MicroRNAs (miRNA) are small 21-22 nucleotide RNAs that regulate expression of genes involved in a variety of cellular processes. miRNAs function by binding to messenger RNAs (mRNA). mRNAs carry the genetic code used for the synthesis of proteins. It has turned out that miRNAs interact with nearly half of all mRNAs in a cell to alter the efficiency of protein synthesis. Although, the mechanism by which miRNAs regulate protein expression remains controversial, studies have shown that miRNAs bind to mRNAs that are in the act of synthesizing protein (translating mRNAs). Surprisingly, we learned that some miRNAs preferentially bind to mRNAs that are not actively synthesizing protein (non-translating mRNAs). We hypothesize that miRNAs that associate with non-translating mRNAs reside at specialized places in the cell, known as P-bodies and stress granules (SG), where they regulate mRNA turnover. On the other hand, miRNAs that associate with translating mRNAs are predicted to regulate protein synthesis. To determine whether factors such as overexpression of mRNA target and abundance of P-bodies or SGs changes the steady-state distribution of miRNAs within the cell, we will subject cell lysates to sucrose gradient fractionation to separate miRNAs associated with translating and non-translating mRNAs. We will measure the abundance of miRNAs in the different fractions using established detection methods, such as northern blot analysis. Moreover, we will use confocal microscopy to visualize the location of miRNAs when processing bodies and stress granules are induced or dispersed. We will also test whether different pools of miRNA have distinct functions, and whether certain components of the mlRNP confer these activities. Growing evidence indicates that several miRNAs are dysregulated in various human diseases, including cancers and viral infections. Unfortunately, little is known about the endogenous function of miRNAs. Our studies will reveal whether the unexpected compartmentalization of certain miRNAs have roles in regulating gene expression in human diseases. Learning how miRNAs function normally will lend insight into disease progression and contribute to the development of new treatments.
