Paulo P. Amaral

Long noncoding RNAs in mouse embryonic stem cell pluripotency and differentiation

The transcriptional networks that regulate embryonic stem (ES) cell pluripotency and lineage specification are the subject of considerable attention. To date such studies have focused almost exclusively on protein-coding transcripts. However, recent transcriptome analyses show that the mammalian genome contains thousands of long noncoding RNAs (ncRNAs), many of which appear to be expressed in a developmentally regulated manner. The functions of these remain untested. To identify ncRNAs involved in ES cell biology, we used a custom-designed microarray to examine the expression profiles of mouse ES cells differentiating as embryoid bodies (EBs) over a 16-d time course. We identified 945 ncRNAs expressed during EB differentiation, of which 174 were differentially expressed, many correlating with pluripotency or specific differentiation events. Candidate ncRNAs were identified for further characterization by an integrated examination of expression profiles, genomic context, chromatin state, and promoter analysis. Many ncRNAs showed coordinated expression with genomically associated developmental genes, such as Dlx1, Dlx4, Gata6, and Ecsit. We examined two novel developmentally regulated ncRNAs, Evx1as and Hoxb5/6as, which are derived from homeotic loci and share similar expression patterns and localization in mouse embryos with their associated protein-coding genes. Using chromatin immunoprecipitation, we provide evidence that both ncRNAs are associated with trimethylated H3K4 histones and histone methyltransferase MLL1, suggesting a role in epigenetic regulation of homeotic loci during ES cell differentiation. Taken together, our data indicate that long ncRNAs are likely to be important in processes directing pluripotency and alternative differentiation programs, in some cases through engagement of the epigenetic machinery.

The Eukaryotic Genome as an RNA Machine

The past few years have revealed that the genomes of all studied eukaryotes are almost entirely transcribed, generating an enormous number of non–protein-coding RNAs (ncRNAs). In parallel, it is increasingly evident that many of these RNAs have regulatory functions. Here, we highlight recent advances that illustrate the diversity of ncRNA control of genome dynamics, cell biology, and developmental programming.

lncRNAdb: a reference database for long noncoding RNAs

Large numbers of long RNAs with little or no protein-coding potential [long noncoding RNAs (lncRNAs)] are being identified in eukaryotes. In parallel, increasing data describing the expression profiles, molecular features and functions of individual lncRNAs in a variety of systems are accumulating. To enable the systematic compilation and updating of this information, we have developed a database (lncRNAdb) containing a comprehensive list of lncRNAs that have been shown to have, or to be associated with, biological functions in eukaryotes, as well as messenger RNAs that have regulatory roles. Each entry contains referenced information about the RNA, including sequences, structural information, genomic context, expression, subcellular localization, conservation, functional evidence and other relevant information. lncRNAdb can be searched by querying published RNA names and aliases, sequences, species and associated protein-coding genes, as well as terms contained in the annotations, such as the tissues in which the transcripts are expressed and associated diseases. In addition, lncRNAdb is linked to the UCSC Genome Browser for visualization and Noncoding RNA Expression Database (NRED) for expression information from a variety of sources. lncRNAdb provides a platform for the ongoing collation of the literature pertaining to lncRNAs and their association with other genomic elements. lncRNAdb can be accessed at: http://www.lncrnadb.org/.

Noncoding RNA in development

Non-protein-coding sequences increasingly dominate the genomes of multicellular organisms as their complexity increases, in contrast to protein-coding genes, which remain relatively static. Most of the mammalian genome and indeed that of all eukaryotes is expressed in a cell- and tissue-specific manner, and there is mounting evidence that much of this transcription is involved in the regulation of differentiation and development. Different classes of small and large noncoding RNAs (ncRNAs) have been shown to regulate almost every level of gene expression, including the activation and repression of homeotic genes and the targeting of chromatin-remodeling complexes. ncRNAs are involved in developmental processes in both simple and complex eukaryotes, and we illustrate this in the latter by focusing on the animal germline, brain, and eye. While most have yet to be systematically studied, the emerging evidence suggests that there is a vast hidden layer of regulatory ncRNAs that constitutes the majority of the genomic programming of multicellular organisms and plays a major role in controlling the epigenetic trajectories that underlie their ontogeny.

RNA regulation of epigenetic processes.

There is increasing evidence that dynamic changes to chromatin, chromosomes and nuclear architecture are regulated by RNA signalling. Although the precise molecular mechanisms are not well understood, they appear to involve the differential recruitment of a hierarchy of generic chromatin modifying complexes and DNA methyltransferases to specific loci by RNAs during differentiation and development. A significant fraction of the genome‐wide transcription of non‐protein coding RNAs may be involved in this process, comprising a previously hidden layer of intermediary genetic information that underpins developmental ontogeny and the differences between species, ecotypes and individuals. It is also evident that RNA editing is a primary means by which hardwired genetic information in animals can be altered by environmental signals, especially in the brain, indicating a dynamic RNA‐mediated interplay between the transcriptome, the environment and the epigenome. Moreover, RNA‐directed regulatory processes may also transfer epigenetic information not only within cells but also between cells and organ systems, as well as across generations.

The Reality of Pervasive Transcription

Despite recent controversies, the evidence that the majority of the human genome is transcribed into RNA remains strong.

