You are here: Home » Archives for RNAi
RNAi - Articles and news items
Issue 2 2008, Past issues / 19 March 2008 /
The early 21st century has seen a revolution in RNA biology, bringing with it the prospect of a new class of medicines based on RNA. What are the prospects for developing these RNA-based medicines for the growing medical problem of neurodegenerative disease and what are the challenges to making these new medicines work successfully within the complex environment of the nervous system? Recent progress on RNA silencing of neurodegenerative disease targets and RNAi delivery to the nervous system is encouraging and suggests that clinical evaluation of these therapeutic agents is realistic within the next few years.
A spectacular revolution in RNA biology over the last decade has created new tools and opportunities for advancing basic biomedical science as well as the tantalising prospect of RNA-based medicines to treat human disease. The discovery of RNA interference (RNAi) in 1998 by Fire and Mello1 led rapidly to elucidating the biochemical mechanism underlying the phenomenon of RNA-based gene silencing. (more…)
Issue 5 2007, Past issues / 21 September 2007 / Eric A. Miska, The Wellcome Trust/Cancer Research UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge, UK
Over the last ten years a small RNA revolution has swept biology. In 1998 RNA interference (RNAi) was discovered as an experimental tool by Andy Fire and Craig Mello1, a finding that was awarded with the 2006 Nobel Prize for Physiology or Medicine. Although the biology of RNAi is still not understood, it has become a powerful experimental tool and is currently being developed for human gene therapy2. During a similar time-frame and linked in some aspects to RNAi, microRNAs (miRNAs) were discovered as a new class of regulatory RNAs in animals, plants and viruses3.
MicroRNAs are transcribed from endogenous genes as long, primary RNA transcripts and are processed to their mature form: a single-stranded RNA with a length of approximately 22 nucleotides, indistinguishable from a small-interfering RNA (siRNA), the mediator of RNAi (Figure 1). In animals these long RNA precursors (pri-miRNAs)4 are processed in the nucleus by the RNase III enzyme Drosha and Pasha/DGCR8 to form the approximately 70-base pre-miRNAs5. Pre-miRNAs are exported from the nucleus by Exportin-56, processed by the RNase III enzyme Dicer, and incorporated into an Argonaute-containing silencing complex (RISC)7. MicroRNAs are thought to regulate gene expression post-transcriptionally by forming Watson-Crick base pairs with target miRNAs. Their mechanism of action is still under debate, but likely includes inhibition of translation and mRNA degradation8. In animals, most miRNAs are thought to form imperfect base-pairs with their target mRNA(s) and these interaction sites are enriched in 3’ un-translated regions (3’UTRs)3. As a consequence, miRNA target identification using computational approaches is non-trivial9. The public database for miRNAs, miRBase release 9.2, currently lists 533 human microRNAs10 and estimates for the total number of human microRNAs range from over one thousand11 to tens of thousands12. Although miRNAs have only been studied intensely for the last five years, important functions for miRNAs in animal development and, potentially, human disease, have already emerged13. (more…)
Issue 4 2007 / 21 July 2007 / Jost Seibler, Head of Technology Development, Artemis Pharmaceuticals and Frieder Schwenk, Principal Scientist, University of Applied Science, Department of Applied Natural Sciences, Gelsenkirchen, Germany
Among the genetic model organisms, the laboratory mouse (Mus musculus) has a predominant role in the study of human disease and in pre-clinical drug development. Apart from the high degree of sequence homology of mouse and human genomes, and similarities in many physiological aspects, advanced targeting technologies make the crucial difference; providing unique tools for elucidating gene function in vivo.
