Delineating the \u3cem\u3eC. elegans\u3c/em\u3e MicroRNA Regulatory Network: A Dissertation

Metazoan genomes contain thousands of protein-coding and non-coding RNA genes, most of which are differentially expressed, i.e., at different locations, at different times during development, or in response to environmental signals. Differential gene expression is achieved through complex regulatory networks that are controlled in part by two types of trans-regulators: transcription factors (TFs) and microRNAs (miRNAs). TFs bind to cis-regulatory DNA elements that are often located in or near their target genes, while microRNAs hybridize to cis-regulatory RNA elements mostly located in the 3’ untranslated region (3’UTR) of their target mRNAs. My work in the Walhout lab has centered on understanding how these trans-regulators interact with each other in the context of gene regulatory networks to coordinate gene expression at the genome-scale level. Our model organism is the free-living nematode Caenorahbditis elegans, which possess approximately 950 predicted TFs and more than 100 miRNAs Whereas much attention has focused on finding the protein-coding target genes of both miRNAs and TFs, the transcriptional networks that regulate miRNA expression remain largely unexplored. To this end, we have embarked in the task of mapping the first genome-scale miRNA regulatory network. This network contains experimentally mapped transcriptional TF=\u3emiRNA interactions, as well as computationally predicted post-transcriptional miRNA=\u3eTF interactions. The work presented here, along with data reported by other groups, have revealed the existence of reciprocal regulation between these two types of regulators, as well as extensive coordination in the regulation of shared target genes. Our studies have also identified common mechanisms by which miRNAs and TFs function to control gene expression and have suggested an inherent difference in the network properties of both types of regulators. Reverse genetic approaches have been extensively used to delineate the biological function of protein-coding genes. For instance, genome-wide RNAi screens have revealed critical roles for TFs in C. elegans development and physiology. However, reverse genetic approaches have not been very insightful in the case of non-coding genes: A single null mutation does not result in an easily detectable phenotype for most C. elegans miRNA genes. To help delineate the biological function of miRNAs we sought to determine when and where they are expressed. Specifically, we generated a collection of transgenic C. elegans strains, each containing a miRNA promoter::gfp (Pmir::gfp) fusion construct. The particular pattern of expression of each miRNA gene should help to identify potential genetic interactors that exhibit similar expression patterns, and to design experiments to test the phenotypes of miRNA mutants