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Small double-stranded
RNA (dsRNA) has been found to
silence gene expression by an evolutionally conserved mechanism known
as RNA interference or RNAi. Such dsRNAs are
called small interfering RNAs
or siRNA.
RNAi can occur at both transcriptional and post-transcriptional levels.
Surprisingly, various recent studies (see below) have found that dsRNA
can also activate
gene expression, a mechanism that has been termed "small
RNA-induced gene activation" or "saRNA" or "RNAa".
It has been shown that dsRNAs targeting gene promoters induce potent
transcriptional activation of associated genes. Both studies
demonstrate RNAa in human cells using synthetic dsRNAs termed small
activating RNAs (saRNAs). Endogenous microRNAs
(miRNA) that cause RNAa has also
been found in humans.
It is currently unknown if RNAa is conserved in other organisms.
saRNA-DIRECTED
TRANSCRIPTIONAL ACTIVATION
UCSF, University of California, San Francisco Currently, there is no dependable and generalizable method for the targeted activation of endogenous genes. Efforts in gene therapy have been forestalled by problems of gene mutagenesis and oncogene activation, resulting in cancer. Yet the pursuit of a method for gene activation remains an important goal because the ability to selectively upregulate genes acting against a diseased state would have far reaching impact in almost every therapeutic realm. One of the most promising current approaches to combating disease at the genetic level employs small dsRNA molecules as therapeutic compounds to achieve gene silencing by RNA interference ("RNAi"). With the ever-increasing amount of research and development dollars being spent on RNAi therapeutics, solutions to potential specificity and toxicity issues have been addressed, and a large number of delivery methods have been developed. These advances have given gene silencing a distinct advantage over gene therapy but does not satisfy the need for gene activation methods. UCSF scientists have addressed this void by developing a gene activation approach which overcomes obstacles in gene therapy by utilizing the advances of RNAi. Researchers at UCSF have discovered a method using small activating RNA ("saRNA") for inducing sequence-specific transcriptional activation. Human cell studies conducted at UCSF have shown that this method induced an 8-fold increase in transcriptional activation of tumor suppressor gene E-cadherin. Transcriptional activation lasted up to 10 days and resulted in the anticipated phenotypic readout. http://www.otm.ucsf.edu/tech/otm2005066.asp External links
RNA interference: hitting the ON switch. Researchers in San Francisco have findings that suggest a whole new side to RNA interference. Erika Check reports on their attempts to make a revolutionary field more revolutionary still.  Turn genes on, turn diseases off Bob Holmes, New Scientist, 2007 http://www.science.org.au/nova/newscientist/098ns_001.htm  News and Views Q&A (by Helge Großhans and Witold Filipowicz) Molecular biology: The expanding world of small RNAs Molecular cell biology has long been dominated by a protein-centric view. But the emergence of small, non-coding RNAs challenges this perception. These plentiful RNAs regulate gene expression at different levels, and have essential roles in health and disease. References: Small dsRNAs induce transcriptional activation in human cells. Li LC, Okino ST, Zhao H, Pookot D, Place RF, Urakami S, Enokida H, Dahiya R. Proc Natl Acad Sci U S A. 2006 103(46): 17337-17342 Department of Urology, Veterans Affairs Medical Center and University of California, San Francisco, CA 94121, USA. Recent studies have
shown that small noncoding RNAs, such as microRNAs and siRNAs, regulate
gene expression at multiple levels including chromatin architecture,
transcription, RNA editing, RNA stability, and translation. Each form
of RNA-dependent regulation has been generally found to silence
homologous sequences and collectively called RNAi. To further study the
regulatory role of small RNAs at the transcriptional level, we designed
and synthesized 21-nt dsRNAs targeting selected promoter regions of
human genes E-cadherin, p21(WAF1/CIP1) (p21), and VEGF. Surprisingly,
transfection of these dsRNAs into human cell lines caused long-lasting
and sequence-specific induction of targeted genes. dsRNA mutation
studies reveal that the 5' end of the antisense strand, or "seed"sequence,
is critical for activity. Mechanistically, the dsRNA-induced gene
activation requires the Argonaute 2 (Ago2) protein and is associated
with a loss of lysine-9 methylation on histone 3 at dsRNA-target sites.
