 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. |