Artificial tethering of Argonaute proteins for studying their role in translational repression of target mRNAs.

ABSTRACT

Small RNAs such as microRNAs (miRNAs) and small-interfering RNAs (siRNAs) associate with members of the RNA-binding Argonaute family proteins. Together they participate in transcriptional and posttranscriptional gene silencing mechanisms. The fate of the target mRNA is determined, in part, by the degree of complementarity with the small RNA. To examine the exact role of the Argonaute protein in the silencing complex, human Argonautes were artificially recruited to reporter mRNAs in a small RNA-independent manner by the BoxB-N-peptide tethering system. Tethering of Argonaute proteins to a reporter mRNA leads to the inhibition of translation, mimicking the repression seen with miRNAs. Similar tethering experiments were performed with fly and fission yeast Argonaute proteins and other components of the small RNP (ribonucleoprotein) complex, uncovering their specific roles in the silencing complexes containing them.