Michael B. Clark

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/.

The Reality of Pervasive Transcription

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

Genome-wide analysis of long noncoding RNA stability

Transcriptomic analyses have identified tens of thousands of intergenic, intronic, and cis-antisense long noncoding RNAs (lncRNAs) that are expressed from mammalian genomes. Despite progress in functional characterization, little is known about the post-transcriptional regulation of lncRNAs and their half-lives. Although many are easily detectable by a variety of techniques, it has been assumed that lncRNAs are generally unstable, but this has not been examined genome-wide. Utilizing a custom noncoding RNA array, we determined the half-lives of ∼800 lncRNAs and ∼12,000 mRNAs in the mouse Neuro-2a cell line. We find only a minority of lncRNAs are unstable. LncRNA half-lives vary over a wide range, comparable to, although on average less than, that of mRNAs, suggestive of complex metabolism and widespread functionality. Combining half-lives with comprehensive lncRNA annotations identified hundreds of unstable (half-life < 2 h) intergenic, cis-antisense, and intronic lncRNAs, as well as lncRNAs showing extreme stability (half-life > 16 h). Analysis of lncRNA features revealed that intergenic and cis-antisense RNAs are more stable than those derived from introns, as are spliced lncRNAs compared to unspliced (single exon) transcripts. Subcellular localization of lncRNAs indicated widespread trafficking to different cellular locations, with nuclear-localized lncRNAs more likely to be unstable. Surprisingly, one of the least stable lncRNAs is the well-characterized paraspeckle RNA Neat1, suggesting Neat1 instability contributes to the dynamic nature of this subnuclear domain. We have created an online interactive resource (http://stability.matticklab.com) that allows easy navigation of lncRNA and mRNA stability profiles and provides a comprehensive annotation of ~7200 mouse lncRNAs.

lncRNAdb v2.0: expanding the reference database for functional long noncoding RNAs

Despite the prevalence of long noncoding RNA (lncRNA) genes in eukaryotic genomes, only a small proportion have been examined for biological function. lncRNAdb, available at http://lncrnadb.org, provides users with a comprehensive, manually curated reference database of 287 eukaryotic lncRNAs that have been described independently in the scientific literature. In addition to capturing a great proportion of the recent literature describing functions for individual lncRNAs, lncRNAdb now offers an improved user interface enabling greater accessibility to sequence information, expression data and the literature. The new features in lncRNAdb include the integration of Illumina Body Atlas expression profiles, nucleotide sequence information, a BLAST search tool and easy export of content via direct download or a REST API. lncRNAdb is now endorsed by RNAcentral and is in compliance with the International Nucleotide Sequence Database Collaboration.

Long noncoding RNAs in cell biology

Whole genome transcriptomic analyses have identified large numbers of dynamically expressed long non-protein-coding RNAs (lncRNAs) in mammals and other animals whose functions are, as yet, largely unknown. Here we summarize the growing evidence that lncRNAs, like mRNAs, can be trafficked to and function in a wide variety of subcellular locations. Investigation of the subcellular distribution of lncRNAs has the potential to greatly expand our knowledge not only of the function of lncRNAs but also of cell biology by identifying previously unknown subcellular structures and novel constituents of known cellular organelles.

Targeted sequencing for gene discovery and quantification using RNA CaptureSeq

RNA sequencing (RNAseq) samples the majority of expressed genes infrequently, owing to the large size, complex splicing and wide dynamic range of eukaryotic transcriptomes. This results in sparse sequencing coverage that can hinder robust isoform assembly and quantification. RNA capture sequencing (CaptureSeq) addresses this challenge by using oligonucleotide probes to capture selected genes or regions of interest for targeted sequencing. Targeted RNAseq provides enhanced coverage for sensitive gene discovery, robust transcript assembly and accurate gene quantification. Here we describe a detailed protocol for all stages of RNA CaptureSeq, from initial probe design considerations and capture of targeted genes to final assembly and quantification of captured transcripts. Initial probe design and final analysis can take less than 1 d, whereas the central experimental capture stage requires ∼7 d.

Quantitative gene profiling of long noncoding RNAs with targeted RNA sequencing

We compared quantitative RT-PCR (qRT-PCR), RNA-seq and capture sequencing (CaptureSeq) in terms of their ability to assemble and quantify long noncoding RNAs and novel coding exons across 20 human tissues. CaptureSeq was superior for the detection and quantification of genes with low expression, showed little technical variation and accurately measured differential expression. This approach expands and refines previous annotations and simultaneously generates an expression atlas.

RNAcentral: an international database of ncRNA sequences

Abstract The field of non-coding RNA biology has been hampered by the lack of availability of a comprehensive, up-to-date collection of accessioned RNA sequences. Here we present the first release of RNAcentral, a database that collates and integrates information from an international consortium of established RNA sequence databases. The initial release contains over 8.1 million sequences, including representatives of all major functional classes. A web portal (http://rnacentral.org) provides free access to data, search functionality, cross-references, source code and an integrated genome browser for selected species.

Mammalian Gene PEG10 Expresses Two Reading Frames by High Efficiency -1 Frameshifting in Embryonic-associated Tissues

Paternally expressed gene 10 (PEG10) is a mammalian gene that is essential for embryonic development in mice. The gene contains two overlapping open reading frames (ORF1 and ORF2) and is derived from a retroelement that acquired a cellular function. It is not known if both reading frames are required for PEG10 function. Synthesis of ORF2 would be possible only if programmed –1 frameshifting occurred during ORF1 translation. In this study the frameshifting activity of PEG10 was analyzed in vivo, and a potential role for ORF2 was investigated. Phylogenetic analysis demonstrated that PEG10 is highly conserved in therian mammals, with all species retaining the elements necessary for frameshifting as well as functional motifs in each ORF. The frameshift site of PEG10 was highly active in cultured cells and produced the ORF1-2 protein. In mice, endogenous ORF1 and an ORF1-2 frameshift protein were detected in the developing placenta and amniotic membrane from 9.5 days post-coitus through to term with a very high frameshift efficiency (>60%). Mutagenesis of the active site motif of a putative protease within ORF2 showed that this enzyme is active and participates in post-translational processing of PEG10 ORF1-2. Both PEG10 proteins were also detected in first trimester human placenta. By contrast, neither protein expression nor frameshifting was detected in adult mouse tissues. These studies imply that the ORF1-2 protein, synthesized utilizing the most efficient –1 frameshift mechanism yet documented in vivo, will have an essential function that is intrinsic to the importance of PEG10 in mammals.

The dark matter rises: the expanding world of regulatory RNAs

The ability to sequence genomes and characterize their products has begun to reveal the central role for regulatory RNAs in biology, especially in complex organisms. It is now evident that the human genome contains not only protein-coding genes, but also tens of thousands of non-protein coding genes that express small and long ncRNAs (non-coding RNAs). Rapid progress in characterizing these ncRNAs has identified a diverse range of subclasses, which vary widely in size, sequence and mechanism-of-action, but share a common functional theme of regulating gene expression. ncRNAs play a crucial role in many cellular pathways, including the differentiation and development of cells and organs and, when mis-regulated, in a number of diseases. Increasing evidence suggests that these RNAs are a major area of evolutionary innovation and play an important role in determining phenotypic diversity in animals.