In conclusion, we have identified several dsRNAs that activate gene
expression by targeting noncoding regulatory regions in gene promoters.
These findings reveal a more diverse role for small RNA molecules in
the regulation of gene expression than previously recognized and
identify a potential therapeutic use for dsRNA in targeted gene
activation.
PATENT: (WO/2006/113246) SMALL ACTIVATING RNA MOLECULES AND METHODS OF USE A small modulatory dsRNA specifies the fate of adult neural stem cells. Kuwabara T, Hsieh J, Nakashima K, Taira K, Gage FH. Cell. 2004 116(6): 779-793 Laboratory of Genetics, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. Discovering the molecular mechanisms that regulate neuron-specific gene expression remains a central challenge for CNS research. Here, we report that small, noncoding double-stranded (ds) RNAs play a critical role in mediating neuronal differentiation. The sequence defined by this dsRNA is NRSE/RE1, which is recognized by NRSF/REST, known primarily as a negative transcriptional regulator that restricts neuronal gene expression to neurons. The NRSE dsRNA can trigger gene expression of neuron-specific genes through interaction with NRSF/REST transcriptional machinery, resulting in the transition from neural stem cells with neuron-specific genes silenced by NRSF/REST into cells with neuronal identity that can express neuronal genes. The mechanism of action appears to be mediated through a dsRNA/protein interaction, rather than through siRNA or miRNA. The discovery of small modulatory dsRNAs (smRNAs) extends the important contribution of noncoding RNAs as key regulators of cell behavior at both transcriptional and posttranscriptional levels. Regulation of endothelial nitric oxide synthase by small RNA. Ming-Xiang Zhang *, Hesheng Ou *, Ying H. Shen, Jing Wang, Jian Wang, Joseph Coselli, and Xing Li Wang PNAS 2005 vol. 102 (47) 16967-16972 Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, and Texas Heart Institute at St Luke's Episcopal Hospital, Houston, TX 77030 Repeats (27-nt) in intron 4 have been shown to play a cis-acting role in endothelial nitric oxide synthase (eNOS) promoter activity. We hypothesize that the 27-nt repeats could be the source of small nuclear RNA specifically regulating eNOS expression. In this study, we used synthesized 27-nt RNA duplex and found that the eNOS gene transcriptional efficiency was reduced 63% (0.047 ± 0.009 vs. 0.126 ± 0.015, P < 0.01) by nuclear run-on assay. In endothelial cells transfected with the 27-nt small RNA duplex, we found that the eNOS mRNA and protein levels were decreased by >64% (P < 0.01). Conversely, a randomly selected 27-nt from luciferase gene had no effect on the eNOS expression. Furthermore, this eNOS silencing effect appeared to be reversible under the stimulation of vascular endothelial growth factor (10 ng/ml), which is known to up-regulate eNOS expression. Using in situ hybridization and Northern blotting, we observed the presence of endogenous eNOS intron 4-derived 27-nt small RNA, which was confined to the nucleus. In summary, we demonstrated that intron-based microRNAs in eNOS can induce significant gene specific transcriptional suppression, which could be an effective negative feedback regulator for gene expression. Activating gene expression in mammalian cells with promoter-targeted duplex RNAs. Janowski BA, Younger ST, Hardy DB, Ram R, Huffman KE, Corey DR. Nat Chem Biol. 2007 3(3): 166-173 Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA. The ability to selectively activate or inhibit gene expression is fundamental to understanding complex cellular systems and developing therapeutics. Recent studies have demonstrated that duplex RNAs complementary to promoters within chromosomal DNA are potent gene silencing agents in mammalian cells. Here we report that chromosome-targeted RNAs also activate gene expression. We have identified multiple duplex RNAs complementary to the progesterone receptor (PR) promoter that increase expression of PR protein and RNA after transfection into cultured T47D or MCF7 human breast cancer cells. Upregulation of PR protein reduced expression of the downstream gene encoding cyclooygenase 2 but did not change concentrations of estrogen receptor, which demonstrates that activating RNAs can predictably manipulate physiologically relevant cellular pathways. Activation decreased over time and was sequence specific. Chromatin immunoprecipitation assays indicated that activation is accompanied by reduced acetylation at histones H3K9 and H3K14 and by increased di- and trimethylation at histone H3K4. These data show that, like proteins, hormones and small molecules, small duplex RNAs interact at promoters and can activate or repress gene expression. Transcriptional activation by small RNA duplexes. John J. Rossi Division of Molecular Biology, Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA. Short double-stranded RNA duplexes are the triggers for post-transcriptional gene silencing and can also induce epigenetic silencing of genes at the level of transcription. A surprising new finding is that short RNA duplexes targeted to promoter regions can also mediate potent enhancement of transcription. When microRNAs acivate translation. Contrary to their
traditional role, microRNAs (miRNAs) contribute to an
increase in translation during cell quiescence. This function may be
exploited for microRNAmediated regulation of protein expression.
