Functional genomics - Articles and news items

Putting the ‘fun’ into functional genomics: a review of RNAi genomewide cellular screens

Issue 6 2012 / 18 December 2012 / Dr. Stephen Brown, Sheffield RNAi Screening Facility, Biomedical Sciences, University of Sheffield

As RNA interference (RNAi) enters its teenage years from the first critical observations, it has now reached a multi-billion pound industry. There are few research areas that have expanded as quickly and spectacularly as the field of RNAi. The potential of RNAi initially sparked a functional genomics gold rush. Different uses of this technology in genomewide screens have identified genes involved in fundamental biological processes. There are now hundreds of research papers reporting genome-wide screens using cell culture to investigate the building blocks of the cell. However tempting it may be to speculate that this technology could be the new magic bullet to all our research needs, especially after some of the previous successes, some basic aspects of the RNAi technology and screening process still need to be addressed and improved upon. This review will investigate the strengths and weaknesses of our current technology, suggesting improvements and highlighting some of the novel growth areas in this field.

Our foundations of cell biology rely upon an understanding of cellular pathways, the components of which have been investigated over the last 40 years or so. Recent embellish – ment of the pathways has been carried out using models in cell culture with RNAi technology1. Many techniques have been used to reveal the functions of core pathway proteins, but few have sparked the imagination like the RNAi screen with the potential to systematically knock down the expression of every gene in the genome. (more…)

MicroRNAs for high-throughput functional analysis

Issue 3 2008, Past issues / 19 June 2008 /

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules, which are potent post-transcriptional gene expression regulators. They have been shown to participate in the regulation of numerous cellular processes, the list of which is still growing. miRNAs affect numerous targets that can be determined by direct experiments or predicted by bioinformatics approaches, and are presented in several online databases. Feasibility of miRNA for high-throughput experimentation is becoming possible due to the availability of commercially produced molecules, which are able to alter the levels of endogenous miRNAs. miRNA functional analysis will help to validate predicted targets and reveal the role of these small molecules in biological pathways. miRNAs have a high potential to be used as a new gene expression regulating reagent for microscopy based assays.

The discovery of the RNA interference (RNAi) phenomenon a decade ago₁ not only truly revolutionised our understanding about gene regulation processes, but also became a superior tool for unravelling the function of unknown genes_2,3. Around the same time as RNAi, miRNA(s) were discovered₄, and were shown to be potent regulators of gene expression in viruses, animals and plants_5,6. Their regulatory effect is exerted through the pairing to complementing mRNA molecules and inducing translational repression₇. Most miRNAs are thought to make imperfect matching₈, what is largely the reason for their ability to regulate expression of numerous mRNAs simultaneously₉. (more…)

RNAi therapeutics for neurodegenerative disease: challenges and prospects

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…)

Size does matter: macro ncRNAs and the regulation of imprinted gene clusters

Issue 1 2008, Past issues / 23 January 2008 /

The availability of the human and the mouse sequence has allowed genome-wide analysis of transcription to produce ‘transcriptomes’ that list all RNA transcripts in specific cell types or tissues. These studies have identified a surprisingly large number of ncRNAs that were not recognised by gene annotation programs applied to the genomic sequence. The earliest mouse transcriptome based on sequence annotation of full-length cDNA clones demonstrated that more than 70% of mapped cDNAs arose from non-coding transcripts and 15% of all transcripts formed sense/antisense pairs1. This surprising finding has been confirmed by genomic tiling arrays that allow whole chromosomes or genomes to be simultaneously analysed at a high resolution1-6 and showed that ncRNA transcripts constitutes the bulk of the mammalian transcriptome. To date, studies that quantify non-coding transcription have been performed in most model organisms7. Non-coding transcription is more prominent in higher eukaryotes, indicating that higher genome complexity made it necessary to make use of an additional layer of gene regulation. It should be noted that a recent yeast study showed that some antisense transcription might be an artefact arising from spurious synthesis of second-strand cDNA during reverse transcription reactions8. This indicates the amount of antisense non-coding transcription identified using strategies that did not include Actinomycin D may have been overestimated. (more…)

In vivo drug target validation using RNAi

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…)

How will MicroRNAs affect the drug discovery landscape?

