Transcriptional and Post-transcriptional Gene Regulation by Long Non-coding RNA.

Advances in genomics technology over recent years have led to the surprising discovery that the genome is far more pervasively transcribed than was previously appreciated. Much of the newly-discovered transcriptome appears to represent long non-coding RNA (lncRNA), a heterogeneous group of largely uncharacterised transcripts. Understanding the biological function of these molecules represents a major challenge and in this review we discuss some of the progress made to date. One major theme of lncRNA biology seems to be the existence of a network of interactions with microRNA (miRNA) pathways. lncRNA has been shown to act as both a source and an inhibitory regulator of miRNA. At the transcriptional level, a model is emerging whereby lncRNA bridges DNA and protein by binding to chromatin and serving as a scaffold for modifying protein complexes. Such a mechanism can bridge promoters to enhancers or enhancer-like non-coding genes by regulating chromatin looping, as well as conferring specificity on histone modifying complexes by directing them to specific loci

Roles of Non-Coding RNAs in Transcriptional Regulation

Non-coding RNAs (ncRNAs) are functional RNA molecules that are transcribed from mammalian genome but lack protein coding capacity. Nearly 80% of the human genome constitutes non-coding elements such as small non-coding RNAs, sncRNAs (miRNA, piRNA, SiRNA, SnRNA) and long non-coding RNAs, lncRNAs (linc RNA, NAT, eRNA, circ RNA, ceRNAs, PROMPTS). These ncRNAs have been extensively studied and are known to mediate the regulation of gene expression. In recent decades, lncRNAs have emerged as pivotal molecules that participate in the post-transcriptional regulation by acting as a signal, guide, scaffold and decoy molecules in addition to their role(s) in transcription. ncRNAs are known to play critical roles in defining DNA methylation patterns, imprinting as well as chromatin remodeling, thus having a substantial effect in epigenetic signaling. The expression of lncRNAs is regulated in a tissue specific and developmental stage specific manner and their mis-regulation is often associated with tumorigenesis. Henceforth, this chapter focuses mainly on the role(s) of ncRNAs in transcriptional and post-transcriptional regulation and their relevance in cancers

Microprocessor mediates transcriptional termination of long noncoding RNA transcripts hosting microRNAs

MicroRNA (miRNA) play a major role in the post-transcriptional regulation of gene expression. Mammalian miRNA biogenesis begins with co-transcriptional cleavage of RNA polymerase II (Pol II) transcripts by the Microprocessor complex. While most miRNA are located within introns of protein coding genes, a substantial minority of miRNA originate from long non coding (lnc) RNA where transcript processing is largely uncharacterized. Here, by detailed characterization of liver-specific lnc-pri-miR-122 and genome-wide analysis, we show that most lnc-pri-miRNA do not use the canonical cleavage and polyadenylation (CPA) pathway but instead use Microprocessor cleavage to terminate transcription. Microprocessor inactivation leads to extensive transcriptional readthrough of lnc-pri-miRNA and transcriptional interference with downstream genes. Consequently we define a novel RNase III-mediated, polyadenylation-independent mechanism of Pol II transcription termination in mammalian cells

Probing the limits to microRNA-mediated control of gene expression

According to the `ceRNA hypothesis', microRNAs (miRNAs) may act as mediators of an effective positive interaction between long coding or non-coding RNA molecules, carrying significant potential implications for a variety of biological processes. Here, inspired by recent work providing a quantitative description of small regulatory elements as information-conveying channels, we characterize the effectiveness of miRNA-mediated regulation in terms of the optimal information flow achievable between modulator (transcription factors) and target nodes (long RNAs). Our findings show that, while a sufficiently large degree of target derepression is needed to activate miRNA-mediated transmission, (a) in case of differential mechanisms of complex processing and/or transcriptional capabilities, regulation by a post-transcriptional miRNA-channel can outperform that achieved through direct transcriptional control; moreover, (b) in the presence of large populations of weakly interacting miRNA molecules the extra noise coming from titration disappears, allowing the miRNA-channel to process information as effectively as the direct channel. These observations establish the limits of miRNA-mediated post-transcriptional cross-talk and suggest that, besides providing a degree of noise buffering, this type of control may be effectively employed in cells both as a failsafe mechanism and as a preferential fine tuner of gene expression, pointing to the specific situations in which each of these functionalities is maximized.Comment: 16 page

