RNA molecules are uniquely capable of encoding and controlling the expression of genetic information. We are interested in understanding RNA-mediated regulation of the genome.
Internal Ribosome Entry Site RNAs
Most eukaryotic and viral messages initiate translation by a mechanism involving recognition of a 7-methylguanosine cap at the 5' end of the mRNA; however, translation sometimes occurs via a cap-independent mechanism in which an internal ribosome entry site (IRES) in the 5'-untranslated region of the mRNA recruits the ribosome. In collaboration with Eva Nogales (HHMI, University of California, Berkeley), we determined structures of the hepatitis C virus (HCV) IRES in complexes with the human translational machinery. The use of affinity-purified samples and mass spectrometry has revealed the full composition and posttranslational modification states of IRES-bound complexes that assemble in human cell extracts (in collaboration with Julie Leary, University of California, Davis).
RNA Recognition by Dicer Enzymes
RNA interference (RNAi) begins with the processing of endogenous or introduced precursor RNA into micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) 21–25 nucleotides in length by the enzyme Dicer. Determining the crystal structure of an intact Dicer enzyme revealed how Dicer functions as a molecular ruler to measure and cleave duplex RNAs of a specific length. Ongoing work focuses on investigating how Dicer interacts with other components of the RNAi pathway.
CRISPR Systems in Bacterial Immunity
Bacteria employ RNA molecules to defend themselves against viruses and plasmids by using sequences stored within clustered regularly interspaced short palindromic repeats (CRISPRs). Our research into the role of small RNAs in bacterial adaptive immunity has provided groundbreaking insights into a set of biological pathways that have profound implications for the way that genomes can be edited and reengineered. In particular, our lab's work on the CRISPR-associated (Cas) enzyme Cas9, in collaboration with the lab of Emmanuelle Charpentier (Helmholtz Centre for Infection Research), revealed how this protein and its guide RNA can be used to change genetic information using RNA-programmed DNA cleavage.
Our lab has studied CRISPRs for the past nine years, leading to many insights into the molecular mechanisms that enable these RNA-guided systems to provide acquired immunity to viral infections in bacteria. This work led to the transformative discovery of DNA cleavage by Cas9, a dual RNA-guided enzyme whose ability to cut double-stranded DNA can be programmed by changing the guide RNA sequence. Recognizing that this enzyme-RNA complex could be employed for precision genome engineering in various kinds of cells, our lab redesigned the natural dual-RNA guide as a single-guide RNA (sgRNA), creating a simple-to-use, two-component system. This work triggered an ongoing revolution in the fields of molecular genetics and genomics. The CRISPR-Cas9 technology is being used in laboratories around the world to advance biological research by engineering cells and organisms in precise ways. This fundamental technology will almost certainly lead to development of new therapeutics, biofuels and agricultural products.
Our lab's research showed how Cas9 functions as an RNA-guided endonuclease that uses RNA-DNA base-pairing to recognize and cleave DNA. Using single-molecule and bulk biochemical experiments, our lab, in collaboration with Eric Greene (HHMI, Columbia University), determined how Cas9-RNA complexes interrogate DNA to find specific cleavage sites. We found that both binding and cleavage of DNA by Cas9-RNA complexes require recognition of a short trinucleotide protospacer adjacent motif (PAM) next to the DNA target sequence, and that DNA-binding affinity scales with PAM density. Furthermore, PAM interactions trigger Cas9 catalytic activity. This work revealed how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules and closely regulates scission of double-stranded DNA.
Ongoing projects are focused on delivery of Cas9 protein-RNA complexes into specific tissues, as well as discovery of the mechanisms of target search and binding in live cells. We are also working on other aspects of CRISPR biology, including the pathway for acquisition of new sequences into CRISPR loci, and the structures and mechanisms of other CRISPR targeting complexes, including the RNA-targeting Cmr and Csm complexes.
This work is supported in part by the National Institutes of Health and the National Science Foundation.
As of April 17, 2015