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Validation of shRNA clones for gene silencing in 293FT cells

von Dr. Wen Wang

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[1.] Ww/Fragment 003 01 - Diskussion
Zuletzt bearbeitet: 2014-10-28 05:08:22 Hindemith
Fragment, Gesichtet, SMWFragment, Schutzlevel sysop, Verschleierung, Wikipedia microRNA 2007, Ww

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SleepyHollow02
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Seite: 3, Zeilen: 1ff (komplett)
Quelle: Wikipedia microRNA 2007
Seite(n): 1 (Internetquelle), Zeilen: -
[This] processing is performed in animals by a protein complex known as the Microprocessor complex, consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha (Denli et al, 2004). These pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC) (Bernstein et al, 2001). This complex is responsible for the gene silencing observed due to miRNA expression and RNA interference. The pathway in plants varies slightly due to the lack of Drosha homologs; instead, Dicer homologs alone affect several processing steps (Kurihara and Watanabe, 2004).

It has been shown that the efficient processing of pre-miRNAs by Drosha requires the presence of extended single-stranded RNAs on both 3’- and 5’-ends of hairpin molecules (Zeng et al, 2005). This study showed that these motifs could be of different composition while their defined length is of high importance for processing to take place. Findings were confirmed in another work by Han et al (2004). Using bioinformatics tools the folding of 321 human and 68 fly pri-miRNAs was analysed. 280 human and 55 fly pri-miRNAs were selected for further study excluding those molecules where folding showed the presence of multiple loops. All human and fly pri-miRNAs contained very similar structural regions, which the authors called ‘’basal segments’’, ‘’lower stem’’, ’’upper stem’’ and ‘’terminal loop’’. Based on the encoding position of miRNAs, in the 5’-strand (5’-donors) or 3’-strand (3’-donors), thermodynamic profiles of pri-miRNAs were determined (Zeng et al, 2005). Subsequent experiments showed that Drosha complex cleaves RNA molecules ~2 helical turns away from the terminal loop and ~1 turn away from basal segments. In most analyzed molecules this region contains unpaired nucleotides and the free energy of the duplex is relatively high compared to lower and upper stem regions (Zeng and Cullen, 2005).

Most pre-miRNAs don’t have a perfect double-stranded RNA (dsRNA) structure topped by a terminal loop. There are few possible explanations for [such selectivity.]

This processing is performed in animals by a protein complex known as the Microprocessor complex, consisting of the nuclease Drosha and the double-stranded RNA binding protein Pasha.[3] These pre-miRNAs are then processed to mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC).[4] This complex is responsible for the gene silencing observed due to miRNA expression and RNA interference. The pathway in plants varies slightly due to their lack of Drosha homologs; instead, Dicer homologs alone effect several processing steps.[5]

Zeng et al. have shown that efficient processing of pre-miRNA by Drosha requires presence of extended single-stranded RNA on both 3'- and 5'-ends of hairpin molecule. They demonstrated that these motifs could be of different composition while their length is of high importance if processing is to take place at all. Their findings were confirmed in another work by Han et al. Using bioinformatical tools Han et al. analysed folding of 321 human and 68 fly pri-miRNAs. 280 human and 55 fly pri-miRNAs were selected for further study, excluding those molecules which folding showed presence of multiple loops. All human and fly pri-miRNA contained very similar structural regions, which authors called 'basal segments', 'lower stem', 'upper stem' and 'terminal loop'. Based on the encoding position of miRNA, i.e. in the 5'-strand (5'-donors) or 3'-strand (3'-donors), thermodynamical profiles of pri-miRNA were determined. Following experiments have shown that Drosha complex cleaves RNA molecule ~2 helical turns away from the terminal loop and ~1 turn away from basal segments. In most analysed molecules this region contains unpaired nucleotides and the free energy of the duplex is relatively high compared to lower and upper stem regions.

Most pre-miRNAs don't have a perfect double-stranded RNA (dsRNA) structure topped by a terminal loop. There are few possible explanations for such selectivity. One could be that dsRNAs longer than 21 base pairs activate interferon response and anti-viral machinery in the cell.


3. Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ. (2004). Nature 432(7014):231-5.

4. Bernstein E, Caudy AA, Hammond SM, Hannon GJ. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409(6818):363-6.

5. Kurihara Y, Watanabe Y. (2004). Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions. Proc Natl Acad Sci USA 101(34):12753-8.

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(SleepyHollow02), Hindemith


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