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Validation of shRNA clones for gene silencing in 293FT cells

von Wen Wang

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[1.] Ww/Fragment 004 01 - Diskussion
Zuletzt bearbeitet: 2014-10-28 05:09:55 Hindemith
Fragment, Gesichtet, SMWFragment, Schutzlevel sysop, Verschleierung, Wikipedia microRNA 2007, Ww

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SleepyHollow02
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Quelle: Wikipedia microRNA 2007
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[There are few possible explanations for] such selectivity. One could be that dsRNAs longer than 21 base pairs activate an interferon response and the anti-viral machinery in the cell. Another plausible explanation could be that the thermodynamical profile of

pre-miRNAs determines which strand will be incorporated into the Dicer complex. Indeed, the aforementioned study by Han et al. demonstrated very clear similarities between pri-miRNAs encoded in respective (5’- or 3’-) strands.

When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one becomes integrated into the RISC complex. This strand is known as the guide strand and is selected by the argonaute protein, the catalytically active RNase in the RISC complex, on the basis of the stability of the 5’ end (Preall et al, 2006). The remaining strand, known as the anti-guide or passenger strand is degraded as a RISC complex substrate (Gregory et al, 2005). After integration into the active RISC complex, miRNAs base pair with their complementary mRNA molecules and induce mRNA degradation by argonaute proteins. It is as yet unclear how the activated RISC complex locates the mRNA targets in the cell, though it has been shown that the process is not coupled to ongoing protein translation from the mRNA (Sen et al, 2005).

1.1.3.2 Cellular functions of miRNA

The miRNAs appear to be important for gene regulation. An individual miRNA is complementary to a part of one or more messenger RNAs (mRNAs). Animal miRNAs are usually complementary to a site in the 3’ UTR whereas plant miRNAs are usually complementary to coding regions of mRNAs. The annealing of the miRNA to the mRNA then inhibits protein translation, but sometimes facilitates cleavage of the mRNA. This is thought to be the primary mode of action of plant miRNAs. In such cases, the formation of the double-stranded RNA through the binding of the miRNA triggers the degradation of the mRNA transcript through a process similar to RNAi, though [in other cases it is believed that the miRNA complex blocks the protein translation machinery or otherwise prevents protein translation without causing the mRNA to be degraded.]

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. Another plausible explanation could be that thermodynamical profile of pre-miRNA determines which strand will be incorporated into Dicer complex. Indeed, aforementioned study by Han et al. demonstrated very clear similarities between pri-miRNAs encoded in respective (5'- or 3'-) strands.

When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNA molecules are formed, but only one is integrated into the RISC complex. This strand is known as the guide strand and is selected by the argonaute protein, the catalytically active RNase in the RISC complex, on the basis of the stability of the 5' end.[6] The remaining strand, known as the anti-guide or passenger strand, is degraded as a RISC complex substrate.[7] After integration into the active RISC complex, miRNAs base pair with their complementary mRNA molecules and induce mRNA degradation by argonaute proteins, the catalytically active members of the RISC complex. It is as yet unclear how the activated RISC complex locates the mRNA targets in the cell, though it has been shown that the process is not coupled to ongoing protein translation from the mRNA.[8]

Cellular functions

The function of miRNAs appears to be in gene regulation. For that purpose, a miRNA is complementary to a part of one or more messenger RNAs (mRNAs). Animal miRNAs are usually complementary to a site in the 3' UTR whereas plant miRNAs are usually complementary to coding regions of mRNAs. The annealing of the miRNA to the mRNA then inhibits protein translation, but sometimes facilitates cleavage of the mRNA. This is thought to be the primary mode of action of plant miRNAs. In such cases, the formation of the double-stranded RNA through the binding of the miRNA triggers the degradation of the mRNA transcript through a process similar to RNA interference (RNAi), though in other cases it is believed that the miRNA complex blocks the protein translation machinery or otherwise prevents protein translation without causing the mRNA to be degraded.


6. Preall JB, He Z, Gorra JM, Sontheimer EJ. (2006). Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila. Curr Biol 16(5):530-5.

7. Gregory RI, Chendrimada TP, Cooch N, Shiekhattar R. (2005). Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123(4):631-40.

8. Sen GL, Wehrman TS, Blau HM. (2005). mRNA translation is not a prerequisite for small interfering RNA-mediated mRNAs cleavage. Differentiation 73(6):287-93.

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