Anton J. Enright

miRBase: tools for microRNA genomics

miRBase is the central online repository for microRNA (miRNA) nomenclature, sequence data, annotation and target prediction. The current release (10.0) contains 5071 miRNA loci from 58 species, expressing 5922 distinct mature miRNA sequences: a growth of over 2000 sequences in the past 2 years. miRBase provides a range of data to facilitate studies of miRNA genomics: all miRNAs are mapped to their genomic coordinates. Clusters of miRNA sequences in the genome are highlighted, and can be defined and retrieved with any inter-miRNA distance. The overlap of miRNA sequences with annotated transcripts, both protein- and non-coding, are described. Finally, graphical views of the locations of a wide range of genomic features in model organisms allow for the first time the prediction of the likely boundaries of many miRNA primary transcripts. miRBase is available at http://microrna.sanger.ac.uk/.

miRBase: microRNA sequences, targets and gene nomenclature

The miRBase database aims to provide integrated interfaces to comprehensive microRNA sequence data, annotation and predicted gene targets. miRBase takes over functionality from the microRNA Registry and fulfils three main roles: the miRBase Registry acts as an independent arbiter of microRNA gene nomenclature, assigning names prior to publication of novel miRNA sequences. miRBase Sequences is the primary online repository for miRNA sequence data and annotation. miRBase Targets is a comprehensive new database of predicted miRNA target genes. miRBase is available at .

Protein interaction maps for complete genomes based on gene fusion events

A large-scale effort to measure, detect and analyse protein–protein interactions using experimental methods is under way. These include biochemistry such as co-immunoprecipitation or crosslinking, molecular biology such as the two-hybrid system or phage display, and genetics such as unlinked noncomplementing mutant detection. Using the two-hybrid system, an international effort to analyse the complete yeast genome is in progress. Evidently, all these approaches are tedious, labour intensive and inaccurate. From a computational perspective, the question is how can we predict that two proteins interact from structure or sequence alone. Here we present a method that identifies gene-fusion events in complete genomes, solely based on sequence comparison. Because there must be selective pressure for certain genes to be fused over the course of evolution, we are able to predict functional associations of proteins. We show that 215 genes or proteins in the complete genomes of Escherichia coli, Haemophilus influenzae and Methanococcus jannaschii are involved in 64 unique fusion events. The approach is general, and can be applied even to genes of unknown function.

Genomic analysis of human microRNA transcripts

MicroRNAs (miRNAs) are important genetic regulators of development, differentiation, growth, and metabolism. The mammalian genome encodes ≈500 known miRNA genes. Approximately 50% are expressed from non-protein-coding transcripts, whereas the rest are located mostly in the introns of coding genes. Intronic miRNAs are generally transcribed coincidentally with their host genes. However, the nature of the primary transcript of intergenic miRNAs is largely unknown. We have performed a large-scale analysis of transcription start sites, polyadenylation signals, CpG islands, EST data, transcription factor-binding sites, and expression ditag data surrounding intergenic miRNAs in the human genome to improve our understanding of the structure of their primary transcripts. We show that a significant fraction of primary transcripts of intergenic miRNAs are 3–4 kb in length, with clearly defined 5′ and 3′ boundaries. We provide strong evidence for the complete transcript structure of a small number of human miRNAs.

An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice

Progressive hearing loss is common in the human population, but little is known about the molecular basis. We report a new N-ethyl-N-nitrosurea (ENU)-induced mouse mutant, diminuendo, with a single base change in the seed region of Mirn96. Heterozygotes show progressive loss of hearing and hair cell anomalies, whereas homozygotes have no cochlear responses. Most microRNAs are believed to downregulate target genes by binding to specific sites on their mRNAs, so mutation of the seed should lead to target gene upregulation. Microarray analysis revealed 96 transcripts with significantly altered expression in homozygotes; notably, Slc26a5, Ocm, Gfi1, Ptprq and Pitpnm1 were downregulated. Hypergeometric P-value analysis showed that hundreds of genes were upregulated in mutants. Different genes, with target sites complementary to the mutant seed, were downregulated. This is the first microRNA found associated with deafness, and diminuendo represents a model for understanding and potentially moderating progressive hair cell degeneration in hearing loss more generally.

