John S. Mattick

Long non-coding RNAs: insights into functions

In mammals and other eukaryotes most of the genome is transcribed in a developmentally regulated manner to produce large numbers of long non-coding RNAs (ncRNAs). Here we review the rapidly advancing field of long ncRNAs, describing their conservation, their organization in the genome and their roles in gene regulation. We also consider the medical implications, and the emerging recognition that any transcript, regardless of coding potential, can have an intrinsic function as an RNA.

Specific expression of long noncoding RNAs in the mouse brain

A major proportion of the mammalian transcriptome comprises long RNAs that have little or no protein-coding capacity (ncRNAs). Only a handful of such transcripts have been examined in detail, and it is unknown whether this class of transcript is generally functional or merely artifact. Using in situ hybridization data from the Allen Brain Atlas, we identified 849 ncRNAs (of 1,328 examined) that are expressed in the adult mouse brain and found that the majority were associated with specific neuroanatomical regions, cell types, or subcellular compartments. Examination of their genomic context revealed that the ncRNAs were expressed from diverse places including intergenic, intronic, and imprinted loci and that many overlap with, or are transcribed antisense to, protein-coding genes of neurological importance. Comparisons between the expression profiles of ncRNAs and their associated protein-coding genes revealed complex relationships that, in combination with the specific expression profiles exhibited at both regional and subcellular levels, are inconsistent with the notion that they are transcriptional noise or artifacts of chromatin remodeling. Our results show that the majority of ncRNAs are expressed in the brain and provide strong evidence that the majority of processed transcripts with no protein-coding capacity function intrinsically as RNAs.

Non-coding RNAs: regulators of disease.

For 50 years the term ‘gene’ has been synonymous with regions of the genome encoding mRNAs that are translated into protein. However, recent genome‐wide studies have shown that the human genome is pervasively transcribed and produces many thousands of regulatory non‐protein‐coding RNAs (ncRNAs), including microRNAs, small interfering RNAs, PIWI‐interacting RNAs and various classes of long ncRNAs. It is now clear that these RNAs fulfil critical roles as transcriptional and post‐transcriptional regulators and as guides of chromatin‐modifying complexes. Here we review the biology of ncRNAs, focusing on the fundamental mechanisms by which ncRNAs facilitate normal development and physiology and, when dysfunctional, underpin disease. We also discuss evidence that intergenic regions associated with complex diseases express ncRNAs, as well as the potential use of ncRNAs as diagnostic markers and therapeutic targets. Taken together, these observations emphasize the need to move beyond the confines of protein‐coding genes and highlight the fact that continued investigation of ncRNA biogenesis and function will be necessary for a comprehensive understanding of human disease. Copyright

Structure and function of long noncoding RNAs in epigenetic regulation

Genomes of complex organisms encode an abundance and diversity of long noncoding RNAs (lncRNAs) that are expressed throughout the cell and fulfill a wide variety of regulatory roles at almost every stage of gene expression. These roles, which encompass sensory, guiding, scaffolding and allosteric capacities, derive from folded modular domains in lncRNAs. In this diverse functional repertoire, we focus on the well-characterized ability for lncRNAs to function as epigenetic modulators. Many lncRNAs bind to chromatin-modifying proteins and recruit their catalytic activity to specific sites in the genome, thereby modulating chromatin states and impacting gene expression. Considering this regulatory potential in combination with the abundance of lncRNAs suggests that lncRNAs may be part of a broad epigenetic regulatory network.

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.

Non-coding RNAs: the architects of eukaryotic complexity.

