Abstract
BackgroundIdentifying the endogenous RNA induced silencing complex(RISC)-associated RNAs is essential for understanding the cellular regulatory networks by miRNAs. Recently, isolation of RISC-associated mRNAs using antibody was reported, but their method needs a large amount of initial materials. We tried to improve the protocol and constructed an efficient and convenient system for analyzing miRNA and mRNA contents in RISC.FindingsWith our protocol, it is possible to clone both miRNAs and mRNAs from the endogenous RISC-associated RNAs immunoprecipitated from less than 107 cells, and we show the ability of our system to isolate the particular target mRNAs for a specific miRNA from the RISC-associated mRNAs using well-characterized miR-122 as an example. After introduction of miR-122 into HepG2 cells, we found several cDNA clones that have miR-122 target sequences. Four of these clones that were concentrated in RISC but decreased in total RNA fraction are expected to be miR-122 target candidates. Interestingly, we found substantial amounts of Alu-related sequences, including both free Alu RNA and Alu-embedded mRNA, which might be one of the general targets for miRNA, in the cDNA clones from the RISC-associated mRNAs.ConclusionOur method thus enables us to examine not only dynamic changes in miRNA and mRNA contents in RISC but also the relationship of miRNA and target mRNA. We believe that our method can contribute to understanding cellular regulatory networks by miRNAs.
Highlights
Identifying the endogenous RNA induced silencing complex(RISC)-associated RNAs is essential for understanding the cellular regulatory networks by miRNAs
We believe that our method can contribute to understanding cellular regulatory networks by miRNAs
After being transcribed and processed, mature miRNAs are incorporated into the Argonaute protein family, the core component of the RNA-induced silencing complex (RISC), for targeting mRNAs based on sequence complementation in 3'UTRs [2,3,4]
Summary
MicroRNAs (miRNAs) are approximately 22-nucleotide endogenous non-coding RNAs that play important roles in post-transcriptional regulation of gene expression by base-pairing to their target mRNAs [1]. After being transcribed and processed, mature miRNAs are incorporated into the Argonaute protein family, the core component of the RNA-induced silencing complex (RISC), for targeting mRNAs based on sequence complementation in 3'UTRs [2,3,4]. The Argonaute family consists of eight members, divided into the Ago subfamily (Ago1-Ago4) and Piwi subfamily (PIWIL1-PIWIL4) [5]. BMC Research Notes 2009, 2:169 http://www.biomedcentral.com/1756-0500/2/169 that Ago is the central RISC component, capable of cleaving target mRNA when perfect complementarity with it exists [7,8,9,10,11,12]. Isolation of RISC-associated mRNA has mainly been performed using exogenously introduced Tagged-Ago or GW182 in combination with antibodies to the Tag [1821], and the protocol needs improvement for the efficient analysis of the RISC-associated mRNA without using large amounts of initial materials [17]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