Complex architecture and regulated expression of the Sox2ot locus during vertebrate development

The Sox2 gene is a key regulator of pluripotency embedded within an intron of a long noncoding RNA (ncRNA), termed Sox2 overlapping transcript (Sox2ot), which is transcribed in the same orientation. However, this ncRNA remains uncharacterized. Here we show that Sox2ot has multiple transcription start sites associated with genomic features that indicate regulated expression, including highly conserved elements (HCEs) and chromatin marks characteristic of gene promoters. To identify biological processes in which Sox2ot may be involved, we analyzed its expression in several developmental systems, compared to expression of Sox2. We show that Sox2ot is a stable transcript expressed in mouse embryonic stem cells, which, like Sox2, is down-regulated upon induction of embryoid body (EB) differentiation. However, in contrast to Sox2, Sox2ot is up-regulated during EB mesoderm-lineage differentiation. In adult mouse, Sox2ot isoforms were detected in tissues where Sox2 is expressed, as well as in different tissues, supporting independent regulation of expression of the ncRNA. Sox2dot, an isoform of Sox2ot transcribed from a distal HCE located >500 kb upstream of Sox2, was detected exclusively in the mouse brain, with enrichment in regions of adult neurogenesis. In addition, Sox2ot isoforms are transcribed from HCEs upstream of Sox2 in other vertebrates, including in several regions of the human brain. We also show that Sox2ot is dynamically regulated during chicken and zebrafish embryogenesis, consistently associated with central nervous system structures. These observations provide insight into the structure and regulation of the Sox2ot gene, and suggest conserved roles for Sox2ot orthologs during vertebrate development.

Pervasive transcription of the eukaryotic genome: functional indices and conceptual implications

Genome-wide analyses of the eukaryotic transcriptome have revealed that the majority of the genome is transcribed, producing large numbers of non-protein-coding RNAs (ncRNAs). This surprising observation challenges many assumptions about the genetic programming of higher organisms and how information is stored and organized within the genome. Moreover, the rapid advances in genomics have given little opportunity for biologists to integrate these emerging findings into their intellectual and experimental frameworks. This problem has been compounded by the perception that genome-wide studies often generate more questions than answers, which in turn has led to confusion and controversy. In this article, we address common questions associated with the phenomenon of pervasive transcription and consider the indices that can be used to evaluate the function (or lack thereof) of the resulting ncRNAs. We suggest that many lines of evidence, including expression profiles, conservation signatures, chromatin modification patterns and examination of increasing numbers of individual cases, argue in favour of the widespread functionality of non-coding transcription. We also discuss how informatic and experimental approaches used to analyse protein-coding genes may not be applicable to ncRNAs and how the general perception that protein-coding genes form the main informational output of the genome has resulted in much of the misunderstanding surrounding pervasive transcription and its potential significance. Finally, we present the conceptual implications of the majority of the eukaryotic genome being functional and describe how appreciating this perspective will provide considerable opportunity to further understand the molecular basis of development and complex diseases.

Non-coding RNAs in homeostasis, disease and stress responses: an evolutionary perspective

Cells and organisms are subject to challenges and perturbations in their environment and physiology in all stages of life. The molecular response to such changes, including insulting conditions such as pathogen infections, involves coordinated modulation of gene expression programmes and has not only homeostatic but also ecological and evolutionary importance. Although attention has been primarily focused on signalling pathways and protein networks, non-coding RNAs (ncRNAs), which comprise a significant output of the genomes of prokaryotes and especially eukaryotes, are increasingly implicated in the molecular mechanisms of these responses. Long and short ncRNAs not only regulate development and cell physiology, they are also involved in disease states, including cancers, in host-pathogen interactions, and in a variety of stress responses. Indeed, regulatory RNAs are part of genetically encoded response networks and also underpin epigenetic processes, which are emerging as key mechanisms of adaptation and transgenerational inheritance. Here we present the growing evidence that ncRNAs are intrinsically involved in cellular and organismal adaptation processes, in both robustness and protection to stresses, as well as in mechanisms generating evolutionary change.

Pinstripe: a suite of programs for integrating transcriptomic and proteomic datasets identifies novel proteins and improves differentiation of protein-coding and non-coding genes.

MOTIVATION Comparing transcriptomic data with proteomic data to identify protein-coding sequences is a long-standing challenge in molecular biology, one that is exacerbated by the increasing size of high-throughput datasets. To address this challenge, and thereby to improve the quality of genome annotation and understanding of genome biology, we have developed an integrated suite of programs, called Pinstripe. We demonstrate its application, utility and discovery power using transcriptomic and proteomic data from publicly available datasets. RESULTS To demonstrate the efficacy of Pinstripe for large-scale analysis, we applied Pinstripes reverse peptide mapping pipeline to a transcript library including de novo assembled transcriptomes from the human Illumina Body Atlas (IBA2) and GENCODE v10 gene annotations, and the EBI Proteomics Identifications Database (PRIDE) peptide database. This analysis identified 736 canonical open reading frames (ORFs) supported by three or more PRIDE peptide fragments that are positioned outside any known coding DNA sequence (CDS). Because of the unfiltered nature of the PRIDE database and high probability of false discovery, we further refined this list using independent evidence for translation, including the presence of a Kozak sequence or functional domains, synonymous/non-synonymous substitution ratios and ORF length. Using this integrative approach, we observed evidence of translation from a previously unknown let7e primary transcript, the archetypical lncRNA H19, and a homolog of RD3. Reciprocally, by exclusion of transcripts with mapped peptides or significant ORFs (>80 codon), we identify 32 187 loci with RNAs longer than 2000 nt that are unlikely to encode proteins. AVAILABILITY AND IMPLEMENTATION Pinstripe (pinstripe.matticklab.com) is freely available as source code or a Mono binary. Pinstripe is written in C# and runs under the Mono framework on Linux or Mac OS X, and both under Mono and .Net under Windows. CONTACT m.dinger@garvan.org.au or j.mattick@garvan.org.au SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.