{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T04:37:53Z","timestamp":1760243873475,"version":"build-2065373602"},"reference-count":17,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2010,2,12]],"date-time":"2010-02-12T00:00:00Z","timestamp":1265932800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/3.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>RNA interference (RNAi) is a post-transcriptional gene-silencing process that occurs in many eukaryotic organisms upon intracellular exposure to double-stranded RNA. Argonaute 2 (Ago2) protein is the catalytic engine of mammalian RNAi. It contains a PIWI domain that is structurally related to RNases H and possibly shares with them a two-metal-ion catalysis mechanism. Here we describe the expression in E. coli of mouse Ago2 and testing of its enzymatic activity in a RISC assay, i.e., for the ability to cleave a target RNA in a single position specified by a complementary small interfering RNA (siRNA). The results show that the enzyme can load the siRNA and cleave the complementary RNA in absence of other cellular factors, as described for human Ago2. It was also found that mutation of Arg669, a residue previously proposed to be involved in substrate and\/or B metal ion binding, doesn\u2019t affect the enzymatic activity, suggesting that this residue doesn\u2019t belong to the active site.<\/jats:p>","DOI":"10.3390\/ijms11020745","type":"journal-article","created":{"date-parts":[[2010,2,16]],"date-time":"2010-02-16T08:28:43Z","timestamp":1266308923000},"page":"745-753","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Bacterial Expression of Mouse Argonaute 2 for Functional and Mutational Studies"],"prefix":"10.3390","volume":"11","author":[{"given":"Vincenzo","family":"Salvatore","sequence":"first","affiliation":[{"name":"Department of Life Sciences, Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicoletta","family":"Potenza","sequence":"additional","affiliation":[{"name":"Department of Life Sciences, Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Umberto","family":"Papa","sequence":"additional","affiliation":[{"name":"Department of Life Sciences, Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Valentina","family":"Nobile","sequence":"additional","affiliation":[{"name":"Department of Life Sciences, Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aniello","family":"Russo","sequence":"additional","affiliation":[{"name":"Department of Life Sciences, Second University of Naples, Via Vivaldi 43, 81100 Caserta, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2010,2,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"806","DOI":"10.1038\/35888","article-title":"Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans","volume":"391","author":"Fire","year":"1998","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"343","DOI":"10.1038\/nature02873","article-title":"Mechanisms of gene silencing by double-stranded RNA","volume":"431","author":"Meister","year":"2004","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"5875","DOI":"10.1093\/emboj\/cdf582","article-title":"Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP","volume":"21","author":"Zhang","year":"2002","journal-title":"EMBO J"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/j.cell.2004.06.017","article-title":"Single processing center models for human Dicer and bacterial RNase III","volume":"118","author":"Zhang","year":"2004","journal-title":"Cell"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"975","DOI":"10.1101\/gad.1187904","article-title":"RISC is a 5\u2032 phosphomonoester producing RNA endonuclease","volume":"18","author":"Martinez","year":"2004","journal-title":"Genes Dev"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1038\/35005107","article-title":"An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells","volume":"404","author":"Hammond","year":"2000","journal-title":"Nature"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"787","DOI":"10.1016\/j.cub.2004.03.008","article-title":"The RNA induced silencing complex is a Mg2+-dependent endonuclease","volume":"14","author":"Schwarz","year":"2004","journal-title":"Curr. 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