Ryan J. Taft

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

Tiny RNAs associated with transcription start sites in animals

It has been reported that relatively short RNAs of heterogeneous sizes are derived from sequences near the promoters of eukaryotic genes. As part of the FANTOM4 project, we have identified tiny RNAs with a modal length of 18 nt that map within −60 to +120 nt of transcription start sites (TSSs) in human, chicken and Drosophila. These transcription initiation RNAs (tiRNAs) are derived from sequences on the same strand as the TSS and are preferentially associated with G+C-rich promoters. The 5′ ends of tiRNAs show peak density 10–30 nt downstream of TSSs, indicating that they are processed. tiRNAs are generally, although not exclusively, associated with highly expressed transcripts and sites of RNA polymerase II binding. We suggest that tiRNAs may be a general feature of transcription in metazoa and possibly all eukaryotes.

The Reality of Pervasive Transcription

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

The long non-coding RNA Gomafu is acutely regulated in response to neuronal activation and involved in schizophrenia-associated alternative splicing

Schizophrenia (SZ) is a complex disease characterized by impaired neuronal functioning. Although defective alternative splicing has been linked to SZ, the molecular mechanisms responsible are unknown. Additionally, there is limited understanding of the early transcriptomic responses to neuronal activation. Here, we profile these transcriptomic responses and show that long non-coding RNAs (lncRNAs) are dynamically regulated by neuronal activation, including acute downregulation of the lncRNA Gomafu, previously implicated in brain and retinal development. Moreover, we demonstrate that Gomafu binds directly to the splicing factors QKI and SRSF1 (serine/arginine-rich splicing factor 1) and dysregulation of Gomafu leads to alternative splicing patterns that resemble those observed in SZ for the archetypal SZ-associated genes DISC1 and ERBB4. Finally, we show that Gomafu is downregulated in post-mortem cortical gray matter from the superior temporal gyrus in SZ. These results functionally link activity-regulated lncRNAs and alternative splicing in neuronal function and suggest that their dysregulation may contribute to neurological disorders.

MicroRNAs in β-cell biology, insulin resistance, diabetes and its complications

MicroRNAs (miRNAs) are small 19–23 nucleotide RNA molecules that act as regulators of protein expression in eukaryotic cells by inducing the translational arrest and degradation of messenger RNAs (1). They are potent drivers of differentiation and development (1), and their dysregulation has been linked to many diseases. Here, we present an overview of the known and proposed roles and effects of miRNAs in type 1 and type 2 diabetes (T1D and T2D), focusing on β-cell biology, insulin resistance, and diabetes complications. Specifically, we discuss miRNAs in β-cell biology, altered expression of miRNAs in adipose tissue in response to obesity, and miRNA dysfunction in organs and tissues that may be affected in later stages of the disease. Additionally, we propose a set of research directions that may yield novel diagnostic and therapeutic approaches for this chronic illness. T2D is characterized by hyperglycemia resultant from impaired insulin secretion and/or impaired insulin action in peripheral tissues (2). T2D constitutes one of the greatest pandemics of our time, with 220 million people currently diagnosed (3), and 366 million people expected to be affected by 2030 (4). A number of lines of evidence support a key role for pancreatic β-cell dysfunction in T2D (in addition to T1D), in which it is the major pathology. For example, recent genome-wide association studies have strongly implicated genes involved in insulin secretion as etiological factors in the development of T2D (5). A role for miRNAs in T2D was first established in 2004 by Poy et al. (6) who showed that miR-375 is directly involved in the regulation of insulin secretion. This study was one of the first to demonstrate that a miRNA could be tightly linked to a disease phenotype. In recent years, dozens of additional miRNAs have been identified as components of pathways triggered by, or contributing …

Nuclear-localized tiny RNAs are associated with transcription initiation and splice sites in metazoans

We have recently shown that transcription initiation RNAs (tiRNAs) are derived from sequences immediately downstream of transcription start sites. Here, using cytoplasmic and nuclear small RNA high-throughput sequencing datasets, we report the identification of a second class of nuclear-specific ∼17- to 18-nucleotide small RNAs whose 3′ ends map precisely to the splice donor site of internal exons in animals. These splice-site RNAs (spliRNAs) are associated with highly expressed genes and show evidence of developmental stage– and region–specific expression. We also show that tiRNAs are localized to the nucleus, are enriched at chromatin marks associated with transcription initiation and possess a 3′-nucleotide bias. Additionally, we find that microRNA-offset RNAs (moRNAs), the miR-15/16 cluster previously linked to oncosuppression and most small nucleolar RNA (snoRNA)-derived small RNAs (sdRNAs) are enriched in the nucleus, whereas most miRNAs and two H/ACA sdRNAs are cytoplasmically enriched. We propose that nuclear-localized tiny RNAs are involved in the epigenetic regulation of gene expression.