The ability to manipulate the genome in ES cells and mice was developed in the late 1980s; since then, gene targeting has been used extensively to study gene function in genetically modified mouse strains. As initially designed; the technique allows the disruption of a target gene in the murine germline by the insertion of a selectable marker. About 4.000 “knock-out” (KO) mice have been described in the literature, demonstrating the wide use of this approach. The application of Cre/loxP recombination has refined the tools for manipulating the mouse genome; detecting site and timing of gene alteration in the living animal1-3. An inherent feature of these recombinase-based approaches, however, is a non-reversible gene switch that does not allow modulating gene expression in a given cell. In addition, the derivation of conditional mouse mutants is costly and time consuming due to extensive vector construction, ES cell manipulation, and breeding. The finding that RNAi can mediate potent gene knockdown in transgenic animals provides the opportunity to significantly reduce time and effort for the generation of genetically modified mouse models4-6. (more…)
Issue 4 2007 / 21 July 2007 / Dr. Neil Clarke and Dr. Mark Edbrooke, GlaxoSmithKline Research and Development, Hertfordshire, UK
The archetypal microRNAs, lin-4 and let-7, were discovered in the nematode worm Caenorhabditis elegans over a decade ago and, at that time, no one would have predicted that they would be anything other than an interesting feature of worm developmental biology. However, in recent years there has been an explosion of research activity in the field of microRNAs (miRNAs), so much so that the number of publications has almost doubled every year over the last five years (see Figure 1).
Fuelling this activity was the identification of miRNAs in many more organisms including humans, and especially the discovery that they are linked to human disease. In this article we review recent progress and developments that are advancing our understanding of the role of miRNAs in several human disease settings and therapeutic arenas, and how this may affect the drug discovery landscape.
MicroRNAs are evolutionarily conserved, noncoding RNA molecules, approximately 22 nucleotides in size, that negatively regulate target gene expression at the post-transcriptional level1-3. Originally ignored as that fraction of the genome colloquially termed “Junk DNA”, examples are now known in many species. Almost 40% of miRNAs are encoded in introns, 50% are intergenic and 10% are in exons. Release 9.2 (May 2007) of miRBase (the miRNA sequence database) http://microrna.sanger.ac.uk/4-6, contains 4584 entries representing hairpin precursor miRNAs, expressing 4430 mature miRNA products in primates, rodents, birds, fish, worms, flies, plants and viruses. The database contains 475 human miRNA sequences, just under half of which have been experimentally verified. The number of miRNA encoding genes is rapidly expanding, and this number may constitute between 1-4% of expressed genes in the genome7-9. Recent literature suggests that between 20-30% of all human genes may be subject to regulation by miRNAs and that each miRNA may, on average, contribute to the regulation of 200 or more mRNA targets10. Most miRNAs bind the 3’UTR of their target genes with imperfect sequence complementarities and repress translation initiation, though perfect complementarity can lead to mRNA cleavage in a process similar to that mediated in RNA interference (RNAi). (more…)
Issue 3 2007 / 23 May 2007 / John J. Rossi, Division of Molecular Biology, Beckman Research Institute of the City of Hope, Graduate School of Biological Sciences, Duarte, United States
RNA interference (RNAi) is a regulatory mechanism of most eukaryotic cells that uses small double stranded RNA (dsRNA) molecules as triggers to direct homology-dependent control of gene activity (Almeida and Allshire 2005).
Known as small interfering RNAs (siRNA) these ~21-22 bp long dsRNA molecules have characteristic 2 nucleotide 3’ overhangs that allows them to be recognised by the enzymatic machinery of RNAi that eventually leads to homology-dependent degradation of the target mRNA. In mammalian cells siRNAs are produced from cleavage of longer dsRNA precursors by the RNaseIII endonuclease Dicer (Zhang et al. 2004). Dicer is complexed with two RNA binding proteins; the TAR-RNA binding protein (TRBP) and PACT, which are involved in the hand off of siRNAs to the RNA-induced silencing complex (RISC)(Lee et al. 2006). The core components of RISC are the Argonaute (Ago) family members, In humans there are eight members of this family but only Ago-2 possesses an active catalytic domain for cleavage activity (Liu et al. 2004; Meister et al. 2004). While siRNAs loaded into RISC are double-stranded, Ago-2 cleaves and releases the “passenger” strand leading to an activated form of RISC with a single-stranded “guide” RNA molecule that directs the specificity of the target recognition by intermolecular base pairing (Tang 2005). Rules that govern selectivity of strand loading into RISC are based upon differential thermodynamic stabilities of the ends of the siRNAs (Khvorova et al. 2003; Schwarz et al. 2003). The less thermodynamically stable end is favoured for binding to the PIWI domain of Ago-2. (more…)
Login to access exclusive content