Network visualization and analysis of gene expression data using BioLayout Express(3D)

Network analysis has an increasing role in our effort to understand the complexity of biological systems. This is because of our ability to generate large data sets, where the interaction or distance between biological components can be either measured experimentally or calculated. Here we describe the use of BioLayout Express3D, an application that has been specifically designed for the integration, visualization and analysis of large network graphs derived from biological data. We describe the basic functionality of the program and its ability to display and cluster large graphs in two- and three-dimensional space, thereby rendering graphs in a highly interactive format. Although the program supports the import and display of various data formats, we provide a detailed protocol for one of its unique capabilities, the network analysis of gene expression data and a more general guide to the manipulation of graphs generated from various other data types.

The miR-144/451 locus is required for erythroid homeostasis

The process of erythropoiesis must be efficient and robust to supply the organism with red bloods cells both under condition of homeostasis and stress. The microRNA (miRNA) pathway was recently shown to regulate erythroid development. Here, we show that expression of the locus encoding miR-144 and miR-451 is strictly dependent on Argonaute 2 and is required for erythroid homeostasis. Mice deficient for the miR-144/451 cluster display a cell autonomous impairment of late erythroblast maturation, resulting in erythroid hyperplasia, splenomegaly, and a mild anemia. Analysis of gene expression profiles from wild-type and miR-144/451–deficient erythroblasts revealed that the miR-144/451 cluster acts as a “tuner” of gene expression, influencing the expression of many genes. MiR-451 imparts a greater impact on target gene expression than miR-144. Accordingly, mice deficient in miR-451 alone exhibited a phenotype indistinguishable from miR-144/451–deficient mice. Thus, the miR-144/451 cluster tunes gene expression to impart a robustness to erythropoiesis that is critical under conditions of stress.

Detecting microRNA binding and siRNA off-target effects from expression data.

Sylamer is a method for detecting microRNA target and small interfering RNA off-target signals in 3′ untranslated regions from a ranked gene list, sorted from upregulated to downregulated, after a microRNA perturbation or RNA interference experiment. The output is a landscape plot that tracks occurrence biases using hypergeometric P-values for all words across the gene ranking. We demonstrated the utility, speed and accuracy of this approach on several datasets.

Construction, Visualisation, and Clustering of Transcription Networks from Microarray Expression Data

Network analysis transcends conventional pairwise approaches to data analysis as the context of components in a network graph can be taken into account. Such approaches are increasingly being applied to genomics data, where functional linkages are used to connect genes or proteins. However, while microarray gene expression datasets are now abundant and of high quality, few approaches have been developed for analysis of such data in a network context. We present a novel approach for 3-D visualisation and analysis of transcriptional networks generated from microarray data. These networks consist of nodes representing transcripts connected by virtue of their expression profile similarity across multiple conditions. Analysing genome-wide gene transcription across 61 mouse tissues, we describe the unusual topography of the large and highly structured networks produced, and demonstrate how they can be used to visualise, cluster, and mine large datasets. This approach is fast, intuitive, and versatile, and allows the identification of biological relationships that may be missed by conventional analysis techniques. This work has been implemented in a freely available open-source application named BioLayout Express 3D.

The endonuclease activity of Mili fuels piRNA amplification that silences LINE1 elements.

Piwi proteins and Piwi-interacting RNAs (piRNAs) have conserved functions in transposon silencing. The murine Piwi proteins Mili and Miwi2 (also called Piwil2 and Piwil4, respectively) direct epigenetic LINE1 and intracisternal A particle transposon silencing during genome reprogramming in the embryonic male germ line. Piwi proteins are proposed to be piRNA-guided endonucleases that initiate secondary piRNA biogenesis; however, the actual contribution of their endonuclease activities to piRNA biogenesis and transposon silencing remain unknown. To investigate the role of Piwi-catalysed endonucleolytic activity, we engineered point mutations in mice that substitute the second aspartic acid to an alanine in the DDH catalytic triad of Mili and Miwi2, generating the MiliDAH and Miwi2DAH alleles, respectively. Analysis of Mili-bound piRNAs from homozygous MiliDAH fetal gonadocytes revealed a failure of transposon piRNA amplification, resulting in the marked reduction of piRNA bound within Miwi2 ribonuclear particles. We find that Mili-mediated piRNA amplification is selectively required for LINE1, but not intracisternal A particle, silencing. The defective piRNA pathway in MiliDAH mice results in spermatogenic failure and sterility. Surprisingly, homozygous Miwi2DAH mice are fertile, transposon silencing is established normally and no defects in secondary piRNA biogenesis are observed. In addition, the hallmarks of piRNA amplification are observed in Miwi2-deficient gonadocytes. We conclude that cycles of intra-Mili secondary piRNA biogenesis fuel piRNA amplification that is absolutely required for LINE1 silencing.