Detection of the argonaute protein Ago2 and microRNAs in the RNA induced silencing complex (RISC) using a monoclonal antibody

Abstract

MicroRNAs (miRNAs) are short RNA molecules responsible for post-transcriptional gene silencing by the degradation or translational inhibition of their target messenger RNAs (mRNAs). This process of gene silencing, known as RNA interference (RNAi), is mediated by highly conserved Argonaute (Ago) proteins which are the key components of the RNA induced silencing complex (RISC). In humans, Ago2 is responsible for the endonuclease cleavage of targeted mRNA and it interacts with the mRNAbinding protein GW182, which is a marker for cytoplasmic foci referred to as GW bodies (GWBs). We demonstrated that the antiAgo2 monoclonal antibody 4F9 recognized GWBs in a cell cycle dependent manner and was capable of capturing miRNAs associated with Ago2. Since Ago2 protein is the effector protein of RNAi, anti-Ago2 monoclonal antibody may be useful in capturing functional miRNAs.

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

“…Interaction between GW182 and AGO-2 proteins is crucial for miRNA-mediated silencing and appears to take place directly after the passenger strand is removed by C3PO [96,97] . Both AGO-2 and GW182/TNRC6A have been shown to co-localize with siRNA and miRNAs within GW bodies (GWB) [98] , another term for P-bodies. Based on these observations, it has been proposed that silencing by miRNAs requires an effector complex formed of at least one AGO and one GW182/TNRC6A protein [99] ( Figure 1).…”

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

“…A key limitation in the study of Argonaute protein function is the lack of methods to purify RISC complexes assembled through natural pathways and that contain a single, unique guide sequence. Purification of RISC using antibodies against endogenous or epitope-tagged Argonautes allows the selection of specific Argonaute proteins, but these contain a complex mixture of siRNA and miRNA guides (Hutvágner and Zamore 2002;Mourelatos et al 2002;Liu et al 2004;Meister et al 2004a;Ikeda et al 2006;Beitzinger et al 2007;Azuma-Mukai et al 2008). RISC has also been purified using a guide strand with a 3 ′ biotin joined to the siRNA through a UV-sensitive linker, which was cleaved by photolysis (Martinez et al 2002;Martinez and Tuschl 2004;Ameres et al 2007) and using tethered siRNAs from which proteins were recovered with denaturing buffers (Gerbasi et al 2010).…”

MicroRNA: Mechanism of gene regulation and application to livestock1

“…For example, trinucleotide repeat containing 6A (GW182) and a dipeptidyl carboxypeptidase (Dcp1/Dcp2) decapping complex colocalize in P-bodies and bind to argonaute proteins (Rehwinkel et al, 2005;Behm-Ansmant et al, 2006;Ikeda et al, 2006). The GW182 protein is an RNA-binding protein, whereas the Dcp1/Dcp2 decapping complex allows for removal of the 5´ cap and degradation of the mRNA sequence (Rehwinkel et al, 2005;Behm-Ansmant et al, 2006;Ikeda et al, 2006). However, evidence also indicates that P-bodies may merely serve as a storage unit for mRNA, because disrupting the P-bodies does not alter the miRNA pathway or the degree of translational repression (Chu and Rana, 2006;Eulalio et al, 2007).…”