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Autor     Stephan Geley, Christiane Müller
Titel    RNAi: ancient mechanism with a promising future
Zeitschrift    Experimental Gerontology
Verlag    Elsevier
Ausgabe    39
Jahr    2004
Seiten    985-998
DOI    10.1016/j.exger.2004.03.040
URL    http://www.sciencedirect.com/science/article/pii/S0531556504001445

Literaturverz.   

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Fragmente    3


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[1.] Ww/Fragment 007 22 - Diskussion
Zuletzt bearbeitet: 2014-10-29 17:07:02 Hindemith
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A major breakthrough in the elucidation of the underlying mechanism was the biochemical analysis of RNAi using Drosophila embryo or cell extracts (Tuschl et al., 1999; Hammond et al., 2000; Zamore et al., 2000), which led to the identification of the dsRNA processing enzyme Dicer (Bernstein et al., 2001a) as well as the RNA induced silencing complex, RISC (Hammond et al., 2000), which executes RNAi by using the small dsRNA species generated by Dicer as guidance molecules to target the homologous, endogenous mRNA for [degradation (Elbashir et al., 2001b,c; Zamore et al., 2000).] A major breakthrough in the elucidation of the underlying mechanism was the biochemical analysis of RNAi using Drosophila embryo or cell extracts (Hammond et al., 2000; Tuschl et al., 1999; Zamore et al., 2000), which led to the identification of the dsRNA processing enzyme Dicer (Bernstein et al., 2001a) as well as the RNA induced silencing complex, RISC (Hammond et al., 2000), which executes RNAi by using the small dsRNA species generated by Dicer as guidance molecules to target the homologous, endogenous mRNA for degradation (Elbashir et al., 2001b,c; Zamore et al., 2000).
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[2.] Ww/Fragment 008 01 - Diskussion
Zuletzt bearbeitet: 2014-10-29 17:06:58 Hindemith
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These discoveries led to the rapid improvement of RNAi tools, tailored to the needs of the various experimental systems, and triggered intense genetic and biochemical research into the molecular basis and regulation of RNAi (Hammond et al., 2001b; Tijsterman et al., 2002). It became clear that RNAi is a highly conserved mechanism that functions in many different cellular pathways from regulating gene expression to fighting infection and the dangers of mobile genetic elements.

1.2.1 Mechanism of RNAi

The genetic and biochemical analysis of RNAi has led to a model, in which RNAi can be divided into two distinct phases: an initiation and an execution phase. The initiation phase involves the processing of dsRNA into siRNA. In the execution phase, siRNAs are then incorporated into large ribonucleoprotein complexes. These effector complexes interfere with gene expression by using the small RNA strand to identify their complementary mRNA, which becomes cleaved and degraded. In a related pathway, short non-coding single stranded RNAs, which are derived from partially complementary dsRNA precursor molecules, are used to regulate the translation of mRNAs harbouring complementary sequences in their 3’'UTRs (Fig. 1).

These discoveries led to the rapid improvement of RNAi tools, tailored to the needs of the various experimental systems, and triggered intense genetic and biochemical

[Seite 986]

research into the molecular basis and regulation of RNAi (Hammond et al., 2001b: Tijsterman et al., 2002). It became clear that RNAi is a highly conserved mechanism that functions in many different cellular pathways from regulating gene expression to fighting infection and the dangers of mobile genetic elements.

[...]

2. The RNAi mechanism

The genetic and biochemical analysis of RNAi has led to a model, in which RNAi can be divided into two distinct phases: an initiation and an execution phase. The initiation phase involves processing of dsRNA into small RNA molecules, called small interfering RNAs (siRNA). In the execution phase, siRNAs are then incorporated into large ribonucleoprotein complexes. These effector complexes interfere with gene expression by using the small RNA strand to identify their complementary mRNA, which becomes cleaved and degraded. In a related pathway, short non-coding single stranded RNAs, which are derived from partially complementary dsRNA precursor molecules, are used to regulate the translation of mRNAs harbouring complementary sequences in their 3' UTRs.

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[3.] Ww/Fragment 011 11 - Diskussion
Zuletzt bearbeitet: 2014-10-29 17:06:55 Hindemith
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1.2.1.2 Execution phase: assembly of siRNA containing silencing complexes

Dicer-generated siRNAs are then incorporated into a large multiprotein complex, which is involved in various gene-silencing modes, and is called the RNA induced silencing complex, or RISC (Hammond et al, 2000; Nykanen et al, 2001). Processing of dsRNA and assembly of a functional RISC likely occurs in the cytoplasm, as Dicer is a cytosolic enzyme and RISC activity can be purified from the cytosol (Billy et al, 2001). R2D2, a Drosophila gene related to the C. elegans RNAi gene RDE-4, has been implicated in the transfer of siRNAs into the RISC (Liu et al, 2003). Generation of siRNAs from dsRNA in Drosophila embryo extracts, unwinding of the siRNA duplex, and incorporation into the RISC requires ATP (Nykanen et al, 2001). In contrast, human Dicer does not seem to rely on ATP for processing of dsRNA into siRNA molecules (Zhang et al., 2002).

2.2. The execution phase: assembly of siRNA containing silencing complexes

Dicer-generated siRNAs are then incorporated into a large multiprotein complex, which is involved in various gene-silencing modes, and is called the RNA induced silencing complex, or RISC (Hammond et al., 2000; Nykanen et al., 2001). Processing of dsRNA and assembly of a functional RISC likely occurs in the cytoplasm, as Dicer is a cytosolic enzyme and RISC activity can be purified from cytosol (Billy et al., 2001). R2D2, a Drosophila gene related to the C. elegans RNAi gene RDE-4, has been implicated in the transfer of siRNAs into the RISC (Liu et al., 2003). Generation of siRNAs from dsRNA in Drosophila embryo extracts, unwinding of the siRNA duplex, and incorporation into the RISC require ATP (Nykanen et al., 2001). In contrast, human Dicer does not seem to require ATP for processing of dsRNA into siRNA molecules (Zhang et al., 2002).

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