Cleavage of the siRNA passenger strand during RISC assembly in human cells

Leuschner, Philipp J.F.; Ameres, Stefan L.; Kueng, Stephanie; Martı́nez, Javier

doi:10.1038/sj.embor.7400637

Cited by 350 publications

(295 citation statements)

References 27 publications

Supporting

Mentioning

285

Contrasting

Order By: Relevance

“…The PAZ domain is involved in the binding of siRNA (Song et al 2003), whereas the PIWI domain acts as the nuclease to cleave mRNA substrates when guided by single-stranded siRNA (Liu et al 2004;Rand et al 2004;Song et al 2004;Rivas et al 2005). Previous biochemical studies have suggested a model in which the Argonautes are also responsible for the generation of single-stranded siRNA through cleavage of the passenger strand of siRNA duplex (Matranga et al 2005;Miyoshi et al 2005;Rand et al 2005;Leuschner et al 2006). Here, we presented strong in vivo evidence to support this model.…”

Section: Qde-2 Is Required For the Cleavage Of The Sirna Passenger Stsupporting

confidence: 60%

See 1 more Smart Citation

QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands

Maiti¹,

Lee²,

Liu³

2007

Genes Dev.

114

149

View full text Add to dashboard Cite

Single-stranded small interfering RNA (siRNA) guides the cleavage of homologous mRNA by Argonaute proteins, the catalytic core of the RNA-induced silencing complex (RISC), in the conserved RNA interference (RNAi) pathway. The separation of the siRNA duplex into single strands is essential for the activation of RISC. Previous biochemical studies have suggested that Argonaute proteins cleave and remove the passenger strand of siRNA duplex from RISC, but the in vivo importance of this process and the mechanism for the removal of the nicked passenger strand are not known. Here, we show that in the filamentous fungus Neurospora, the Argonaute homolog QDE-2 and its slicer function are required for the generation of single-stranded siRNA and gene silencing in vivo. Biochemical purification of QDE-2 led to the identification of QIP, a QDE-2-interacting protein, with an exonuclease domain. The disruption of qip in Neurospora impaired gene silencing and siRNA accumulated, mostly in nicked duplex form. Furthermore, our results suggest that QIP acts as an exonuclease that cleaves and removes the nicked passenger strand from siRNA duplex in a QDE-2-dependent manner. Together, these results suggest that both the cleavage and removal of the passenger strand from the siRNA duplex are important steps in RNAi pathways.[Keywords: RNAi; Neurospora; Argonaute; siRNA; exonuclease; RISC] Supplemental material is available at http://www.genesdev.org.

show abstract

Section: Qde-2 Is Required For the Cleavage Of The Sirna Passenger Stsupporting

confidence: 60%

“…The identity of such a factor(s), if it exists, is not known. A similar conclusion was also drawn in human cells and it was demonstrated that the introduction of a nick in the passenger strand can bypass the requirement of passenger strand cleavage by RISC (Leuschner et al 2006).…”

mentioning

confidence: 54%

QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands

Maiti¹,

Lee²,

Liu³

2007

Genes Dev.

114

149

View full text Add to dashboard Cite

show abstract

“…The composition and phosphorylation status of the 5′ nucleotide of small RNA duplexes influences their binding by the different Agos (14,15). In mammalian cells, several studies have shown that miRNAs are similarly associated with four human Agos (7,8,16), suggesting the absence of a specific sorting mechanism.Similar to what has been reported in flies (12,(17)(18)(19)(20), mammalian Ago loading is a stepwise process (21,22). First, small RNAs associate with Agos as a duplex with the help of chaperon proteins and consume ATP, presumably like their Drosophila homologs (23, 24).…”

mentioning

confidence: 56%

Thermodynamic stability of small hairpin RNAs highly influences the loading process of different mammalian Argonautes

