TY - JOUR
T1 - High-throughput in vivo mapping of RNA accessible interfaces to identify functional sRNA binding sites
AU - Mihailovic, Mia K.
AU - Vazquez-Anderson, Jorge
AU - Li, Yan
AU - Fry, Victoria
AU - Vimalathas, Praveen
AU - Herrera, Daniel
AU - Lease, Richard A.
AU - Powell, Warren Buckler
AU - Contreras, Lydia M.
N1 - Funding Information:
The authors would like to acknowledge Dhivya Arasappan (UT Bioinformatics Consulting Group) for RNA-seq mapping advice, Erika Hale (UT Austin Department of Statistics and Data Sciences) for statistical consultation, Kristofer Reyes (University at Buffalo Department of Materials Design and Innovation) for code consultation, Samuel Stimple (The Ohio State University Department of Chemical and Biomolecular Engineering) for cloning relevant sRNA-and mRNA-coding DNA sequences into plasmids for use in electrophoretic mobility shift assays, as well as Matthew Lab (UT Austin McCombs School of Business), Angela Chen and Runhua Han (UT Austin McKetta Dept. of Chemical Engineering) for their assistance with biochemical assays. We further thank Abigail N. Leistra and Katie Haning (UT Austin McKetta Department of Chemical Engineering) for useful discussions of the manuscript as well as the Texas Advanced Computing Center (TACC) at The University of Texas at Austin for providing high-performance computing resources. We also acknowledge Welch Foundation [F-1756], Air Force Office of Scientific Research Young Investigator program (FA9550-13-1-0160), Consejo Nacional de Ciencia y Tecnología for the graduate fellowship [CONACYT-194638 to J.V.-A.], National Science Foundation (NSF) (CAREER CBET-1254754 to L.M.C.; DGE-1610403 to M.K.M.).
Publisher Copyright:
© 2018, The Author(s).
PY - 2018/12/1
Y1 - 2018/12/1
N2 - Herein we introduce a high-throughput method, INTERFACE, to reveal the capacity of contiguous RNA nucleotides to establish in vivo intermolecular RNA interactions for the purpose of functional characterization of intracellular RNA. INTERFACE enables simultaneous accessibility interrogation of an unlimited number of regions by coupling regional hybridization detection to transcription elongation outputs measurable by RNA-seq. We profile over 900 RNA interfaces in 71 validated, but largely mechanistically under-characterized, Escherichia coli sRNAs in the presence and absence of a global regulator, Hfq, and find that two-thirds of tested sRNAs feature Hfq-dependent regions. Further, we identify in vivo hybridization patterns that hallmark functional regions to uncover mRNA targets. In this way, we biochemically validate 25 mRNA targets, many of which are not captured by typically tested, top-ranked computational predictions. We additionally discover direct mRNA binding activity within the GlmY terminator, highlighting the information value of high-throughput RNA accessibility data.
AB - Herein we introduce a high-throughput method, INTERFACE, to reveal the capacity of contiguous RNA nucleotides to establish in vivo intermolecular RNA interactions for the purpose of functional characterization of intracellular RNA. INTERFACE enables simultaneous accessibility interrogation of an unlimited number of regions by coupling regional hybridization detection to transcription elongation outputs measurable by RNA-seq. We profile over 900 RNA interfaces in 71 validated, but largely mechanistically under-characterized, Escherichia coli sRNAs in the presence and absence of a global regulator, Hfq, and find that two-thirds of tested sRNAs feature Hfq-dependent regions. Further, we identify in vivo hybridization patterns that hallmark functional regions to uncover mRNA targets. In this way, we biochemically validate 25 mRNA targets, many of which are not captured by typically tested, top-ranked computational predictions. We additionally discover direct mRNA binding activity within the GlmY terminator, highlighting the information value of high-throughput RNA accessibility data.
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U2 - 10.1038/s41467-018-06207-z
DO - 10.1038/s41467-018-06207-z
M3 - Article
C2 - 30287822
AN - SCOPUS:85054427741
SN - 2041-1723
VL - 9
JO - Nature communications
JF - Nature communications
IS - 1
M1 - 4084
ER -