BG, BMMB, CHEM, MCIBS, PLBIO
Overview: Biological Chemistry of RNA
The Bevilacqua lab is interested in the folding and catalysis of ribonucleic acid (RNA), and its interactions with proteins. RNA-protein complexes carry out structural and functional roles central to the execution and regulation of many biological processes. Our laboratory focuses on biologically important systems including viral replication and the human viral response, as well as how RNA mediates responses to abiotic stresses in plants. The laboratory is problem based and uses a variety of experimental approaches, some in collaboration as described below, including rapid mixing kinetics, fluorescence spectroscopy, UV melting, site-directed mutagenesis, combinatorial selection of RNA (or SELEX), Raman spectroscopy, NMR, SAXS, and X-ray crystallography. We also apply theory in a number of ways to these problems, including statistical thermodynamics, molecular dynamics (MD), and quantum mechanics. In general, the problems we study lie at the interface of chemistry, biochemistry, biology, and physics.
In the early 1980's, Tom Cech and Sidney Altman showed that RNA could be an enzyme--a so-called 'ribozyme'-- catalyzing the making and breaking of covalent bonds. This led to the 1989 Nobel Prize in Chemistry. We have pioneered a number of advances in this field including establishing roles for nucleobases in proton transfer, determination of driving forces for pKa shifting by Raman spectroscopy, and establishing pathways for RNA cleavage by a combination of experiments and classical and quantum mechanical theory. We are currently working in the HDV and glmS ribozymes to determine roles of metal ions, nucleobases, and cofactors in the mechanism of cleavage of the catalytic RNAs. We are also collaborating with Sharon Hammes-Schiffer's lab to perform quantum calculations on these ribozymes. In addition, the lab is focused on how these RNAs fold during in vivo conditions.
RNA Folding In Vivo
Folding of RNA in the cell is not well understood nor has it been integrated into a cohesive mechanistic framework. The broad objectives of this project are to develop comprehensive molecular mechanisms for how functional RNAs fold in vivo and to relate these mechanisms to the evolutionary forces that help shape them. We are taking a comprehensive approaching which both the biophysical and evolutionary driving forces that give rise to RNA folding mechanism in vivo are being identified. We are establishing biophysical principles for in vivo RNA folding by examining the folding mechanisms of several naturally occurring riboswitches and ribozymes in both model cytoplasms and in cells. In addition we are elucidating evolutionary principles that guide RNA folding in vivo by testing the folding mechanisms of sequences that will emerge from several neutral drift selections.
In addition, we recently developed a new approach to probe the structure of RNAs across an entire transcriptome. (Ding et al. Nature, 2014) This work is funded by an NSF PGRP grant that is collaborative between our lab, Sally Assmann’s lab (Penn State Biology), and David Mathews' lab (U Rochester). Our goal in this work is to determine the folds of all the RNA in an entire transcriptome (tens of thousands of RNAs at the single nucleotide leve) in vivo and how they change during stress. We are identifying new paradigms for RNA folding in gene regulation. We are working with rice and Arabidopsis, but are moving into other organisms in the tree of life.
Early Earth and RNA
A major question in biology is how life began. It is thought that RNA may have played a major role in the process through the so-called RNA world hypothesis. We are collaborating with the Christine Keating's lab at Penn State to address physical means that may have aided the emergence of life through the localization and improvement of catalysis. We are evaluating compartmentalization driven by aqueous phase separation as a potential physicochemical mechanism to concentrate and help chaperone the folding, multiple turnover, and evolution of rare catalytic RNA molecules. We are also addressing how compartmentalization may facilitate the assembly of progenitor membranes as a step towards protocell formation. Accomplishing these goals will provide insight into the early evolution of life on this and other planets.
RNA In Innate Immunity
PKR is the RNA-activated protein kinase and mediates an anti-viral response in humans. PKR is activated by long stretches of perfect dsRNA to autophosphorylate and then phosphorylate eIF-2a, thereby inhibiting the initiation of translation. We have shown that the dsRNA binding domain (dsRBD), which consists of two dsRBMs, has a site size of about 16 bp and that PKR is activated by non-canonical RNA structures. We also demonstrated that the PKR can be activated by short dsRNAs, as long as they have single-stranded tails, and that the dsRBD is capable of modulating the topology of RNA by straightening bent RNA. Our lab demonstrated that the 5'-triphosphate acts as a pathogen associated molecular patter (PAMP) to activate PKR and initiate translation inhibition. Additional studies showed roles for RNA dimerization and PKR dimerization and activation, and roles for RNA modifications and bulges in PKR regulation. We are currently working to understand how RNAs for new and novel pathogens activate PKR both in the cell and under in vivo like conditions in the lab.