Regulatory Mechanism of the RNAi Pathway

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Title: Regulatory Mechanism of the RNAi Pathway
Author: Liu, Ying
Abstract: RNA interference (RNAi ) is post -transcriptional gene silencing initiated by Dicer , a RNase III that processes double -stranded RNA (dsRNA ) precursors into small interfering RNA (siRNA ) . In Drosophila , Dicer2 and R2D2 coordinately recruit duplex siRNA to the effector RNA -induced silencing complex (RISC ) , wherein single -stranded siRNA guides the endoribonuclease Argonaute (Ago ) to catalyze sequence -specific cleavage of complementary mRNA . It remains unclear as to what constitutes holo -RISC , how is RISC assembled and how is RISC regulated . Here we took a candidate approach to reconstitute for the first time the long double -stranded RNA - and duplex siRNA -initiated RISC activities with the use of recombinant Drosophila Dicer -2 , R2D2 , and Ago2 proteins . We further employed this core reconstitution system to purify a RNAi regulator that we named C3PO (component 3 promoter of RISC ) , a complex of Translin and Trax . C3PO is a novel Mg2+ -dependent endoribonuclease that promotes RISC activation by removing the siRNA passenger strand cleavage products . Similar as Drosophila C3PO , human C3PO also degrades passenger strand fragments and facilitates RISC activation . RISC is a multiple -turnover enzyme , wherein single -stranded (ss ) -siRNA guides Ago2 to catalyze sequence -specific cleavage of the target mRNA at the effector step . We employed human minimal RISC reconstitution system to purify antoantigen La as a novel activator of the RISC effector step . Biochemical studies indicated that La promotes the multiple -turnover of RISC catalysis by facilitating the release of RISC cleaved products . Moreover , we demonstrated that La is required for efficient RNAi , antiviral defense , and transposon silencing in mammalian and Drosophila cells . Taken together , our findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2 -cleaved products to assemble or restore active RISC . The robust reconstitution system establishes a powerful platform for in -depth studies of the assembly , function , and regulation of RISC . Similar to the discovery of C3PO and La , it can be used to identify novel regulators and study post -translational regulations of RNAi , therefore , connecting RNAi to other cellular signaling pathways . As such , these biomedical studies could have a major and lasting impact on the biological understanding and therapeutic application of RNAi .
URI: http : / /hdl .handle .net /2152 .5 /947
Date: 2011-12-14


Regulatory Mechanism of the RNAi Pathway. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /947 .

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