Switching from repression to activation: microRNAs can up-regulate translation. Vasudevan S, Tong Y, Steitz JA. Science. 2007 318(5858): 1931-1934 Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536, USA. AU-rich elements (AREs)
and microRNA target sites are conserved
sequences in messenger RNA (mRNA) 3' untranslated regions (3'UTRs) that
control gene expression posttranscriptionally. Upon cell cycle arrest,
the ARE in tumor necrosis factor-alpha (TNFalpha) mRNA is transformed
into a translation activation signal, recruiting Argonaute (AGO) and
fragile X mental retardation-related protein 1 (FXR1), factors
associated with micro-ribonucleoproteins (microRNPs). We show that
human microRNA miR369-3 directs association of these proteins with the
AREs to activate translation. Furthermore, we document that two
well-studied microRNAs-Let-7 and the synthetic microRNA
miRcxcr4-likewise induce translation up-regulation of target
mRNAs on cell cycle arrest, yet they repress translation in
proliferating cells. Thus, activation is a common function of microRNPs
on cell cycle arrest. We propose that translation regulation by
microRNPs oscillates between repression and activation during the cell
cycle.
Activation of an oncogenic microRNA cistron by provirus integration. Wang CL, Wang BB, Bartha G, Li L, Channa N, Klinger M, Killeen N, Wabl M. Proc Natl Acad Sci U S A. 2006 103(49): 18680-18684 Department of Microbiology and Immunology, University of California-San Francisco, San Francisco, CA 94143, USA. Retroviruses can cause
tumors when they integrate near a protooncogene
or tumor suppressor gene of the host. We infected >2,500 mice with
the SL3-3 murine leukemia virus; in 22 resulting tumors, we found
provirus integrations nearby or within the gene that contains the
mir-17-92 microRNA (miRNA) cistron. Using quantitative real-time PCR,
we showed that expression of miRNA was increased in these tumors,
indicating that retroviral infection can induce expression of oncogenic
miRNAs. Our results demonstrate that retroviral mutagenesis can be a
potent tool for miRNA discovery.
