Divergent GW182 functional domains in the regulation of translational silencing.
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, Δ12 (amino acids 896-1219) containing the Ago hook and Δ5 (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 Δ5, but not the Ago hook in Δ12, were important for silencing. Repression mediated by Δ5 or Δ12 was not differentially affected when known endogenous repressors RCK/p54, GW182/TNGW1, TNRC6B were depleted. Transfected Δ5, but not Δ12, enhanced Ago2-mediated repression in a tethering assay. Transfected Δ12, but not Δ5, released endogenous miRNA reporter silencing without affecting siRNA function. Alanine substitution showed that GW/WG motifs in Δ12 (Δ12a, amino acids 896-1045) were important for silencing activity. Although Δ12 appeared to bind PABPC1 more efficiently than Δ5, neither Δ5 nor Δ12 significantly enhanced reporter mRNA degradation. These different functional characteristics of Δ5 and Δ12 suggest that their roles are distinct, and possibly dynamic, in human GW182-mediated silencing.
Department of Oral Biology, University of Florida, Gainesville, FL 32610-0424, USA.
Bing
B
Yao
Songqing
S
Li
Hyun Min
HM
Jung
Shang L
SL
Lian
Grant X
GX
Abadal
Frank
F
Han
Marvin J
MJ
Fritzler
Edward K L
EK
Chan
D020816:Amino Acid Motifs; D060565:Argonaute Proteins; D001324:Autoantigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism; D039122:Poly(A)-Binding Protein I / Q000378:metabolism; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D001324:Autoantigens; D015852:Eukaryotic Initiation Factor-2; D035683:MicroRNAs; D039122:Poly(A)-Binding Protein I; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C465663:TNRC6A protein, human