Jin

Zhang

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

MicroRNAs and siRNAs interact with target sequences in mRNAs, inducing cleavage-and non-cleavage-based gene repression through the RNA-induced silencing complex (RISC) that consists of one of four mammalian Argonaute proteins, Ago1-Ago4. The process of how Dicer substrate small hairpin RNAs (shRNAs) are loaded into different mammalian Agos in vivo is not well established. Here we report that shRNAs are loaded into mammalian Agos in two stepwise processes, physical association and activation, with the latter being the rate-limiting step with noncleaving RISC. We establish that, although RNA duplexes processed from shRNAs bind to Agos in cells with similar affinity, the degree by which the complexes are activated (coupled with the removal of the passenger strand) correlates with the thermodynamic instability of RNA duplexes being loaded rather than the structure of the RNA, as was previously demonstrated in Drosophila. Interestingly, Ago loading of siRNAs is less sensitive to thermostability than that of their shRNA equivalents. These results may have important implications for the future design of RNAi-based therapeutics.RNA interference | gene silencing M icroRNAs (miRNAs) and 21-to 23-nt siRNAs regulate gene expression in almost all eukaryotic organisms. Small RNA-mediated gene regulation is sequence-specific and believed to regulate at least one-half of all protein-encoding mRNAs (reviewed in refs. 1-4).Argonaute proteins (Agos), which directly associate with small RNAs, are the core of all known RNA-induced silencing complexes (RISCs). These proteins are functionally specialized into distinct RNA silencing pathways. In Drosophila, Ago2-RISC is involved in target mRNA cleavage, and Ago1-RISC mediates translational repression (5, 6). The human genome encodes four Ago-like subfamily proteins (Ago1-Ago4) that have redundant functions as negative regulators of gene expression, but only Ago2 has cleavage activity (7-9).In Drosophila, siRNAs and miRNAs are actively sorted into functionally distinct Ago-RISCs based on differences in structure rather than on their biogenesis (10, 11). Perfectly matched duplexes (siRNA-like) are preferentially incorporated into Ago2, and the loading is achieved by cleaving the passenger strand, whereas duplexes with central mismatched bulges (miRNA-like) are sorted to Ago1, and the unselected strand (asterisk strand) is eliminated through a "bypass" pathway, which is sensitive to the structure of the small RNA duplex and independent of the weak cleavage activity of Ago1 (12). A similar sorting mechanism exists in Caenorhabditis elegans, whereby small RNA duplexes with perfectly matched or bulged stems are channeled into RDE-1 and ALG-1, respectively (13). In plants, a different sorting mechanism has been reported. The composition and phosphorylation status of the 5′ nucleotide of small RNA duplexes influences their binding by the different Agos (14,15). In mammalian cells, several studies have shown that miRNAs are similarly associated with four human Agos (7,8,16), suggesting the abse...

show abstract

“…In spite of the fact, that the recombinant hAgo2 are not capable of unwinding short RNA duplexes, genetic data in flies suggest that Ago2 is indispensable for this process (Okamura et al, 2004). In fact, it has been recently demonstrated that the cleavage competent fly and the human Ago2 actively participate in the unwinding mechanism by cleaving the passenger strand of the perfectly double-stranded siRNA (Matranga et al, 2005;Miyoshi et al, 2005;Leuschner et al, 2006).…”

Section: Risc Formationmentioning

confidence: 99%

Principles and effects of microRNA-mediated post-transcriptional gene regulation

2006

View full text Add to dashboard Cite

MicroRNAs (miRNAs) are abundant regulatory RNAs involved in the regulation of many key biological processes. Recent advances in understanding the mechanism of RNA interference and miRNA-mediated mechanisms shed light on major principals of the formation of the regulatory complex and provide models to explain how these small regulatory RNA species interfere with gene expression and how they influence the translational status of the transcriptome.

show abstract

Cleavage of the siRNA passenger strand during RISC assembly in human cells

Cited by 350 publications

References 27 publications

QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands

QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands

Thermodynamic stability of small hairpin RNAs highly influences the loading process of different mammalian Argonautes

Principles and effects of microRNA-mediated post-transcriptional gene regulation

Contact Info

Product

Resources

About