MicroRNA-373 induces expression of genes with complementary promoter sequences. Robert F. Place, Long-Cheng Li, Deepa Pookot, Emily J. Noonan, and Rajvir Dahiya PNAS (2008) 105(5) 1608-1613 Recent studies have
shown that microRNA (miRNA) regulates gene expression by repressing
translation or directing sequence-specific degradation of complementary
mRNA. Here, we report new evidence in which miRNA may also function to
induce gene expression. By scanning gene promoters in silico for
sequences complementary to known miRNAs, we identified a putative
miR-373 target site in the promoter of E-cadherin. Transfection of
miR-373 and its precursor hairpin RNA (pre-miR-373) into PC-3 cells
readily induced E-cadherin expression. Knockdown experiments confirmed
that induction of E-cadherin by pre-miR-373 required the miRNA
maturation protein Dicer. Further analysis revealed that cold-shock
domain-containing protein C2 (CSDC2), which possesses a putative
miR-373 target site within its promoter, was also readily induced in
response to miR-373 and pre-miR-373. Furthermore, enrichment of RNA
polymerase II was detected at both E-cadherin and CSDC2 promoters after
miR-373 transfection. Mismatch mutations to miR-373 indicated that gene
induction was specific to the miR-373 sequence. Transfection of
promoter-specific dsRNAs revealed that the concurrent induction of
E-cadherin and CSDC2 by miR-373 required the miRNA target sites in both
promoters. In conclusion, we have identified a miRNA that targets
promoter sequences and induces gene expression. These findings reveal a
new mode by which miRNAs may regulate gene expression.
Silencing of microRNAs in vivo with 'antagomirs'. Krützfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M, Stoffel M. Nature. 2005 438(7068): 685-689 Laboratory of Metabolic Diseases, The Rockefeller University, 1230 York Avenue, New York, New York 10021, USA. MicroRNAs (miRNAs) are
an abundant class of non-coding RNAs that are believed to be important
in many biological processes through regulation of gene expression. The
precise molecular function of miRNAs in mammals is largely unknown and
a better understanding will require loss-of-function studies in vivo.
Here we show that a novel class of chemically engineered
oligonucleotides, termed 'antagomirs', are efficient and specific
silencers of endogenous miRNAs in mice. Intravenous administration of
antagomirs against miR-16, miR-122, miR-192 and miR-194 resulted in a
marked reduction of corresponding miRNA levels in liver, lung, kidney,
heart, intestine, fat, skin, bone marrow, muscle, ovaries and adrenals.
The silencing of endogenous miRNAs by this novel method is specific,
efficient and long-lasting. The biological significance of silencing
miRNAs with the use of antagomirs was studied for miR-122, an abundant
liver-specific miRNA.Gene expression and bioinformatic analysis of
messenger RNA from antagomir-treated animals revealed that the 3'
untranslated regions of upregulated genes are strongly
enriched in miR-122 recognition motifs, whereas downregulated genes are
depleted in these motifs. Analysis of the functional annotation of
downregulated genes specifically predicted that cholesterol
biosynthesis genes would be affected by miR-122, and plasma cholesterol
measurements showed reduced levels in antagomir-122-treated mice. Our
findings show that antagomirs are powerful tools to silence specific
miRNAs in vivo and may represent a therapeutic strategy for silencing
miRNAs in disease.
Specificity, duplex degradation and subcellular localization of antagomirs. Krützfeldt J, Kuwajima S, Braich R, Rajeev KG, Pena J, Tuschl T, Manoharan M, Stoffel M. Nucleic Acids Res. 2007;35(9): 2885-2892 Laboratory of Metabolic Diseases, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA. MicroRNAs (miRNAs) are
an abundant class of 20-23-nt long regulators of gene expression. The
study of miRNA function in mice and potential therapeutic approaches
largely depend on modified oligonucleotides. We recently demonstrated
silencing miRNA function in mice using chemically modified and
cholesterol-conjugated RNAs termed 'antagomirs'. Here, we further
characterize the properties and function of antagomirs in mice. We
demonstrate that antagomirs harbor optimized phosphorothioate
modifications, require >19-nt length for highest efficiency and can
discriminate between single nucleotide mismatches of the targeted
miRNA. Degradation of different chemically protected miRNA/antagomir
duplexes in mouse livers and localization of antagomirs in a cytosolic
compartment that is distinct from processing (P)-bodies indicates a
degradation mechanism independent of the RNA interference (RNAi)
pathway. Finally, we show that antagomirs, although
incapable of silencing miRNAs in the central nervous system (CNS) when
injected systemically, efficiently target miRNAs when injected locally
into the mouse cortex. Our data further validate the effectiveness of
antagomirs in vivo and should facilitate future studies to silence
miRNAs for functional analysis and in clinically relevant settings.
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