Function of the Trypanosome Argonaute 1 protein in RNA interference requires the N-terminal RGG domain and arginine 735 in the Piwi domain.
Argonaute proteins are central components of RNA interference (RNAi) and related phenomena in a wide variety of eukaryotes, including the early diverging protozoan Trypanosoma brucei. The single T. brucei Argonaute protein (TbAGO1) is in a complex with small interfering RNAs (siRNAs), and a fraction of this ribonucleoprotein particle is associated with polyribosomes. In this study, we generated a panel of insertion, deletion, and single point mutants of TbAGO1 and assayed them in vivo for their function in RNAi. In addition to the signature domains of Argonaute proteins, PAZ and Piwi, TbAGO1 has an N-terminal domain with a high abundance of RGG repeats. Deletion of the N-terminal domain blocked association of AGO1 with polyribosomes and severely affected mRNA cleavage. Nevertheless, the mutant protein was in a complex with siRNAs. In contrast, deletion of the Piwi domain led to a loss of siRNAs but did not abolish polyribosome association. Site-directed mutagenesis of conserved amino acids in the Piwi domain identified arginine 735 as essential for RNAi. Although the R735A mutant bound siRNAs and associated with polyribosomes, it displayed a severe defect in the cleavage of target mRNA.
Shi
Huafang
H
Department of Internal Medicine, Yale University Medical School, New Haven, Connecticut 06536-0812, USA.
Ullu
Elisabetta
E
Tschudi
Christian
C
D000818:Animals; D001120:Arginine / Q000235:genetics / Q000502:physiology*; D060565:Argonaute Proteins; D029721:Drosophila Proteins / Q000235:genetics / Q000502:physiology*; D039642:Eukaryotic Initiation Factors; D009154:Mutation; D017434:Protein Structure, Tertiary; D034622:RNA Interference / Q000502:physiology*; D034741:RNA, Small Interfering / Q000502:physiology; D016601:RNA-Binding Proteins / Q000502:physiology*; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000502:physiology*
C489654:AGO1 protein, Drosophila; C495987:AGO1 protein, Trypanosoma brucei; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D039642:Eukaryotic Initiation Factors; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D001120:Arginine