Abstract
Many non-coding RNAs form structures that interact with cellular machinery to control gene expression. A central goal of molecular and synthetic biology is to uncover design principles linking RNA structure to function to understand and engineer this relationship. Here we report a simple, high-throughput method called in-cell SHAPE-Seq that combines in-cell probing of RNA structure with a measurement of gene expression to simultaneously characterize RNA structure and function in bacterial cells. We use in-cell SHAPE-Seq to study the structure-function relationship of two RNA mechanisms that regulate translation in Escherichia coli. We find that nucleotides that participate in RNA-RNA interactions are highly accessible when their binding partner is absent and that changes in RNA structure due to RNA-RNA interactions can be quantitatively correlated to changes in gene expression. We also characterize the cellular structures of three endogenously expressed non-coding RNAs: 5S rRNA, RNase P and the btuB riboswitch. Finally, a comparison between in-cell and in vitro folded RNA structures revealed remarkable similarities for synthetic RNAs, but significant differences for RNAs that participate in complex cellular interactions. Thus, in-cell SHAPE-Seq represents an easily approachable tool for biologists and engineers to uncover relationships between sequence, structure and function of RNAs in the cell.
Original language | English (US) |
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Pages (from-to) | e12 |
Journal | Nucleic acids research |
Volume | 44 |
Issue number | 2 |
DOIs | |
State | Published - Jan 29 2016 |
Funding
National Science Foundation Graduate Research Fellowship Program [DGE-1144153 to K.E.W.]; Cornell University College of Engineering 'Engineering Learning Initiatives' Undergraduate Research Grant Program [to T.R.A.]; Defense Advanced Research Projects Agency Young Faculty Award (DARPA YFA) [N66001-12-1-4254 to J.B.L.]; New Innovator Award through the National Institute of General Medical Sciences of the National Institutes of Health [1DP2GM110838 to J.B.L.]. K.E.W. is a Fleming Scholar in the School of Chemical and Biomolecular Engineering at Cornell University. J.B.L. is an Alfred P. Sloan Research Fellow. Funding for open access charge: National Institutes of Health/1DP2GM110838.
ASJC Scopus subject areas
- Genetics