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…)

Academic contribution to high-content screening for functional and chemical genomics

Issue 4 2007 / 21 July 2007 / Kerstin Korn and Eberhard Krausz (Corresponding author), Head, HT-Technology Development Studio (TDS), Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG)

High-content screening (HCS) is defined as multiplexed functional screening based on imaging multiple markers (e.g. nuclei, mitochondria etc.) in the physiologic context of intact cells by extraction of multicolour fluorescence information1. It is based on a combination of advanced fluorescence-based reagents, modern liquid handling devices, automated imaging systems and data processing, as well as sophisticated image analysis software.

Initially, HCS was mainly used by pharmaceutical and biotech companies to discover new therapeutic targets and characterize new chemical leads against those targets. As this technology provides not only morphological, phenotypic and genotypic information, but functional data, it has been established as a powerful tool in modern research and drug development. Today, HCS has the potential to be used in a wide range of applications, such as target identification and validation, primary and secondary screening, mode-of-action studies, the hit-to-lead process, the identification of biomarkers, the exploitation of cytotoxicity and genotoxicity, and the tracking of cellular processes applying living cells to support basic research as well as pharmaceutical R&D. While the primary HCS literature still remains rare, a considerable number of review articles summarizing strategies, progress and developments in HCS have been already published, e.g.2-7. Within this review we are going to be focusing on HCS screens published by academic groups; moreover we would like to give a short overview of new technology developments driven by academic groups. (more…)

Inducible systemic RNA silencing in Caenorhabditis elegans

Issue 2 2007, Past issues / 27 March 2007 / Lisa Timmons, Department of Molecular Biosciences, The University of Kansas, Hiroaki Tabara, University of Tokushima, Japan, Craig C. Mello, Howard Hughes Medical Institutes and Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts and Andrew Fire, Stanford School of Medicine, Stanford University

Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed.

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Cancer immunotherapy using RNAi

Issue 1 2007, Past issues / 25 January 2007 / Chih-Ping Mao, Department of Pathology, Chien-Fu Hung, Ph.D, Department of Pathology and Oncology and T-C Wu, Ph.D., Department of Pathology, Oncology, Obstetrics and Gynecology and Molecular Microbiology and Immunology, Johns Hopkins Medical Institutions

Immunotherapy has recently emerged as an attractive form of treatment for cancer due to the potential of the immune system to eradicate tumours without inflicting damage on normal tissue. However, natural immune responses are usually inadequate to control cancer progression and require enhancement by vaccines.

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A new field in biomarker discovery and target identification

Issue 1 2007, Past issues / 25 January 2007 / Dr. Ina K. Dahlsveen, Exiqon

The last few years have seen a rush of discoveries within a new field of post-transcriptional gene regulation. microRNAs, or miRNAs for short, are small regulating RNAs akin to small interfering RNAs (siRNA), but which are naturally expressed in vivo. Originally discovered in C. elegans 14 years ago, these small 20-22 nucleotide non-coding RNA molecules bind specifically to target messenger RNAs (mRNA) blocking their translation into protein or causing their degradation.

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Challenges in High-Content siRNA Screening

Issue 6 2006, Past issues / 28 November 2006 / Dr. Eberhard Krausz, HT-Technology Development Studio (TDS), Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG)

Huge progress has been made, both in RNA interference technology applied to mammalian cells and in automated microscopy to analyse gene functions upon silencing in the cellular context. Large-scale siRNA screens have been published recently, mainly applying assays that gain multi-parametric information on biological processes. It is a long way to establish an infrastructure that allows high-content siRNA screening, and in this article the major challenges are summarised.

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The right approach to human therapy

Issue 5 2006, Past issues / 28 September 2006 / Dr Simone Hess, Max-Planck-Institute for Infection Biology

The RIGHT (RNA Interference Technology as Human Therapeutic Tool) consortium consists of 18 research institutions and four companies from nine European countries. The project has been funded as an integrated project by the European Commission’s Sixth Framework Programme for Research and Development (FP6) since January 2005. Thomas F. Meyer from the Max Planck Institute for Infection Biology in Berlin is coordinating this European research project that aims at exploiting the vast potential of RNA interference (RNAi) for human therapy.

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