Title

The regulation of microRNA activity

Date of Completion

January 2009

Keywords

Chemistry, Biochemistry

Degree

Ph.D.

Abstract

MicroRNAs are short, single-stranded, non-protein coding RNAs that negatively regulate gene expression. MicroRNAs have been shown to play a critical role in many biological processes and diseases. Therefore, it is critical to gain an understanding of the factors that can regulate microRNA activity. It has been established that, microRNA activity is exerted by a class of RNA-binding proteins called Argonautes. MicroRNAs arise from a transient duplex (the microRNA duplex) from which only one strand (the microRNA strand) is specifically sorted into an Argonaute protein, whereas the other strand (the microRNA* strand) is not. This observation provokes the question; how is the microRNA specifically sorted into an Argonaute protein? To this effect we have found that the differing 5' terminal nucleotides of the microRNA and microRNA* strand are a critical determinant for strand specific sorting of the microRNA into Argonaute. Knowing that microRNAs proceed through a duplex intermediate we set out to identify a microRNA duplex unwindase. Biochemical purification of a microRNA duplex specific unwinding activity from cells yielded P68 RNA Helicase. We showed that P68 RNA Helicase is sufficient to unwind a microRNA duplex, and also that P68 is necessary for microRNA function in cells. Next we asked if silent mutations could create novel, non-canonical microRNA target sites. We identified a silent variant (G4304A) in the BRCA1 mRNA, creates a novel microRNA target site for microRNA-501. ^