Title

The 5' terminal uracil of let-7 in critical for the recruitment of mRNA to Argonaute2

Date of Completion

January 2009

Keywords

Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Small RNAs modulate gene expression by forming a ribonucleoprotein complex with Argonaute proteins and directing them to specific complementary sites in target nucleic acids. However, the interactions required for the recruitment of target nucleic acid to the ribonucleoprotein complex are poorly understood. Here we have investigated this question using let-7 microRNA, Argonaute2 and a fully complementary target mRNA. Importantly, this model system has allowed us to investigate the mechanistic basis of silencing in vitro and in vivo. In these studies we have identified a sequence specific interaction with Argonaute2 that is critical for silencing activity. This is a novel finding since it is believed that the complementarity between the seed region of a small RNA and its cognate target is the critical determinant of silencing activity. Furthermore, by establishing an assay to directly identify the binary (let-7/Argonaute2) and ternary (let-7/Argonaute2/mRNA) complex intermediates we have found that substitution of the 5' terminal uracil of let-7 has little effect upon the formation of the binary complex but inhibits the formation of the ternary complex. Thus, we have concluded that the interaction of the 5' terminal uracil base with Argonaute2 plays a critical and novel role in the recruitment of mRNA. ^