Around 98% of all transcriptional output in humans is non‐coding RNA. RNA‐mediated gene regulation is widespread in higher eukaryotes and complex genetic phenomena like RNA interference, co‐suppression, transgene silencing, imprinting, methylation, and possibly position‐effect variegation and transvection, all involve intersecting pathways based on or connected to RNA signaling. I suggest that the central dogma is incomplete, and that intronic and other non‐coding RNAs have evolved to comprise a second tier of gene expression in eukaryotes, which enables the integration and networking of complex suites of gene activity. Although proteins are the fundamental effectors of cellular function, the basis of eukaryotic complexity and phenotypic variation may lie primarily in a control architecture composed of a highly parallel system of trans‐acting RNAs that relay state information required for the coordination and modulation of gene expression, via chromatin remodeling, RNA–DNA, RNA–RNA and RNA–protein interactions. This system has interesting and perhaps informative analogies with small world networks and dataflow computing.

The genetic signatures of noncoding RNAs.

The majority of the genome in animals and plants is transcribed in a developmentally regulated manner to produce large numbers of non–protein-coding RNAs (ncRNAs), whose incidence increases with developmental complexity. There is growing evidence that these transcripts are functional, particularly in the regulation of epigenetic processes, leading to the suggestion that they compose a hitherto hidden layer of genomic programming in humans and other complex organisms. However, to date, very few have been identified in genetic screens. Here I show that this is explicable by an historic emphasis, both phenotypically and technically, on mutations in protein-coding sequences, and by presumptions about the nature of regulatory mutations. Most variations in regulatory sequences produce relatively subtle phenotypic changes, in contrast to mutations in protein-coding sequences that frequently cause catastrophic component failure. Until recently, most mapping projects have focused on protein-coding sequences, and the limited number of identified regulatory mutations have been interpreted as affecting conventional cis-acting promoter and enhancer elements, although these regions are often themselves transcribed. Moreover, ncRNA-directed regulatory circuits underpin most, if not all, complex genetic phenomena in eukaryotes, including RNA interference-related processes such as transcriptional and post-transcriptional gene silencing, position effect variegation, hybrid dysgenesis, chromosome dosage compensation, parental imprinting and allelic exclusion, paramutation, and possibly transvection and transinduction. The next frontier is the identification and functional characterization of the myriad sequence variations that influence quantitative traits, disease susceptibility, and other complex characteristics, which are being shown by genome-wide association studies to lie mostly in noncoding, presumably regulatory, regions. There is every possibility that many of these variations will alter the interactions between regulatory RNAs and their targets, a prospect that should be borne in mind in future functional analyses.

The rise of regulatory RNA.

Discoveries over the past decade portend a paradigm shift in molecular biology. Evidence suggests that RNA is not only functional as a messenger between DNA and protein but also involved in the regulation of genome organization and gene expression, which is increasingly elaborate in complex organisms. Regulatory RNA seems to operate at many levels; in particular, it plays an important part in the epigenetic processes that control differentiation and development. These discoveries suggest a central role for RNA in human evolution and ontogeny. Here, we review the emergence of the previously unsuspected world of regulatory RNA from a historical perspective.

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.

Somatic retrotransposition alters the genetic landscape of the human brain

Retrotransposons are mobile genetic elements that use a germline ‘copy-and-paste’ mechanism to spread throughout metazoan genomes. At least 50 per cent of the human genome is derived from retrotransposons, with three active families (L1, Alu and SVA) associated with insertional mutagenesis and disease. Epigenetic and post-transcriptional suppression block retrotransposition in somatic cells, excluding early embryo development and some malignancies. Recent reports of L1 expression and copy number variation in the human brain suggest that L1 mobilization may also occur during later development. However, the corresponding integration sites have not been mapped. Here we apply a high-throughput method to identify numerous L1, Alu and SVA germline mutations, as well as 7,743 putative somatic L1 insertions, in the hippocampus and caudate nucleus of three individuals. Surprisingly, we also found 13,692 somatic Alu insertions and 1,350 SVA insertions. Our results demonstrate that retrotransposons mobilize to protein-coding genes differentially expressed and active in the brain. Thus, somatic genome mosaicism driven by retrotransposition may reshape the genetic circuitry that underpins normal and abnormal neurobiological processes.