A useful approach to total analysis of RISC-associated RNA

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.

Structure-function analysis of Methanobacterium thermoautotrophicum RNA ligase – engineering a thermostable ATP independent enzyme

“…These structures may lead to a significant reduction in ligation efficiency for certain RNAs. Thermostable phage T2126 ligase might be used in these applications [17,18], but not in the ATP independent ligation.…”

Identification of MIWI-associated Poly(A) RNAs by immunoprecipitation with an anti-MIWI monoclonal antibody

“…We previously established a cDNA cloning method utilizing an adaptor ligation to identify poly(A) RNA in immunoprecipitated products, and succeeded in identifying target mRNA candidates of miRNAs in the AGO2-IP products of HeLa cells and HepG2 cells (31). In order to examine whether poly(A) RNAs are contained in the MIWI-IP and AGO2-IP products of adult mouse testes, we performed cDNA cloning of poly(A) RNAs in both products using the same method.…”

“…cDNA synthesis and PCR amplification were performed using a Target mRNA Cloning Kit (Wako, Osaka, Japan) according to the manufacturer's instructions and reference (31). PCR-amplified cDNA fragments were subcloned into the pGEM-T easy vector (Promega), and sequenced using a BigDye Terminator Cycle Sequencing Kit (Life Technologies).…”

Fascin Regulates TLR4/PKC-mediated Translational Activation Through miR-155 and miR-125b, which Targets the 3′ Untranslated Region of TNF-α mRNA

“…Transfection of siRNA was performed using DharmaFECT (Dharmacon Inc., Lafayette, CO, USA) according to a previously described method . The procedure used for measurement of miR-155 and miR-125b expression levels was based on a previously described method (Hayashida et al, 2009) and performed using an Ncode VILO miRNA cDNA synthesis kit purchased from Life Technologies (Grand Island, NY, USA).…”