Determination of factors that controls Dicer expression in vivo in C. elegans

Abstract

Aging increases the risk of chronic diseases, being slowed by caloric restriction. Our group and coworkers identified that, in a conserved way, the synthesis of microRNAs decrease with aging, being reverted by caloric restriction. This phenomenon is a consequence of the regulation of the expression of key enzymes of the microRNA processing pathway, particularly Dicer, which cleaves pre-miRNA in mature microRNA. In mammals, these effects have been observed only in adipose tissue. In C. elegans, it is not yet known in which tissues Dicer suffers a major regulation, though it is known that its expression reduces with aging and increases with caloric restriction. We therefore aim to identify, in a tissue-specific manner, how Dicer is regulated in C. elegans under these situations. Commonly used methods have limitations to answer these questions. Real time PCR and Western Blot consider only the whole body expression; while gene reporter extrachromosomal constructs may be randomly propagated in the cells and/or lost after generations. To overcome these limitations, we will integrate to the genome of C. elegans a construct in which the Dicer gene, under the Dicer promoter, codes a protein fused to GFP. Using microscopic analysis we will validate this model under known situations that stimulate Dicer (i.e., caloric restriction and aging). We will then identify in which tissues Dicer is modulated and test genes involved in this modulation. These data would impact our understanding about the biology of aging, since they could outline a fundamental mechanism that would determine the effects of caloric restriction. (AU)