Systematic identification of mirna targets and the steps in gene expression regulated by mirnas [electronic resource]

In the last decade, RNA interference (RNAi), the process by which small RNAs direct the post-transcriptional silencing of cognate mRNA transcripts has revolutionized longstanding paradigms about RNA function. In addition, researchers have harnessed this pathway for experimentally induced gene silencing in what is arguably one of the most important technological advances in modern biology. That disruption of the RNAi pathway results in aberrant development, cancer, and embryonic death suggesting that RNAi is an integral component of eukaryotic gene expression programming. Although RNAi is a vast and diverse pathway with marked distinctions between species, the basic organization has been established largely from work carried out in worms, flies, humans and mice. Double stranded RNA (dsRNA) inputs are "diced" by the class III ribonuclease Dicer into small dsRNA intermediates of ~21-22nt in length which are transferred to the RNA induced silencing complex (RISC) wherein the guide strand is selected and bound to an Argonaute (Ago) family protein. Target mRNAs are then recruited to RISC through Watson-Crick base pairing to the guide strand. Silencing of target transcripts can be directed by Ago mediated cleavage, or through Ago mediated recruitment of factors that induce translational inhibition and mRNA degradation. MicroRNAs (miRNAs) are the most common class of endogenous small silencing RNAs. Despite these advances, many of molecular details of RISC mediated gene silencing are poorly understood as current models are based on only a few miRNA:mRNA target pairs. Here, we present a method for systematic identification of specific miRNA targets. We demonstrate that immuno-affinity purification (IP) of Argonaute proteins is a viable method for isolating RISC associated miRNAs and mRNAs for identification using DNA microarrays. The strong enrichment of mRNAs with binding sites to the experimentally introduced miR-1 and miR-124 in Ago IPs from human embryonic kidney 293T cells (HEK293T) validates the utility of this method. Furthermore, mRNAs classified as targets of miR-1 and miR-124 using this approach behave like bona fide targets in that they exhibit significant down-regulation at the mRNA level. To learn about the steps in gene expression regulated by miRNAs, we simultaneously measured miR-124 mediated changes in Ago enrichment, mRNA abundance, and ribosome occupancy and ribosome density for ~8,000 genes. The translational parameters were used to estimate apparent changes in translational rate and were collected using standard polysome profiling in tandem with DNA microarrays and a novel gradient encoding scheme. We found that for the majority of the miR-124 targets, changes in mRNA concentration and apparent translation rate are concordant and that ~75% of the estimated change in protein levels could be accounted for by changes in mRNA abundance. Our data is most consistent with models of miRNA inhibition of translation initiation. To rule out miRNA mediated repressive mechanisms that would not be visible to our translational profiling (concordant reductions in translational initiation and elongation, co-translational proteolysis) we tested the protein levels for 13 targets by Western blot and found that our estimated changes in protein were nearly identical to the actual changes for 12/13 of the proteins measured. In addition, we observed a large dynamic range for miR-124 mediated down-regulation of mRNA abundance and apparent translation rate, and estimated protein abundance demonstrating the versatility of miRNA mediated regulation. The concordance between miR-124 specific changes in mRNA level and translation supports a model wherein these two regulatory outcomes are functionally linked in a sequential process or regulated by the same cis factors. We have also sought to learn about the RNAi pathway from a Dicer-centric perspective. We generated a library of Dicer truncations to test the contribution of Dicer's conserved protein domains to in vitro dicing reactions to learn about potentially interesting in vivo function as well as for increasing the efficacy of in vitro dicing as a gene silencing tool. We found that the domain of unknown function 283 (DUF283) may be important for proper spacing in dicing reactions and is part of Dicer's "molecular ruler". In addition we found that the ATPase/Helicase domains may inhibit Dicer activity and are dispensable for in vitro dicing, but may play a role in non-canonical substrate recognition.

Keywords: Chemical and Systems Biology.
Advisor: Ferrell James Ellsworth primary advisor. Thesis advisor
Chen James Kenneth advisor. Thesis advisor
Fire Andrew Zachary advisor. Thesis advisor
Meyer Tobias advisor. Thesis advisor