belong to two different kingdoms). To address this limitation, we developed an efficient tool to predict potential gut bacterial genes targeted by miRNAs from edible plants. We successfully predicted ginger miRNAs that target two genes from a gut bacterial strain called Lactobacillus rhamnosus GG. To maintain the efficiency of our tool while using a larger number of miRNAs and bacterial strains, we used a hash-table to index the sequences of bacterial genes. To predict the function of a miRNA, we start by compiling the list of direct target genes (ones with binding sites) and then we search for biological process in which these genes are enriched. This approach does not include other genes affected by the miRNA but do not necessarily have a physical binding site (indirect targets). An example of an indirect target is the gene that doesn’t have a binding site and is regulated through other direct targets like transcription factors. To overcome this limitation, we developed miRinGO an interactive web application to include these indirect targets in the functional analysis. Our approach showed better performance compared to the existing approach in predicting biological processes known to be targeted by certain miRNAs

Deep sequencing uncovers numerous small RNAs on all four replicons of the plant pathogen Agrobacterium tumefaciens

Agrobacterium species are capable of interkingdom gene transfer between bacteria and plants. The genome of Agrobacterium tumefaciens consists of a circular and a linear chromosome, the At-plasmid and the Ti-plasmid, which harbors bacterial virulence genes required for tumor formation in plants. Little is known about promoter sequences and the small RNA (sRNA) repertoire of this and other α-proteobacteria. We used a differential RNA sequencing (dRNA-seq) approach to map transcriptional start sites of 388 annotated genes and operons. In addition, a total number of 228 sRNAs was revealed from all four Agrobacterium replicons. Twenty-two of these were confirmed by independent RNA gel blot analysis and several sRNAs were differentially expressed in response to growth media, growth phase, temperature or pH. One sRNA from the Ti-plasmid was massively induced under virulence conditions. The presence of 76 cis-antisense sRNAs, two of them on the reverse strand of virulence genes, suggests considerable antisense transcription in Agrobacterium. The information gained from this study provides a valuable reservoir for an in-depth understanding of sRNA-mediated regulation of the complex physiology and infection process of Agrobacterium