| Small noncoding RNAs, including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and endogenous small-interfering RNAs (endo-siRNAs), regulate developmental and defense pathways in animals. While many small RNA silencing protein cofactors have been identified, much more is to be learned from a dynamic and quantitative perspective to reveal the underlying mechanisms and designing principles of each pathway. In this dissertation, I present studies that examine the temporal dynamics of small RNA pathways---one from an evolutionary time scale among the nematode species, and one from finely staged Caenorabditis elegans during the first larval stage. I also describe works identifying new cofactors functions in the miRNA pathway, potentially through regulating the spatial dynamics of the miRNA silencing complex.;To better understand the various small RNA pathways from an evolutionary perspective, I deep sequenced small RNA from several nematode species and examined the conservation and evolution of each class of small RNAs. This reveals an extraordinary sequence fluidity of piRNAs and endo-siRNAs. However, many features such as their genomic distribution and expression patterns are highly conserved. I found that nematodes produce two distinct sex-specific classes of piRNAs, suggesting different roles for piRNAs in male and female germlines.;To reveal the kinetics of miRNA-mediated silencing during the animal cell fate transition, we performed a quantitative analysis of lin-4 miRNA-mediated silencing of its target gene, lin-14. Our results point to two phases of regulation: a fast lin-14 mRNA destabilization phase, and long-term translational inhibition that is important in maintaining the silencing of lin-14 by the lin-4 miRNA.;Lastly, I performed a candidate-based RNAi screen for genes involved in miRNA activity. This study leads to the finding that the mevalonate pathway regulates miRNA activity. Dolichol phosphate, synthesized from the mevalonate pathway, functions as a lipid carrier of the oligosaccharide moiety destined for protein N-linked glycosylation. Inhibition of the dolichol pathway of protein N-glycosylation also causes derepression of miRNA target mRNAs, suggesting proteins that mediate miRNA repression could be regulated by N-glycosylation.;Together, these studies highlight the importance of studying both the temporal and spatial dynamics of small RNA pathways in creating new insights. |
