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      Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.

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          Abstract

          RNA interference (RNAi) is the process of sequence-specific, post-transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The mediators of sequence-specific messenger RNA degradation are 21- and 22-nucleotide small interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer dsRNAs. Here we show that 21-nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells. Therefore, 21-nucleotide siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.

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          RNA interference is mediated by 21- and 22-nucleotide RNAs.

          S. Elbashir (2001)
          Double-stranded RNA (dsRNA) induces sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 21- and 22-nt RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3' ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA-protein complex.
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            Use of double-stranded RNA interference in Drosophila cell lines to dissect signal transduction pathways.

            J. Clemens, C. Worby, N Simonson-Leff … (2000)
            We demonstrate the efficacy of double-stranded RNA-mediated interference (RNAi) of gene expression in generating "knock-out" phenotypes for specific proteins in several Drosophila cell lines. We prove the applicability of this technique for studying signaling cascades by dissecting the well-characterized insulin signal transduction pathway. Specifically, we demonstrate that inhibiting the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) prevents the activation of the downstream ERK-A (MAPK). In contrast, blocking ERK-A expression results in increased activation of DSOR1. We also show that Drosophila AKT (DAKT) activation depends on the insulin receptor substrate, CHICO (IRS1-4). Finally, we demonstrate that blocking the expression of Drosophila PTEN results in the activation of DAKT. In all cases, the interference of the biochemical cascade by RNAi is consistent with the known steps in the pathway. We extend this powerful technique to study two proteins, DSH3PX1 and Drosophila ACK (DACK). DSH3PX1 is an SH3, phox homology domain-containing protein, and DACK is homologous to the mammalian activated Cdc42 tyrosine kinase, ACK. Using RNAi, we demonstrate that DACK is upstream of DSH3PX1 phosphorylation, making DSH3PX1 an identified downstream target/substrate of ACK-like tyrosine kinases. These experiments highlight the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the Drosophila genome sequencing project.
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              CpG methylation, chromatin structure and gene silencing-a three-way connection.

              A Razin (1998)
              The three-way connection between DNA methylation, gene activity and chromatin structure has been known for almost two decades. Nevertheless, the molecular link between methyl groups on the DNA and the positioning of nucleosomes to form an inactive chromatin configuration was missing. This review discusses recent experimental data that may, for the first time, shed light on this molecular link. MeCP2, which is a known methylcytosine-binding protein, has been shown to possess a transcriptional repressor domain (TRD) that binds the corepressor mSin3A. This corepressor protein constitutes the core of a multiprotein complex that includes histone deacetylases (HDAC1 and HDAC2). Transfection and injection experiments with methylated constructs have revealed that the silenced state of a methylated gene, which is associated with a deacetylated nucleosomal structure, could be relieved by the deacetylase inhibitor, trichostatin A. Thus, methylation plays a pivotal role in establishing and maintaining an inactive state of a gene by rendering the chromatin structure inaccessible to the transcription machinery.
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                Author and article information

                Journal
                Journal ID (iso-abbrev): Nature
                Title: Nature
                Publisher: Springer Science and Business Media LLC
                ISSN: 0028-0836
                ISSN (Print): 0028-0836
                Publication date (Electronic): May 24 2001
                Volume: 411
                Issue: 6836
                Affiliations
                [1 ] Department of Cellular Biochemistry, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
                Article
                Publisher Item ID: 35078107
                DOI: 10.1038/35078107
                PubMed ID: 11373684
                SO-VID: 91299b7d-0a6c-481f-a3c0-88d2917c9511
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