A De Novo Mutation in the β-Tubulin Gene TUBB4A Results in the Leukoencephalopathy Hypomyelination with Atrophy of the Basal Ganglia and Cerebellum

Hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) is a rare hereditary leukoencephalopathy that was originally identified by MRI pattern analysis, and it has thus far defied all attempts at identifying the causal mutation. Only 22 cases are published in the literature to date. We performed exome sequencing on five family trios, two family quartets, and three single probands, which revealed that all eleven H-ABC-diagnosed individuals carry the same de novo single-nucleotide TUBB4A mutation resulting in nonsynonymous change p.Asp249Asn. Detailed investigation of one of the family quartets with the singular finding of an H-ABC-affected sibling pair revealed maternal mosaicism for the mutation, suggesting that rare de novo mutations that are initially phenotypically neutral in a mosaic individual can be disease causing in the subsequent generation. Modeling of TUBB4A shows that the mutation creates a nonsynonymous change at a highly conserved asparagine that sits at the intradimer interface of α-tubulin and β-tubulin, and this change might affect tubulin dimerization, microtubule polymerization, or microtubule stability. Consistent with H-ABCs clinical presentation, TUBB4A is highly expressed in neurons, and a recent report has shown that an N-terminal alteration is associated with a heritable dystonia. Together, these data demonstrate that a single de novo mutation in TUBB4A results in H-ABC.

Cross-mapping and the identification of editing sites in mature microRNAs in high-throughput sequencing libraries

MicroRNAs (miRNAs) are short (20-23 nt) RNAs that are sequence-specific mediators of transcriptional and post-transcriptional regulation of gene expression. Modern high-throughput technologies enable deep sequencing of such RNA species on an unprecedented scale. We find that the analysis of small RNA deep-sequencing libraries can be affected by cross-mapping, in which RNA sequences originating from one locus are inadvertently mapped to another. Similar to cross-hybridization on microarrays, cross-mapping is prevalent among miRNAs, as they tend to occur in families, are similar or derived from repeat or structural RNAs, or are post-transcriptionally modified. Here, we develop a strategy to correct for cross-mapping, and apply it to the analysis of RNA editing in mature miRNAs. In contrast to previous reports, our analysis suggests that RNA editing in mature miRNAs is rare in animals.

An Insect Virus-Encoded MicroRNA Regulates Viral Replication

ABSTRACT MicroRNAs (miRNAs) are small (∼22 nucleotides) noncoding RNAs which play an essential role in gene regulation and affect a wide range of processes, including development, differentiation, and oncogenesis. Here we report the identification of the first miRNA from an insect virus, derived from the major capsid protein (MCP) gene in Heliothis virescens ascovirus (HvAV) (HvAV-miR-1). Although MCP was abundantly expressed at all time points 24 h after infection, HvAV-miR-1 expression was strictly regulated and specifically detected from 96 h postinfection. HvAV-miR-1 expression coincided with a marked reduction of the expression of HvAV DNA polymerase I, which is a predicted target. Ectopic expression of full-length and truncated versions of MCP retaining the miRNA sequence significantly reduced DNA polymerase I transcript levels and inhibited viral replication. Our results indicate that HvAV-miR-1 directs transcriptional degradation of DNA polymerase I and regulates HvAV replication. These findings are congruent with recent reports that miR-BART-2 regulates Epstein-Barr virus DNA polymerase expression and suggest that virus-encoded miRNA regulation of virus replication may be a general phenomenon.

Mutations in DARS Cause Hypomyelination with Brain Stem and Spinal Cord Involvement and Leg Spasticity

Inherited white-matter disorders are a broad class of diseases for which treatment and classification are both challenging. Indeed, nearly half of the children presenting with a leukoencephalopathy remain without a specific diagnosis. Here, we report on the application of high-throughput genome and exome sequencing to a cohort of ten individuals with a leukoencephalopathy of unknown etiology and clinically characterized by hypomyelination with brain stem and spinal cord involvement and leg spasticity (HBSL), as well as the identification of compound-heterozygous and homozygous mutations in cytoplasmic aspartyl-tRNA synthetase (DARS). These mutations cause nonsynonymous changes to seven highly conserved amino acids, five of which are unchanged between yeast and man, in the DARS C-terminal lobe adjacent to, or within, the active-site pocket. Intriguingly, HBSL bears a striking resemblance to leukoencephalopathy with brain stem and spinal cord involvement and elevated lactate (LBSL), which is caused by mutations in the mitochondria-specific DARS2, suggesting that these two diseases might share a common underlying molecular pathology. These findings add to the growing body of evidence that mutations in tRNA synthetases can cause a broad range of neurologic disorders.