Post-Transcriptional Mechanisms of Neuronal Translational Control in Synaptic Plasticity

The dynamic complexity of synaptic function is matched by extensive multidimensional regulation of neuronal mRNA translation which is achieved by a number of post‐transcriptional mechanisms. The first key aspect of this regulatory capacity is mRNA distal trafficking through RNA‐binding proteins, which governs the transcriptomic composition of post‐synaptic compartments. Small non‐coding microRNA and associated machinery have the capacity to precisely coordinate neural gene networks in space and time by providing a flexible specificity dimension to translational regulation. This RNA‐guided subcellular fine‐tuning of protein synthesis is an exquisite mechanism used in neurons to exert control of synaptic properties. Emerging evidence also implicates brain‐enriched long non‐coding RNA and novel circular RNA in posttranscriptional regulation of gene expression through the modulation of both mRNA and miRNA functions, thereby exemplifying the complex nature of neuronal translation. Herein, we review current knowledge of these regulatory systems and analyse how these mechanisms of transcriptomic regulation may be linked together to achieve high‐order spatiotemporal control of post‐synaptic translation

Hypoxia drives glucose transporter 3 expression through HIF-mediated induction of the long non-coding RNA NICI

Hypoxia inducible transcription factors (HIFs) directly dictate the expression of multiple RNA species including novel and as yet uncharacterized long non-coding transcripts with unknown function. We used pan-genomic HIF-binding and transcriptomic data to identify a novel long non-coding RNA NICI (Non-coding Intergenic Co-Induced transcript) on chromosome 12p13.31 which is regulated by hypoxia via HIF-1 promoter-binding in multiple cell types. CRISPR/Cas9-mediated deletion of the hypoxia-response element revealed co-regulation of NICI and the neighboring protein-coding gene, solute carrier family 2 member 3 (SLC2A3) which encodes the high-affinity glucose transporter 3 (GLUT3). Knock-down or knock-out of NICI attenuated hypoxic induction of SLC2A3 indicating a direct regulatory role of NICI in SLC2A3 expression, which was further evidenced by CRISPR/Cas9-VPR mediated activation of NICI expression. We also demonstrate that regulation of SLC2A3 is mediated through transcriptional activation rather than post-transcriptional mechanisms since knock-out of NICI leads to reduced recruitment of RNA polymerase 2 to the SLC2A3 promoter. Consistent with this we observe NICI-dependent regulation of glucose consumption and cell proliferation. Furthermore, NICI expression is regulated by the VHL tumour suppressor and is highly expressed in clear cell renal cancer, where SLC2A3 expression is associated with patient prognosis, implying an important role for the HIF/NICI/SLC2A3 axis in this malignancy

From Junk to Function: LncRNAs in CNS Health and Disease

Recent advances in RNA sequencing technologies helped to uncover the existence of tens of thousands of long non-coding RNAs (lncRNAs) that arise from the dark matter of the genome. These lncRNAs were originally thought to be transcriptional noise but an increasing number of studies demonstrate that these transcripts can modulate protein-coding gene expression by a wide variety of transcriptional and post-transcriptional mechanisms. The spatiotemporal regulation of lncRNA expression is particularly evident in the central nervous system, suggesting that they may directly contribute to specific brain processes, including neurogenesis and cellular homeostasis. Not surprisingly, lncRNAs are therefore gaining attention as putative novel therapeutic targets for disorders of the brain. In this review, we summarize the recent insights into the functions of lncRNAs in the brain, their role in neuronal maintenance, and their potential contribution to disease. We conclude this review by postulating how these RNA molecules can be targeted for the treatment of yet incurable neurological disorders

Urb-RIP - An adaptable and efficient approach for immunoprecipitation of RNAs and associated RNAs/proteins

