MicroRNA (miRNA)-mediated gene regulation has become a major focus in many biological processes. GW182 and its long isoform TNGW1 are marker proteins of GW/P bodies and bind to Argonaute proteins of the RNA induced silencing complex. The goal of this study is to further define and distinguish the repression domain(s) in human GW182/TNGW1. Two non-overlapping regions, 12 (amino acids 896-1219) containing the Ago hook and 5 (amino acids 1670-1962) containing the RRM, both induced comparable silencing in a tethering assay. Mapping data showed that the RRM and its flanking sequences in 5, but not the Ago hook in 12, were important for silencing. Repression mediated by 5 or 12 was not differentially affected when kn... More
MicroRNA (miRNA)-mediated gene regulation has become a major focus in many biological processes. GW182 and its long isoform TNGW1 are marker proteins of GW/P bodies and bind to Argonaute proteins of the RNA induced silencing complex. The goal of this study is to further define and distinguish the repression domain(s) in human GW182/TNGW1. Two non-overlapping regions, 12 (amino acids 896-1219) containing the Ago hook and 5 (amino acids 1670-1962) containing the RRM, both induced comparable silencing in a tethering assay. Mapping data showed that the RRM and its flanking sequences in 5, but not the Ago hook in 12, were important for silencing. Repression mediated by 5 or 12 was not differentially affected when known endogenous repressors RCK/p54, GW182/TNGW1, TNRC6B were depleted. Transfected 5, but not 12, enhanced Ago2-mediated repression in a tethering assay. Transfected 12, but not 5, released endogenous miRNA reporter silencing without affecting siRNA function. Alanine substitution showed that GW/WG motifs in 12 (12a, amino acids 896-1045) were important for silencing activity. Although 12 appeared to bind PABPC1 more efficiently than 5, neither 5 nor 12 significantly enhanced reporter mRNA degradation. These different functional characteristics of 5 and 12 suggest that their roles are distinct, and possibly dynamic, in human GW182-mediated silencing.
