RNA interference ( RNAi ) & basic and essential siRNA publication (4)
siRNA & quantitative real-time RT-PCR (1)
siRNA & quantitative real-time RT-PCR (2)
siRNA & quantitative real-time RT-PCR (3)
siRNAdb: a database of siRNA sequences
Alistair M. Chalk, Richard E. Warfinge, Patrick Georgii-Hemming and Erik L. L. Sonnhammer
Nucleic Acids Research, 2005, Vol. 33, Database issue
Center for Genomics and Bioinformatics, Karolinska Institutet, Berzelius va¨g 35, S-171 77 Stockholm, Sweden
The database is available at http://siRNA.cgb.ki.se
Short interferingRNAs (siRNAs) are a popular method for gene-knockdown, acting by degrading the target mRNA. Before performing experiments it is invaluable to locate and evaluate previous knockdown experiments for the gene of interest. The siRNA databaseprovides a gene-centric view of siRNA experimental data, including siRNAs of known efficacy and siRNAs predicted to be of high efficacy by a combination of methods. Linked to these sequences is information such as siRNA thermodynamic properties and the potential for sequence-specific off-target effects. The database enables the user to evaluate an siRNA’s potential for inhibition and non-specific effects.
Regulation of gene expression by small non-coding RNAs: a quantitative view.
Yishai Shimoni, Gilgi Friedlander, Guy Hetzroni, Gali Niv, Shoshy Altuvia, Ofer Biham and Hanah Margalit
Molecular Systems Biology 3: 138
The importance of post-transcriptional regulation by small non-coding RNAs has recently been recognized in both pro- and eukaryotes. Small RNAs (sRNAs) regulate gene expression posttranscriptionally by base pairing with the mRNA. Here we use dynamical simulations to characterize this regulation mode in comparison to transcriptional regulation mediated by protein–DNA interaction and to post-translational regulation achieved by protein–protein interaction. We show quantitatively that regulation by sRNA is advantageous when fast responses to external signals are needed, consistent with experimental data about its involvement in stress responses. Our analysis indicates that the half-life of the sRNA–mRNA complex and the ratio of their production rates determine the steady-state level of the target protein, suggesting that regulation by sRNA may provide fine-tuning of gene expression. We also describe the network of regulation by
sRNA in Escherichia coli, and integrate it with the transcription regulation network, uncovering mixed regulatory circuits, such as mixed feed-forward loops. The integration of sRNAs in feedforward loops provides tight repression, guaranteed by the combination of transcriptional and post-transcriptional regulations.
Translational control and target recognition by Escherichia coli small RNAs in vivo.
Johannes H. Urban and Joerg Vogel
Max Planck Institute for Infection Biology, RNA Biology Group, Chariteplatz 1, 10117 Berlin, Germany
Nucleic Acids Research, 2007, Vol. 35, No. 3 1018–1037
Small non-coding RNAs (sRNAs) are an emerging class of regulators of bacterial gene expression. Most of the regulatory Escherichia coli sRNAs known to date modulate translation of transencoded target mRNAs. We studied the specificity of sRNA target interactions using gene fusions to green fluorescent protein (GFP) as a novel reporter of translational control by bacterial sRNAs in vivo. Target sequences were selected from both monocistronic and polycistronic mRNAs. Upon expression of the cognate sRNA (DsrA, GcvB, MicA, MicC,MicF, RprA, RyhB, SgrS and Spot42), we observed highly specific translation repression/activation of target fusions under various growth conditions. Target regulation was also tested in mutants that lacked Hfq or RNase III, or which expressed a truncated RNase E (rne701). We found that translational regulation by these sRNAs was largely independent of full-length RNase E, e.g. despite the fact thatompA fusion mRNA decay could no longer be promoted by MicA. This is the first study in which multiple well-defined E.coli sRNA target pairs have been studied in a uniform manner in vivo. We expect our GFP fusion approach to be applicable to sRNA targets of other bacteria, and also demonstrate that Vibrio RyhB sRNA represses a Vibrio sodB fusion when co-expressed in E.coli.
RNA interference: from gene silencing to gene-specific therapeutics.
Ray K.M. Leung, Paul A. Whittaker
Pharmacology & Therapeutics 107 (2005) 222 – 239
In the past 4 years, RNA interference (RNAi) has become widely used as an experimental tool to analyse the function of mammalian genes, both in vitro and in vivo. By harnessing an evolutionary conserved endogenous biological pathway, first identified in plants and lower
organisms, double-stranded RNA (dsRNA) reagents are used to bind to and promote the degradation of target RNAs, resulting in knockdown of the expression of specific genes. RNAi can be induced in mammalian cells by the introduction of synthetic double-stranded small interfering RNAs (siRNAs) 21–23 base pairs (bp) in length or by plasmid and viral vector systems that express double-stranded short hairpin RNAs (shRNAs) that are subsequently processed to siRNAs by the cellular machinery. RNAi has been widely used in mammalian cells to define the functional roles of individual genes, particularly in disease. In addition, siRNA and shRNA libraries have been developed to allow the systematic analysis of genes required for disease processes such as cancer using high throughput RNAi screens. RNAi has been used for the knockdown of gene expression in experimental animals, with the development of shRNA systems that allow tissue-specific and inducible knockdown of genes promising to provide a quicker and cheaper way to generate transgenic animals than conventional approaches. Finally, because of the ability of RNAi to silence disease-associated genes in tissue culture and animal models, the development of RNAi-based reagents for clinical applications is gathering pace, as technological enhancements that improve siRNA stability and delivery in vivo, while minimising off-target and nonspecific effects, are developed.
