Small regulatory RNAs in mammals : genomics, function and evolution

Abstract

This thesis explores two aspects of small regulatory RNAs in mammals: (1) the genomic origin of mammalian piwi-interacting RNAs (piRNAs), (2) the evolutionary and functional implication of the seed-based target recognition mechanism of microRNAs (miRNAs). First, we participated in the discovery of mammalian piRNAs from adult rat testes. Our initial characterization of mammalian piRNAs by high-throughput sequencing revealed the peculiar features of their genomic origin: they predominantly derive from long singlestranded RNA precursors that are encoded at ~100 loci with no preferential association to repeat elements. Second, we measured the efficacy of polymorphic miRNA target sites in mammals. A large part of the miRNA-target recognition is determined by the 7-8-nt match between the seed region of miRNAs and the 3'UTR of mRNAs. Because of the small informational complexity of the specificity, spontaneous point mutations in 3'UTRs often create or disrupt miRNA target sites. The resulting polymorphisms in the target sites may contribute to gene expression diversity. By experimentally measuring the efficacy of such polymorphic target sites, we were able to conclude that between two unrelated mammalian individuals of the same species more than 100 genes are likely differentially regulated due to the target-site polymorphisms. Some of the expression diversity might translate into phenotypic diversity, providing substrates for the natural selection to act upon. We also constructed a miRNA library covering nearly all ~-16,000 theoretically possible seed sequences. Under the assumption that the functionality of a miRNA is approximately defined by the identity of the seed, the library is a resource that may enable the systematic exploration of the phenotypic consequences of nearly all possible functionally distinct miRNA species.