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Functional genomics - Articles and news items
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.
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 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.
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.
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.
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).
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.
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.
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.
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.
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.
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.