Folding of RNA in the cell is not well understood nor has it been integrated into a cohesive mechanistic framework. We are taking two approaches to understanding RNA folding in vivo: (1) Simulating in vivo conditions and examining the RNA folding pathways and the evolutionary driving forces for these, and (2) Determining RNA folding transcriptome-wide in living organisms (presently plants) and evaluating implications of this folding on gene regulation and RNA processing. The former project has the advantage that one can precisely control experimental variable such as crowding, cosolutes, and ionic conditions on RNA folding, while the latter project has the advantage that observations are being made directly in a living organism.
(1) RNA folding under in vivo-like conditions. One aspect of this project is 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 have a longstanding interest in RNA folding. Presently, we are taking a comprehensive approach in 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. 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. One recent advance has been determining that cellular-like crowding supports ribozyme reactivity by favoring a compact form of the ribozyme, but only under physiological ionic and cosolute conditions. SAXS studies are done in collaboration with Neela Yennawar . Another study from the lab found that molecular crowders and cosolutes promote RNA folding cooperativity in physiological conditions . We collaborate with Martin Gruebele to measure kinetics in vivo.
(2) RNA Folding Transcriptome-wide in Living Organisms. We recently developed a new approach to probe the structure of RNAs across an entire transcriptome in Arabidopsis  and a gene-specific way for low abundance RNAs . In a related study, we demonstrated that RNA G-quadruplex downregulates translation of a DNA repair gene . This work is funded by an NSF PGRP (plant genome research program) grant that is collaborative between our lab, Sally Assmann’s lab (Penn State Biology) on the plant biology, and David Mathews’ lab (U Rochester) on the computational implementation of structure prediction across a genome. 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 currently working with rice and Arabidopsis, but are moving into other organisms in the tree of life.
Thermodynamics: UV-detected thermal denaturations, ITC, DSC; Kinetics: Stopped-flow, T-jump; Structural: structure-mapping, SAXS; Genomics: Structure-seq, NGS, reporter-gene assays.
1. Strulson, C.A., Yennawar, N.H., Rambo, R.P., and Bevilacqua, P.C. “Molecular crowding favors reactivity of a human ribozyme under physiological ionic conditions.” Biochemistry, 52, 8187-8197 (2013).[PubMed]
2. Strulson, C. A., Boyer, J. A., Whitman, E. E. & Bevilacqua, P. C. “Molecular crowders and cosolutes promote folding cooperativity of RNA under physiological ionic conditions.” RNA 20, 331-347 (2014). [PubMed]
3. Ding, Y., Tang, Y., Kwok, C.K., Zhang, Y., Bevilacqua, P.C. & Assmann, S.M. “In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features.” Nature 505, 696-700 (2014). [PubMed]
4. Kwok, C. K., Ding, Y., Tang, Y., Assmann, S. M. & Bevilacqua, P. C. “Determination of in vivo RNA structure in low-abundance transcripts.” Nature Commun. 4, 2971 (2013). [PubMed]
5. Kwok, C. K., Ding, Y., Shahid, S. Assmann, S. M.*, and Bevilacqua, P. C.* “A stable RNA G-quadruplex within the 5’UTR of arabidopsis thaliana ATR mRNA downregulates translation” for Biochem. J. 467, 91-102 (2015). [Biochem. J.]
1. Kwok, C.K., Tang, Y., Assmann, S. M., Bevilacqua, P. C. “The RNA Structurome: Transcriptome-Wide Structure Probing with Next-Gen Sequencing” for Trends Biochem. Sci. (in press). [TiBS]
2. Bevilacqua, P.C., Blose, J.M. “Structures, kinetics, thermodynamics, and biological functions of RNA hairpins.” Annu. Rev. Phys. Chem. 59, 79-103 (2008). [PubMed]
3. The following general interest articles regarding Ding et al. Nature 505, 696 (2014). Nature, Nature Methods, Nature Chemical Biology.