Dissecting Dicer: How Regulatory RNAs Are Made and Used
Two RNA-binding proteins, TRBP and PACT, have been shown to influence human Dicer activity in vivo, but their roles in RNA-silencing pathways remain poorly defined. Quantitative biochemical experiments are proposed to determine effects of TRBP and PACT on human Dicer catalysis, substrate specificity, and target cleavage. We will test the hypothesis that TRBP and PACT affect binding and processing of distinct classes of RNA substrates. These experiments will provide important insights into the behavior and mechanism of Dicer in the context of its natural binding partners.
Understanding and Engineering Viral Immunity in Bacteria
The hallmarks of prokaryotic adaptive immunity are genomic clustered regularly interspaced short palindromic repeats (CRISPRs), containing an ~550 base pair leader sequence followed by a short (24–48 nt) repeat sequence adjacent to a similarly sized "unique" spacer sequence. The spacers, which often match segments from phage and plasmids, confer resistance to propagation of phage or plasmids bearing those sequences. In S. thermophilus, CRISPR-associated (Cas) proteins encoded by open reading frames adjacent to the CRISPR element were found to be required for CRISPR-mediated phage resistance. Experiments in E. coli showed that a large macromolecular complex called Cascade (CRISPR-associated complex for antiviral defense), formed from multiple Cas proteins, recognizes long transcripts from CRISPR arrays and generates short crRNAs consisting of a repeat-spacer unit. These small CRISPR-derived RNAs serve as homing oligos for the targeted interference of DNA-based foreign genetic elements. However, the mechanisms responsible for this novel regulatory system remain undetermined. The overall goal of this project is to provide a comprehensive understanding of microbial adaptive immunity. We will address (1) how foreign DNA is selectively targeted, (2) how genetic silencing is achieved, and (3) how adaptive immunity has evolved across microbial populations. The long-term objective of this work is to enable engineering of adaptive immunity to control gene expression in environmentally important microbes.
Specific projects (1) test the requirements for DNA recognition by Cascade and related complexes from organisms with active CRISPR systems, using a combination of biochemical reconstitution and structure determination; (2) determine the fate of DNA molecules targeted by crRNAs within Cascade and related complexes, and (3) map the coevolution of CRISPR repeat sequences with the associated Cas genes, using sequence- and structure-based computational analysis.