MABE253 Sigma-AldrichAnti-Ago2 Antibody, clone 11A9

Translation of Hepatitis C Virus (HCV) RNA is directed by an internal ribosome entry site (IRES) in the 5'-untranslated region (5'-UTR). HCV translation is stimulated by the liver-specific microRNA-122 (miR-122) that binds to two binding sites between the stem-loops I and II near the 5'-end of the 5'-UTR. Here, we show that Argonaute (Ago) 2 protein binds to the HCV 5'-UTR in a miR-122-dependent manner, whereas the HCV 3'-UTR does not bind Ago2. miR-122 also recruits Ago1 to the HCV 5'-UTR. Only miRNA duplex precursors of the correct length stimulate HCV translation, indicating that the duplex miR-122 precursors are unwound by a complex that measures their length. Insertions in the 5'-UTR between the miR-122 binding sites and the IRES only slightly decrease translation stimulation by miR-122. In contrast, partially masking the miR-122 binding sites in a stem-loop structure impairs Ago2 binding and translation stimulation by miR-122. In an RNA decay assay, also miR-122-mediated RNA stability contributes to HCV translation stimulation. These results suggest that Ago2 protein is directly involved in loading miR-122 to the HCV RNA and mediating RNA stability and translation stimulation.

Epstein-Barr virus (EBV) controls gene expression to transform human B cells and maintain viral latency. High-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) identified mRNA targets of 44 EBV and 310 human microRNAs (miRNAs) in Jijoye (Latency III) EBV-transformed B cells. While 25% of total cellular miRNAs are viral, only three viral mRNAs, all latent transcripts, are targeted. Thus, miRNAs do not control the latent/lytic switch by targeting EBV lytic genes. Unexpectedly, 90% of the 1664 human 3'-untranslated regions targeted by the 12 most abundant EBV miRNAs are also targeted by human miRNAs via distinct binding sites. Half of these are targets of the oncogenic miR-17∼92 miRNA cluster and associated families, including mRNAs that regulate transcription, apoptosis, Wnt signalling, and the cell cycle. Reporter assays confirmed the functionality of several EBV and miR-17 family miRNA-binding sites in EBV latent membrane protein 1 (LMP1), EBV BHRF1, and host CAPRIN2 mRNAs. Our extensive list of EBV and human miRNA targets implicates miRNAs in the control of EBV latency and illuminates viral miRNA function in general.

Recently identified small (20 to 40 bases) RNAs, such as microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs) participate in important cellular pathways. In this report, we systematically characterized several novel features of human and viral RNA products smaller than miRNAs. We found that Kaposi sarcoma-associated herpesvirus K12-1 miRNA (23 bases) associates with a distinct, unusually small (17-base) RNA (usRNA) that can effectively downregulate a K12-1 miRNA target, human RAD21, suggesting that stable degradation-like products may also contribute to gene regulation. High-throughput sequencing reveals a diverse set of human miRNA-derived usRNAs and other non-miRNA-derived usRNAs. Human miRNA-derived usRNAs preferentially match to 5' ends of miRNAs and are also more likely to associate with the siRNA effector protein Ago2 than with Ago1. Many non-miRNA-derived usRNAs associate with Ago proteins and also frequently contain C-rich 3'-specific motifs that are overrepresented in comparison to Piwi-interacting RNAs and transcription start site-associated RNAs. We postulate that approximately 30% of usRNAs could have evolved to participate in biological processes, including gene silencing.

Small regulatory RNAs including small interfering RNAs (siRNAs), microRNAs (miRNAs), or Piwi interacting RNAs (piRNAs) guide regulation of gene expression in many different organisms. The Argonaute (Ago) protein family constitutes the cellular binding partners of such small RNAs and regulates gene expression on the levels of transcription, mRNA stability, or translation. Due to the lack of highly specific and potent monoclonal antibodies directed against the different Ago proteins, biochemical analyses such as Ago complex purification and characterization rely on overexpression of tagged Ago proteins. Here, we report the generation and functional characterization of a highly specific monoclonal anti-Ago2 antibody termed anti-Ago2(11A9). We show that anti-Ago2(11A9) is specific for human Ago2 and detects Ago2 in Western blots as well as in immunoprecipitation experiments. We further demonstrate that Ago2 can be efficiently eluted from our antibody by a competing peptide. Finally, we show that anti-Ago2(11A9) recognizes Ago2 in immunofluorescence experiments, and we find that Ago2 not only localizes to cytoplasmic processing bodies (P-bodies) and the diffuse cytoplasm but also to the nucleus. With the anti-Ago2(11A9) antibody we have generated a potent tool that is useful for many biochemical or cell biological applications.