Analysis of mammalian gene function using small interfering RNAs

Martı́nez, Javier; Patkaniowska, Agnieszka; Elbashir, Sayda M.; Harborth, Jens; Hoßbach, Markus; Urlaub, Henning; Meyer, Jutta; Weber, Klaus; Vandenburgh, Kimberlie; Manninga, Heiko; Scaringe, Stephen A.; Luehrmann, R.; Tuschl, Thomas

doi:10.1093/nass/3.1.333

Cited by 8 publications

(7 citation statements)

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“…Either strand of the siRNA duplex may function as a guide that localizes RISC to the target RNA via Watson-Crick base pairing (Nykanen et al 2001;Martinez et al 2002Martinez et al , 2003. Prior to incorporation into RISC, the strands of the siRNA duplex must be unwound in a step that is thought to require an ATP-dependent helicase.…”

Section: Discussionmentioning

confidence: 99%

Small interfering RNAs containing full 2′-O-methylribonucleotide-modified sense strands display Argonaute2/eIF2C2-dependent activity

Kraynack¹,

Baker²

2005

RNA

126

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RNA interference (RNAi) is a process by which short interfering RNAs (siRNAs) direct the degradation of complementary singlestrand RNAs. In this study, we investigated the effects of full-strand phosphorothioate (PS) backbone and 2¢-O-methyl (2¢-OMe) sugar modifications on RNAi-mediated silencing. In contrast to previous reports, we have identified active siRNA duplexes containing full 2¢-OMe-modified sense strands that display comparable activity to the unmodified analog of similar sequence. The structure of these modified siRNAs is the predominant determinant of their activity, with sequence and backbone composition being secondary. We further show, by using biotin-tagged siRNAs and affinity-tagged hAgo2/eIF2C2, that activity of siRNA duplexes containing full 2¢-OMe substitutions in the sense strand is mediated by the RNA-induced silencing complex (RISC) and that strand-specific loading (or binding) to hAgo2 may be modulated through selective incorporation of these modifications.

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Section: Discussionmentioning

confidence: 99%

Small interfering RNAs containing full 2′-O-methylribonucleotide-modified sense strands display Argonaute2/eIF2C2-dependent activity

Kraynack¹,

Baker²

2005

RNA

126

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“…siRNA-mediated Gene Silencing-siRNA duplexes targeted to sONE mRNA sequences were designed as described (31) (Table II). sONE targeted duplexes were transfected along with scrambled control siRNA (Scramble II siRNA duplex, Dharmacon) into HAoVSMC using TransMessenger (Qiagen, Valencia, CA).…”

Section: Methodsmentioning

confidence: 99%

Post-transcriptional Regulation of Endothelial Nitric-oxide Synthase by an Overlapping Antisense mRNA Transcript

Robb

Carson

Tai

et al. 2004

Journal of Biological Chemistry

133

127

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Endothelial nitric-oxide synthase (eNOS) mRNA levels are abnormal in diseases of the cardiovascular system, but changes in gene expression cannot be accounted for by transcription alone. We found evidence for the existence of an antisense mRNA (sONE) that is derived from a transcription unit (NOS3AS) on the opposite DNA strand from which the human eNOS (NOS3) mRNA is transcribed at human chromosome 7q36. The genes are oriented in a tail-to-tail configuration, and the mRNAs encoding sONE and eNOS are complementary for 662 nucleotides. The mRNA for sONE could be detected in a variety of cell types, both in vivo and in vitro, but not vascular endothelial cells. In contrast, expression of eNOS is highly restricted to vascular endothelium. Most surprisingly, interrogation of transcriptional events across NOS3/NOS3AS genomic regions, using single-and double-stranded probes for nuclear run-off analyses and chromatin immunoprecipitation-based assessments of RNA polymerase II distribution, indicated that NOS3 and NOS3AS gene transcription did not correlate with steady-state mRNA levels. We found strong evidence supporting a role for NOS3AS in the post-transcriptional regulation of NOS3 expression. RNA interference-mediated inhibition of sONE expression in vascular smooth muscle cells increased eNOS expression. Overexpression of sONE in endothelial cells blunted eNOS expression. Finally, the histone deacetylase inhibitor trichostatin A is known to regulate the expression of eNOS via a post-transcriptional mechanism. We found that trichostatin A treatment of vascular endothelial cells increased expression of sONE mRNA levels prior to the observed decrease in eNOS mRNA expression. Taken together, these results indicate that an antisense mRNA (sONE) participates in the post-transcriptional regulation of eNOS and provide a newer model for endothelial cell-specific gene expression.

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“…Gene silencing by siRNAs is a powerful technique to investigate cellular metabolism and the influence of specific genes (Caplen et al, 2001;Elbashir et al, 2001a;Elbashir et al, 2001b;Harborth et al, 2003;Martinez et al, 2003;Timmons et al, 2003). Silencing is a posttranscriptional mechanism that targets a specific mRNA.…”

Section: Introductionmentioning

confidence: 99%

The effect of temperature on gene silencing by siRNAs: Implications for silencing in the anterior chamber of the eye

Russell

Walsh

Chen

et al. 2006

Experimental Eye Research

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Gene silencing by siRNAs offers the potential for reducing the expression of mutated genes that cause disease. It has been shown that the folding or secondary structure of a specific mRNA was significantly correlated to the silencing observed. Since this base pairing is dependent on free energy, the temperature of the cells may influence the effectiveness of a particular siRNA. The aqueous humor of the human eye has been measured to be around 34°C with the lens acting as a thermal barrier in the eye. The trabecular meshwork, bathed by the aqueous humor, probably has a temperature lower than body temperature under normal conditions. Mutated myocilin, that is associated with primary open angle glaucoma, would appear to be a candidate for silencing by siRNAs. Silencing of the mutated myocilin might prevent additional accumulation of this protein in the rough endoplasmic reticulum. These experiments were undertaken to determine the influence of lowered temperatures on the silencing of myocilin by five siRNAs. Three different patterns of silencing emerged when the silencings were compared with cells grown at 33°C, 35°C, and 37°C. For one of the siRNAs, the silencing was increased at lower temperatures. For two siRNAs, no significant changes in silencing were observed with different temperatures. Two of the siRNAs were significantly influenced by temperature with little if any silencing occurring at the lowest temperature. These data indicate that siRNAs directed to tissues in the anterior chamber of the eye should be checked at temperatures lower than 37°C to determine their effectiveness.

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Analysis of mammalian gene function using small interfering RNAs

Cited by 8 publications

References 0 publications

Small interfering RNAs containing full 2′-O-methylribonucleotide-modified sense strands display Argonaute2/eIF2C2-dependent activity

Small interfering RNAs containing full 2′-O-methylribonucleotide-modified sense strands display Argonaute2/eIF2C2-dependent activity

Post-transcriptional Regulation of Endothelial Nitric-oxide Synthase by an Overlapping Antisense mRNA Transcript

The effect of temperature on gene silencing by siRNAs: Implications for silencing in the anterior chamber of the eye

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