C3PO, an Endoribonuclease That Promotes RNAi by Facilitating RISC Activation

Liu, Ying; Ye, Xuecheng; Jiang, Feng; Liang, Chunyang; Chen, Dongmei; Peng, Junmin; Kinch, Lisa N.; Grishin, Nick V.; Liu, Qinghua

doi:10.1126/science.1176325

Cited by 229 publications

(269 citation statements)

References 19 publications

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“…Our mass spectrometry analyses support this view, because we did not find proteins that participate in small RNA biogenesis or RISC assembly. We detected no peptides from Dicer-1, Dicer-2, R2D2, Loquacious, or C3PO (Jiang et al 2005;Liu et al 2006Liu et al , 2007Liu et al , 2009) copurifying with Drosophila Ago1-or Ago2-RISC, nor did we detect Dicer, TRBP, or PACT (Hutvágner et al 2001;Chendrimada et al 2005;Haase et al 2005;Lee et al 2006) copurifying with mouse AGO2-RISC (Supplemental Fig. S3; Supplemental Tables S1-S3).…”

Section: Discussionmentioning

confidence: 91%

Rapid and specific purification of Argonaute-small RNA complexes from crude cell lysates

Flores-Jasso

Salomon

Zamore

2012

RNA

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Small interfering RNAs (siRNAs) direct Argonaute proteins, the core components of the RNA-induced silencing complex (RISC), to cleave complementary target RNAs. Here, we describe a method to purify active RISC containing a single, unique small RNA guide sequence. We begin by capturing RISC using a complementary 2 ′ -O-methyl oligonucleotide tethered to beads. Unlike other methods that capture RISC but do not allow its recovery, our strategy purifies active, soluble RISC in good yield. The method takes advantage of the finding that RISC partially paired to a target through its siRNA guide dissociates more than 300 times faster than a fully paired siRNA in RISC. We use this strategy to purify fly Ago1-and Ago2-RISC, as well as mouse AGO2-RISC. The method can discriminate among RISCs programmed with different guide strands, making it possible to deplete and recover specific RISC populations. Endogenous microRNA:Argonaute complexes can also be purified from cell lysates. Our method scales readily and takes less than a day to complete.

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Section: Discussionmentioning

confidence: 91%

Rapid and specific purification of Argonaute-small RNA complexes from crude cell lysates

Flores-Jasso

Salomon

Zamore

2012

RNA

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“…After nicking, the endonuclease component 3 promoter of RISC complex (C3PO), composed of Trax and Translin proteins in humans, is able to cleave and remove the passenger strand [87,90,91] . This results in activation of pre-RISC into the RISC complex.…”

Section: Ptgsmentioning

confidence: 99%

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

Méndez

2015

WJV

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“…Following AGO2 loading, the duplex strand with the less stable 5′ terminus (the guide strand) is selectively retained, whereas the complementary (passenger) strand is cleaved by the slicer activity of AGO2, a process known as passenger-strand nicking (19)(20)(21)(22). The siRNA duplex is subsequently unwound, and the sliced passenger strand is removed by the C3PO endoribonuclease complex (23), resulting in the formation of the mature RISC that contains the siRNA guide strand. Finally, target mRNAs are recruited to mature RISC by perfectly complementary base pairing with the siRNA guide strand and cleaved (sliced) by AGO2 (24,25).…”

mentioning

confidence: 99%

Core small nuclear ribonucleoprotein particle splicing factor SmD1 modulates RNA interference in Drosophila

Xiong

Kurthkoti

Chang

et al. 2013

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

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RNAi is an evolutionarily conserved gene regulatory process that operates in a wide variety of organisms. During RNAi, long doublestranded RNA precursors are processed by Dicer proteins into ∼21-nt siRNAs. Subsequently, siRNAs are incorporated into the RNAinduced silencing complexes (RISCs) that contain Argonaute-family proteins and guide RISC to target RNAs via complementary base pairing, leading to posttranscriptional gene silencing. Select premRNA splicing factors have been implicated in RNAi in fission yeast, worms, and flies, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle implicated in splicing, is required for RNAi and antiviral immunity in cultured cells and in vivo. SmD1 interacts with both Dicer-2 and dsRNA precursors and is indispensable for optimal siRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the small interfering RISC without significantly affecting the expression of major canonical siRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the small interfering RISC, including Argonaute 2, both in flies and in humans. Notably, RNAi defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the RNAi and splicing machineries are physically and functionally distinct entities. Our results suggest that Drosophila SmD1 plays a direct role in RNAi-mediated gene silencing independently of its premRNA splicing activity and indicate that the dual roles of splicing factors in posttranscriptional gene regulation may be evolutionarily widespread. RNAi is an ancient gene regulatory process (1). RNAi is triggered by ∼21-to 24-nt double-stranded siRNAs and microRNAs (miRNAs) (2-5), which are assembled into RNAinduced silencing complexes (RISCs). Once incorporated into the RISC, one strand of the siRNA or miRNA engages target mRNAs via complementary base pairing and modulates gene expression in a sequence-specific manner. RNAi generally represses gene expression posttranscriptionally by inhibiting translation and/ or promoting destabilization of target mRNAs, or transcriptionally by modulating chromatin structure at the target loci. By regulating the expression of diverse target RNAs, RNAi plays a key role in numerous biological processes, including cancer, development, homeostasis, and antiviral defense.In Drosophila melanogaster, siRNAs are generated from long dsRNA precursors that are either transcribed endogenously or introduced exogenously (6-10). Long dsRNAs are processed into duplex siRNAs by the ribonuclease III Dicer-2 (Dcr-2), which forms a stable complex with the cofactors loquacious (Loqs)-PD and R2D2 (11)(12)(13)(14). The formation of mature RISC is a multistep process. First, the Dcr-2/R2D2 heterodimer gauges the thermodynamic stability of the ends of the siRNA duplex and associates with duplex siRNAs in a polarized pattern to form the RISC-loading complex (RLC) (15, 16). Several othe...

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C3PO, an Endoribonuclease That Promotes RNAi by Facilitating RISC Activation

Cited by 229 publications

References 19 publications

Rapid and specific purification of Argonaute-small RNA complexes from crude cell lysates

Rapid and specific purification of Argonaute-small RNA complexes from crude cell lysates

Post-transcriptional gene silencing, transcriptional gene silencing and human immunodeficiency virus

Core small nuclear ribonucleoprotein particle splicing factor SmD1 modulates RNA interference in Drosophila

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