Cell type–specific delivery of siRNAs with aptamersiRNA chimeras.
James O McNamara, Eran R Andrechek, Yong Wang, Kristi D Viles, Rachel E Rempel, Eli Gilboa, Bruce A Sullenger & Paloma H Giangrande
NATURE BIOTECHNOLOGY VOLUME 24 NUMBER 8 AUGUST 2006 1005
Technologies that mediate targeted delivery of small interfering RNAs (siRNAs) are needed to improve their therapeutic efficacy and safety. Therefore, we have developed aptamer-siRNA chimeric RNAs capable of cell type–specific binding and delivery of functional siRNAs into cells. The aptamer portion of the chimeras mediates binding to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium, whereas the siRNA portion targets the expression of survival genes. When applied to cells expressing PSMA, these RNAs are internalized and processed by Dicer, resulting in depletion of the siRNA target proteins and cell death. In contrast, the chimeras do not bind to or function in cells that do not express PSMA. These reagents also specifically inhibit tumor growth and mediate tumor regression in a xenograft model of prostate cancer. These studies demonstrate an approach for targeted delivery of siRNAs with numerous potential applications, including cancer therapeutics.
RNA interference has second helpings.
Eric A Miska & Julie Ahringer
NATURE BIOTECHNOLOGY 2007 VOLUME 25 NUMBER 3 302
Efficient RNA interference in Caenorhabditis elegans requires a distinct class of secondary short interfering RNA.
Stable suppression of gene expression by RNAi in mammalian cells.
Patrick J. Paddison, Amy A. Caudy, and Gregory J. Hannon
PNAS (2002) vol. 99 no. 3 1443–1448
In a diverse group of organisms including plants, Caenorhabditis elegans, Drosophila, and trypanosomes, double-stranded RNA (dsRNA) is a potent trigger of gene silencing. In several model systems, this natural response has been developed into a powerful tool for the investigation of gene function. Use of RNA interference (RNAi) as a genetic tool has recently been extended to mammalian cells, being inducible by treatment with small, 22-nt RNAs that mimic those produced in the first step of the silencing process. Here, we show that some cultured murine cells specifically silence gene expression upon treatment with long dsRNAs (500 nt). This response shows hallmarks of conventional RNAi including silencing at the posttranscriptional level and the endogenous production of 22-nt small RNAs. Furthermore, enforced expression of long, hairpin dsRNAs induced stable gene silencing. The ability to create stable ‘‘knock-down’’ cell lines expands the utility of RNAi in mammalian cells by enabling examination of phenotypes that develop over long time periods and lays the groundwork for by using RNAi in phenotype-based, forward genetic selections.
Reversible gene knockdown in mice using a tight, inducible shRNA expression system.
Jost Seibler, Andre Kleinridders, Birgit Kuter-Luks, Sandra Niehaves, Jens C. Bruning and Frieder Schwenk
Nucleic Acids Research, 2007, 1–9
RNA interference through expression of short hairpin (sh)RNAs provides an efficient approach for gene function analysis in mouse genetics.
Techniques allowing to control time and degree of gene silencing in vivo, however, are still lacking. Here we provide a generally applicable system for the temporal control of ubiquitous shRNA expression in mice. Depending on the dose of the inductor doxycycline, the knockdown efficiency reaches up to 90%. To demonstrate the feasibility of our tool, a mouse model of reversible insulin resistance wasgenerated by expression of an insulin receptor (Insr)-specific shRNA. Upon induction, mice develop severe hyperglycemia within seven days. The onset and progression of the disease correlates with the concentration of doxycycline, and the phenotype returns to baseline shortly after withdrawal of the inductor. On a broad basis, this approach will enable new insights into gene function and molecular disease mechanisms.
Small non-coding RNAs in animal development.
Giovanni Stefani & Frank J. Slack
Nature Reviews Molecular Cell Biology 9, 219-230 (2008)
The modulation of gene expression by small non-coding RNAs is a recently discovered level of gene regulation in animals and plants. In particular, microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs) have been implicated in various aspects of animal development, such as neuronal, muscle and germline development. During the past year, an improved understanding of the biological functions of small non-coding RNAs has been fostered by the analysis of genetic deletions of individual miRNAs in mammals. These studies show that miRNAs are key regulators of animal development and are potential human disease loci.