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Validation of shRNA clones for gene silencing in 293FT cells

von Wen Wang

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[1.] Ww/Fragment 010 01 - Diskussion
Zuletzt bearbeitet: 2016-01-20 18:13:48 Schumann
Fragment, Gesichtet, Hammond 2005, SMWFragment, Schutzlevel sysop, Verschleierung, Ww

Typus
Verschleierung
Bearbeiter
SleepyHollow02
Gesichtet
Yes
Untersuchte Arbeit:
Seite: 10, Zeilen: 1 ff. (komplett)
Quelle: Hammond 2005
Seite(n): 5823 f, Zeilen: 5823: l.col. 30 ff.; r.col. 4 ff.; 5824 l.col. last lines
[Class III enzymes are further characterized] by a helicase domain and a PAZ (Piwi/Argonaute/Zwille) domain. This last domain is also present in Argonaute family proteins, already known to be essential for RNAi, which led to the proposal that Class III enzymes are the initiator of RNAi (Bass, 2000).

The generation of a siRNA from dsRNA potentially requires four endonucleolytic reactions. It has been revealed that Dicer acts as a monomer, using two endonucleolytic reactions to generate one new terminus (Zhang et al, 2004). This would occur if Dicer binds to an existing terminus and makes a cut ~21 nucleotides from the end (Schütze, 2004). If however the enzyme can not initiate processing from the end and is forced to cut internally, the reaction becomes significantly delayed. Once binding of Dicer occurred and a single new terminus is created, further processing occurrs at normal rates, since the enzyme now has terminal ends from which to process.

MicroRNAs (miRNAs) are transcribed by RNA polymerase II as long primary transcripts (Kim, 2005). The active miRNA species, termed the mature RNA, is present in a stem–loop structure within the primary transcript. The stem–loop can be located in an exon or in an intron. For example, the miRNAs miR-106b, miR-93, and miR-25 are located within an intron of the protein coding gene mcm-7. After transcription, the miRNAs are processed from the primary transcript, and the spliced mRNA is exported and translated . Sequential processing of the primary transcript by the RNase III enzymes Drosha and Dicer liberates the mature RNA. Drosha cleavage releases the stem–loop, termed the precursor, which is exported from the nucleus in an exportin-5/RAN-GTPase-dependent manner. In the cytoplasm, the precursor is processed into a siRNA-like structure by Dicer. Drosha generates a 2 nt 3’ overhang terminus on the precursor which is recognized by the PAZ domain of Dicer, analogous to the recognition of dsRNA termini. The double stranded miRNA is incorporated into RISC in a similar manner as siRNAs.

Drosha is a Class II enzyme. This enzyme assumes a pseudo-dimer catalytic [core similar to Dicer (Han et al, 2004).]

Class III enzymes are further characterized by a helicase domain and a PAZ (Piwi/Argonaute/Zwille) domain. This last domain is also present in Argonaute family proteins, already known to be essential for RNAi, which led to the proposal that Class III enzymes are the initiator of RNAi [12,13]. [...]

The generation of an siRNA from dsRNA potentially requires four endonucleolytic reactions. How does Dicer achieve this? Early models were based on the prediction that Dicer forms a dimer on the substrate and performs four cleavage reactions [15]. Recent data, however, favors a model whereby Dicer acts as a monomer, using two endonucleolytic reactions to generate one new terminus [16,17]. This would occur if Dicer bound to an existing terminus and made a cut ~21 nucleotides from the end. This was first suggested by studies using dsRNA substrates with blocked termini [16]. If the enzyme could not initiate processing from the end and was forced to process internally, the reaction was significantly delayed. The authors' interpretation was that internal binding was less efficient and caused a lag in processing. Once binding occurred and a single new terminus was created, further processing occurred at normal rates, since the enzyme now had terminal ends from which to process.

[Seite 5824]

MicroRNAs are transcribed from RNA polymerase II as long primary transcripts (see [26] for a review). The active microRNA species, termed the mature RNA, is present in a stem–loop structure within the primary transcript. The stem–loop can be located in an exon or an intron. For example, the microRNAs miR-106b, miR-93, and miR-25 are located within an intron of the protein coding gene mcm-7. After transcription, the microRNAs are processed out of the primary transcript, and the spliced mRNA is exported and translated. Whether the microRNA is processed before, during, or after splicing is not known. Sequential processing of the primary transcript by the RNaseIII enzymes Drosha and Dicer liberates the mature RNA. Drosha cleavage releases the stem–loop, termed the precursor, which is exported from the nucleus in an Exportin-5/RAN-GTPase-dependent manner. In the cytoplasm, the precursor is processed into a siRNA-like structure by Dicer. Drosha generates a 2 nt 30 overhang terminus on the precursor which is recognized by the PAZ domain of Dicer, analogous to the recognition of dsRNA termini. The doublestranded microRNA is incorporated into RISC in a similar manner as siRNAs.

Drosha is a Class II enzyme as shown in Fig. 1. This enzyme assumes a pseudo-dimer catalytic core similar to Dicer [27].


[12] Tabara, H., Sarkissian, M., Kelly, W.G., Fleenor, J., Grishok, A., Timmons, L., Fire, A. and Mello, C.C. (1999) The rde-1 gene, RNA interference, and transposon silencing in C. elegans. Cell 99, 123–132.

[13] Bass, B.L. (2000) Double-stranded RNA as a template for gene silencing. Cell 101, 235–238.

[14] Bernstein, E., Caudy, A.A., Hammond, S.M. and Hannon, G.J. (2001) Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366.

[15] Zamore, P.D. (2001) Thirty-three years later, a glimpse at the ribonuclease III active site. Mol. Cell 8, 1158–1160.

[16] Zhang, H., Kolb, F.A., Brondani, V., Billy, E. and Filipowicz, W. (2002) Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. EMBO J. 21, 5875–5885.

[17] Zhang, H., Kolb, F.A., Jaskiewicz, L., Westhof, E. and Filipowicz, W. (2004) Single processing center models for human Dicer and bacterial RNase III. Cell 118, 57–68.

[26] Kim, V.N. (2005) MicroRNA biogenesis: coordinated cropping and dicing. Nat. Rev. Mol. Cell. Biol. 6, 376–385.

[27] Han, J., Lee, Y., Yeom, K.H., Kim, Y.K., Jin, H. and Kim, V.N. (2004) The Drosha-DGCR8 complex in primary microRNA processing. Genes Dev. 18, 3016–3027.

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Sichter
(SleepyHollow02), Hindemith



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Letzte Bearbeitung dieser Seite: durch Benutzer:Hindemith, Zeitstempel: 20141029060534