Simultaneous characterization of cellular RNA structure and function with in-cell SHAPE-Seq.
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.
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
Kyle E
KE
Watters
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA.
Timothy R
TR
Abbott
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853, USA jblucks@cornell.edu.
Julius B
JB
Lucks
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D012341:RNA, Ribosomal, 5S / Q000737:chemistry* / Q000235:genetics; D043262:Ribonuclease P / Q000737:chemistry* / Q000235:genetics; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics; D058928:Riboswitch; D017423:Sequence Analysis, RNA; D013329:Structure-Activity Relationship
D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D012329:RNA, Bacterial; D012341:RNA, Ribosomal, 5S; D058928:Riboswitch; D043262:Ribonuclease P