PRMT1 methylates the single Argonaute of Toxoplasma gondii and is important for the recruitment of Tudor nuclease for target RNA cleavage by antisense guide RNA.
Argonaute (Ago) plays a central role in RNA interference in metazoans, but its status in lower organisms remains ill-defined. We report on the Ago complex of the unicellular protozoan, Toxoplasma gondii (Tg), an obligatory pathogen of mammalian hosts. The PIWI-like domain of TgAgo lacked the canonical DDE/H catalytic triad, explaining its weak target RNA cleavage activity. However, TgAgo associated with a stronger RNA slicer, a Tudor staphylococcal nuclease (TSN), and with a protein Arg methyl transferase, PRMT1. Mutational analysis suggested that the N-terminal RGG-repeat domain of TgAgo was methylated by PRMT1, correlating with the recruitment of TSN. The slicer activity of TgAgo was Mg(2+)-dependent and required perfect complementarity between the guide RNA and the target. In contrast, the TSN activity was Ca(2+) -dependent and required an imperfectly paired guide RNA. Ago knockout parasites showed essentially normal growth, but in contrast, the PRMT1 knockouts grew abnormally. Chemical inhibition of Arg-methylation also had an anti-parasitic effect. These results suggest that the parasitic PRMT1 plays multiple roles, and its loss affects the recruitment of a more potent second slicer to the parasitic RNA silencing complex, the exact mechanism of which remains to be determined.
Department of Biochemistry and Molecular Biology, University of South Alabama, College of Medicine, 307 University Blvd., Mobile, Alabama, USA.
Alla
A
Musiyenko
Tanmay
T
Majumdar
Joel
J
Andrews
Brian
B
Adams
Sailen
S
Barik
D000595:Amino Acid Sequence; D060565:Argonaute Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020029:Base Pairing; D001483:Base Sequence; D002455:Cell Division; D002478:Cells, Cultured; D004722:Endoribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D006801:Humans; D008745:Methylation; D008969:Molecular Sequence Data; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D011484:Protein-Arginine N-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000737:chemistry; D059367:RNA Cleavage; D034622:RNA Interference; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014122:Toxoplasma / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
D060565:Argonaute Proteins; D021122:Protein Subunits; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D012313:RNA; D011484:Protein-Arginine N-Methyltransferases; D004722:Endoribonucleases