2The Kellogg School of Science and Technology, The Scripps Research Institute, La Jolla, CA, USA
Emerging research suggests that long noncoding RNAs (ncRNAs) may play a role in the basic fabric of gene regulation in human cells. Mechanistic studies carried out on a small subset of antisense ncRNAs have begun to link RNA-mediated modifications of DNA and chromatin structure with gene expression, implicating ncRNAs in the regulation of transcription. Meanwhile, genome-wide studies have revealed that transcription of ncRNAs is far more ubiquitous than previously thought and suggest a more pervasive role for ncRNAs. This review will describe the current state of research regarding gene regulation by ncRNAs and highlight major techniques used in the field. Furthermore, the potential for therapeutic applications based on ncRNA regulation will also be discussed.
Gene expression plays a critical role in the normal function of human cells. While changes in gene expression are normal for certain cell processes such as differentiation or activation, unintended changes in gene expression can lead to human disease. Altered gene expression is linked to two main events: (i) a change in the DNA sequence (such as a mutation, deletion, insertion, etc.) or (ii) a change in the local chromatin. While numerous mechanisms are known to cause a change in DNA sequence, the mechanisms that drive changes in chromatin structure are less understood. Chromatin refers to the protein-DNA complexes that interact in order to structure and compact two meters of DNA into one microscopic cell. Chromatin can exist as either euchromatin (uncompressed) or heterochromatin (compressed). These two states also correlate with the transcriptional activity of DNA, as regions of euchromatin tend to be transcriptionally active while heterochromatin tends to be transcriptionally inactive. The structure and transcriptional activity of chromatin is regulated in large part by DNA methylation and posttranslational modifications to histones, the major proteins that interact to form the nucleosomes around which DNA is compacted (1). DNA methylation and histone modifications are capable of recruiting protein complexes that can maintain or change these chromatin marks and thereby alter the transcriptional activity of DNA (1). Recent research suggests that noncoding RNAs (ncRNAs) are playing a role in the regulation of genes at the chromatin level by affecting DNA methylation and histone posttranslational modifications.
Two major pathways of RNA-based gene regulation have been characterized: (i) posttranscriptional gene silencing (PTGS) and (ii) transcriptional gene silencing (TGS). PTGS, originally observed in Caenorhabditis elegans, is a well-described pathway that acts at the mRNA level through an Argonaute-2 (Ago-2)–dependent mechanism and is commonly referred to as RNA interference (RNAi) (2). Small interfering RNAs (siRNAs) act to recruit Ago-2 to a target messenger RNA (mRNA) through sequence complementarity. Recognition of the mRNA results in Ago-2–mediated cleavage or translational repression of the target mRNA and subsequently to decreased gene expression (2). The mechanism of PTGS is transient and depends on the continued presence of the siRNA effector molecule. Loss of the effector molecule in the PTGS pathway eventually results in the return of normal gene expression (2). In contrast to PTGS, TGS acts at the level of DNA and can result in long-term silencing. TGS involves ncRNA-mediated chromatin changes to a gene promoter, resulting in reduced transcription at the targeted locus. Chromatin modifications, including DNA methylation and histone methylation resulting from TGS-inducing RNAs, have been observed in plants (3), Drosophila (4,5), and in the fission yeast Schizosaccharomyces pombe (6). In 2004, TGS was observed in human cells when small RNAs targeted to the promoter of elongation factor 1α were shown to induce gene silencing (7).
Mechanistic details of small RNA-directed TGS in human cells have begun to emerge. Numerous new studies over the past 6 years have significantly advanced our understanding of TGS in mammalian cells while raising an important question: how much do we really know about the role of RNA in gene regulation? Recent genomewide studies have revealed that transcription of ncRNAs is far more ubiquitous than previously thought. Furthermore, new research indicates that ncRNAs may also have the ability to cause gene activation, the exact opposite form of gene regulation observed in PTGS and TGS. This review will detail current research regarding gene regulation by ncRNAs and highlight commonly used techniques in the field that have significantly aided in the study of this molecular process. In addition, the potential for therapeutic applications based on ncRNA regulation and the need for novel delivery methods will also be discussed.TGS by small RNAs
The first studies of TGS used small RNAs targeted to various gene promoters to induce gene silencing. RNAs used to target gene promoters have included synthetic siRNAs (7,8,9,10,11,12,13,14,15,16,17,18) and plasmids expressing short hairpin RNAs (shRNAs) (8,11, 19,20,21) or small antisense RNAs (8,22) from RNA polymerase III (RNA Pol III) promoters (Figure 1). RNA Pol III is responsible for transcribing various ncRNAs in the cell such as ribosomal RNA and transfer RNAs. As such, RNA Pol III binding promoters including the human U6 small nuclear RNA or the human H1 are commonly used to express ncRNAs due to the short recognition and termination sequences and characterized initiation sites. One of the defining characteristics of TGS is the ability of promoter-targeted small RNAs to reduce mRNA levels of a gene by reducing the amount of transcriptional initiation. Levels of transcription are determined by using quantitative real-time PCR (qRT-PCR) to amplify reverse-transcribed cellular RNA, thereby identifying whether a targeted gene has been affected. While a critical tool, qRT-PCR cannot differentiate between decreases in transcription due to less initiation (as in TGS) versus degradation of mRNA (as in PTGS). Therefore, it is critical to initially assess the action of small RNAs involved in TGS using nuclear run-ons, in which active transcription is halted and then resumed in the presence of labeled UTP, often 32P or biotin (7,10,18,20,22,23). The labeled RNA is most commonly analyzed by dot blot to determine the level of transcription at a target gene compared with a control gene. Using qRT-PCR and nuclear run-ons as initial assays, promoter-targeted small RNAs generated de novo have been shown to reduce the transcriptional activity of numerous genes.