Dr. Dipali Sashital, The Scripps Research Institute

Structure, function and assembly of RNA-protein complexes involved in bacterial adaptive immunity and protein synthesis

RNA-protein (RNP) complexes are central to many fundamental processes of gene regulation and genome maintenance in all kingdoms of life. Understanding the biophysical and structural basis for RNA-protein interactions is vital for gaining insight into the assembly and function of these molecular machines. The first part of my talk will focus on the RNA-guided targeting complex, Cascade, a 405 kDa RNP involved in the bacterial CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system. Cascade employs a short CRISPR (cr)RNA as a homing molecule for targeting foreign DNA from plasmids or bacteriophages for destruction. My work has focused on two subunits of Cascade: (1) CasE, the endoribonuclease responsible for the biogenesis of crRNAs from long precursor transcripts and (2) CasA, the largest subunit of Cascade, which is required for specific and non-specific DNA binding. Using a combination of X-ray crystallography, biochemistry and in vivo phage challenge experiments, we have determined how CasE recognizes and cleaves pre-crRNAs, and how CasA facilitates foreign target recognition and binding by Cascade. These results provide a framework for understanding the molecular mechanisms of cellular surveillance by Cascade.

The second part of my talk will focus on one of the most important molecular machines in the cell, the ribosome. The translation of messenger RNA to protein by the ribosome is one of the central tenets of biology. Prior to initiation of this vital process, the ribosome must be assembled from its component parts through a highly ordered series of RNA folding and protein binding events. Although ribosome assembly has been studied extensively in vitro, our current understanding of in vivo assembly pathways, and especially the roles of the numerous factors that assist in ribosome biogenesis, is limited. We have developed a hybrid approach combining quantitative mass spectrometry (qMS) and single-particle electron microscopy (EM) to provide unprecedented insight into the composition and structure of cellular ribosome assembly intermediates. Our approach exploits the exquisite faculty of both techniques for the analysis of heterogeneous samples, here generated by fractionating crude lysates across sucrose gradients. By comparing the populations and conformations of assembly intermediates that accumulate in wild type and ribosome biogenesis factor deletion strains, we have discovered assembly pathways not previously observed in in vitro ribosome assembly studies, providing novel insights into the roles of biogenesis factors in in vivo ribosome assembly. In addition, this approach represents a generalizable toolkit for studying the assembly of supramolecular structures in heterogeneous cellular samples.

 For more information contact Prof. Nick Hud (404-385-1162).