Abstract
Argonaute proteins (AGOs) are central to RNA interference (RNAi) and related silencing pathways. At the core of the RNAi pathway in the ancient parasitic eukaryote Trypanosoma brucei is a single Argonaute protein, TbAGO1, with an established role in the destruction of potentially harmful retroposon transcripts. One notable feature of TbAGO1 is that a fraction sediments with polyribosomes, and this association is facilitated by an arginine/glycine-rich domain (RGG domain) at the N terminus of the protein. Here we report that reducing the size of the RGG domain and, in particular, mutating all arginine residues severely reduced the association of TbAGO1 with polyribosomes and RNAi-induced cleavage of mRNA. However, these mutations did not change the cellular localization of Argonaute and did not affect the accumulation of single-stranded siRNAs, an essential step in the activation of the RNA-induced silencing complex. We further show that mRNA on polyribosomes can be targeted for degradation, although this alliance is not a pre-requisite. Finally, sequestering tubulin mRNAs from translation with antisense morpholino oligonucleotides reduced the RNAi response indicating that mRNAs not engaged in translation may be less accessible to the RNAi machinery. We conclude that the association of the RNAi machinery and target mRNA on polyribosomes promotes an efficient RNAi response. This mechanism may represent an ancient adaptation to ensure that retroposon transcripts are efficiently destroyed, if they become associated with the translational apparatus.
Highlights
Argonaute proteins (AGOs) are central to RNA interference (RNAi) and related silencing pathways
The Size of the TbAGO1 RGG Domain and the Presence of Arginine Residues Are Important Determinants for RNAi Competency—One of the striking features of the T. brucei AGO1 N-terminal domain is the high representation of arginine-glycine-glycine (RGG) motifs, which make up almost 50% of the first 59 amino acids [16, 36]
The findings presented in this work argue that the N-terminal RGG domain significantly contributes to the function of
Summary
We report that reducing the size of the RGG domain and, in particular, mutating all arginine residues severely reduced the association of TbAGO1 with polyribosomes and RNAi-induced cleavage of mRNA These mutations did not change the cellular localization of Argonaute and did not affect the accumulation of single-stranded siRNAs, an essential step in the activation of the RNA-induced silencing complex. RNAs (miRNAs), and PIWI-associated RNAs, have emerged as key mediators of a variety of gene-silencing mechanisms operating both at transcriptional and post-transcriptional levels [1] These small RNAs, in a complex with a member of the Argonaute (AGO) or Piwi protein family, act as guides to identify target transcripts. MiRNAs in a complex with an AGO-family member (miRISC) mostly mediate translational repression of target mRNAs to which they bind, but translational activation has been reported [28]. We show that mRNA cleavage can occur on polyribosomes stabilized by cycloheximide, mRNA association with ribosomes is not a pre-requisite for slicing to occur
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
