Research in the Sontheimer Laboratory
Noncoding RNAs in Gene Regulation
RNA molecules are essential participants in many aspects of cellular function. We aim to understand the roles and mechanisms of regulatory RNAs in gene expression, with an emphasis on genetic interference pathways.
Although genetic interference pathways [in particular, RNA interference (RNAi)] were reported in eukaryotes more than a decade ago, analogous RNA-guided silencing phenomena were thought to be largely absent in bacteria and archaea. In 2007, however, genetic elements known as clustered, regularly interspaced, short palindromic repeat (CRISPR) loci were revealed as sequence-based specificity determinants of an adaptive immune system that defends against bacteriophage infection. Shortly thereafter, CRISPR interference was found to be directed by small RNAs that are transcribed from CRISPR loci. Although this pathway has functional parallels with RNAi in eukaryotes, the mechanisms of RNAi and CRISPR interference are completely distinct. We are working to understand the roles and mechanisms of CRISPR interference in bacteria. Thus far our work has provided three fundamental advances in our understanding of the CRISPR pathway. (i) CRISPR interference is not confined to phage defense but functions more broadly to limit horizontal gene transfer. (ii) The CRISPR RNAs (crRNAs) that specify interference can target DNA rather than RNA, establishing a fundamental distinction between CRISPR interference and RNAi. (iii) The mode of crRNA/target interaction includes a built-in mechanism for distinguishing "self" DNA (the encoding CRISPR locus) from "non-self" DNA (phage or plasmid sequences), leading to selective targeting of the latter. We are now employing biochemical approaches to identify the molecular events that lead to crRNA-directed silencing.
We have also continued our work on eukaryotic silencing pathways by identifying novel factors that associate with the short interfering RNAs (siRNAs) that direct RNAi. Most notably, we identified a protein called Blanks that is expressed in the male germline of Drosophila melanogaster and is essential for spermiogenesis and male fertility. Blanks is localized to the nucleus and associates with additional proteins in a particle that is distinct from canonical forms of the RNA-induced silencing complex (RISC). Our current focus is on defining the regulatory targets and mechanisms of Blanks as well as an apparent Blanks paralog.
Our third research avenue is an analysis of the roles of non-coding RNAs (ncRNAs), as well as non-coding portions of mRNAs, in the budding yeast Saccharomyces cerevisiae. Unlike most other eukaryotes, this model organism lacks the signature components of the RNAi machinery, indicating that it uses other RNA-based systems to regulate gene expression. Our genome-wide analyses of RNAs expressed during meiosis and sporulation has revealed large numbers of ncRNAs, as well as an unanticipated degree of dynamism in transcript architecture. We are now using this dataset as a springboard to identify and characterize novel modes of eukaryotic gene regulation.