Post-transcriptional regulation of gene expression is an important process that is mediated by interactions between mRNAs and RNA binding proteins (RBP), non-coding RNAs (ncRNA) or ribonucleoproteins (RNP). Key to the study of post-transcriptional regulation of mRNAs and the function of ncRNAs such as long non-coding RNAs (lncRNAs) is an understanding of what factors are interacting with these transcripts. While several techniques exist for the enrichment of a transcript whether it is an mRNA or an ncRNA, many of these techniques are cumbersome or limited in their application. Here we present a novel method for the immunoprecipitation of mRNAs and ncRNAs, Urb-RNA immunoprecipitation (Urb-RIP). This method employs the RRM1 domain of the "resurrected" snRNA-binding protein Urb to enrich messages containing a stem-loop tag. Unlike techniques which employ the MS2 protein, which require large repeats of the MS2 binding element, Urb-RIP requires only one stem-loop. This method routinely provides over ~100-fold enrichment of tagged messages. Using this technique we have shown enrichment of tagged mRNAs and lncRNAs as well as miRNAs and RNA-binding proteins bound to those messages. We have confirmed, using Urb-RIP, interaction between RNA PolIII transcribed lncRNA BC200 and polyA binding protein

RNA interference-mediated co-transcriptional gene silencing in fission yeast

In the last decade or so, RNA interference (RNAi) has gained unanticipated recognition in the fields of RNA biology and gene regulation. It exists in a wide variety of eukaryotic organisms, and various forms of RNAi are involved in diverse biological processes. At its core, RNAi comprises small non-coding RNAs (sRNAs) in association with Argonaute proteins. The sRNAs are usually produced by cleavage of long double-stranded RNA by the endoribonuclease Dicer enzymes. The sRNAs guide Argonautes to target transcripts via complementary base-pairing, resulting in repression that can occur at various stages of the RNA production process. Perhaps the most well-studied mechanisms of RNAi-mediated repression are those occurring in the cytoplasm at a post-transcriptional level, whereby the target transcript is subject to degradation and/or inhibition of translation. However, well-characterised examples of nuclear RNAi also exist, and usually involve RNAi-mediated chromatin modification such as DNA methylation in plants and histone methylation in protozoa and fungi. These modifications can contribute to heterochromatin formation and inhibit RNA production at the level of transcription. In addition to mediating post-transcriptional and transcriptional gene silencing, recent evidence from several organisms suggests that RNAi can mediate co-transcriptional gene silencing (CTGS), whereby physical association of the RNAi machinery with chromatin can promote degradation of the nascent transcripts and/or inhibit transcription. Such a mode of silencing was first proposed in the fission yeast Schizosaccharomyces pombe (S. pombe), where the RNAi machinery is thought to repress heterochromatic RNA at a transcriptional and co-transcriptional level. During my PhD, I focused on the association of the RNAi machinery with chromatin in S. pombe. Using a sensitive chromatin profiling technique called DamID, I was able to provide the first direct evidence that S. pombe Dicer functions in cis on chromatin. Secondly, I uncovered a novel role for RNAi in gene regulation outside of the well-studied heterochromatic regions. The evidence presented here shows that the S. pombe RNAi machinery is concentrated at nuclear pores where it acts to co-transcriptionally degrade euchromatic RNAs, particularly those from retrotransposon long-terminal repeats, non-coding RNAs and stress response genes bound by the activating transcription factor Atf1. This may keep such features ‘poised’ for expression, allowing more rapid upregulation under inducing conditions. Of particular note, Argonaute is not required for targeting the other RNAi components to euchromatin, suggesting that in this case guidance by the sRNA is not responsible for recognition of substrates. I discuss the implications of these results, particularly in the context of RNAi in other eukaryotes

Non-Coding RNAs in Neural Networks, REST-Assured

In the nervous system, several key steps in cellular complexity and development are regulated by non-coding RNAs (ncRNAs) and the repressor element-1 silencing transcription factor/neuron-restrictive silencing factor (REST/NRSF). REST recruits gene regulatory complexes to regulatory sequences, among them the repressor element-1/neuron-restrictive silencer element, and mediates developmental stage-specific gene expression or repression, chromatin (re-)organization or silencing for protein-coding genes as well as for several ncRNAs like microRNAs, short interfering RNAs or long ncRNAs. NcRNAs are far from being just transcriptional noise and are involved in chromatin accessibility, transcription and post-transcriptional processing, trafficking, or RNA editing. REST and its cofactor CoREST are both highly regulated through various ncRNAs. The importance of the correct regulation within the ncRNA network, the ncRNAome, is demonstrated when it comes to a deregulation of REST and/or ncRNAs associated with molecular pathophysiology underlying diverse disorders including neurodegenerative diseases or brain tumors