Comprehensive analysis reveals how single nucleotides contribute to noncoding RNA function in bacterial quorum sensing.

TitleComprehensive analysis reveals how single nucleotides contribute to noncoding RNA function in bacterial quorum sensing.
Publication TypeJournal Article
Year of Publication2015
AuthorsRutherford, ST, Valastyan, JS, Taillefumier, T, Wingreen, NS, Bassler, BL
JournalProc Natl Acad Sci U S A
Volume112
Issue44
PaginationE6038-47
Date Published2015 Nov 3
ISSN1091-6490
KeywordsEscherichia coli, Nucleotides, Quorum Sensing, RNA, Untranslated, Vibrio
Abstract

<p>Five homologous noncoding small RNAs (sRNAs), called the Qrr1-5 sRNAs, function in the Vibrio harveyi quorum-sensing cascade to drive its operation. Qrr1-5 use four different regulatory mechanisms to control the expression of ∼ 20 mRNA targets. Little is known about the roles individual nucleotides play in mRNA target selection, in determining regulatory mechanism, or in defining Qrr potency and dynamics of target regulation. To identify the nucleotides vital for Qrr function, we developed a method we call RSort-Seq that combines saturating mutagenesis, fluorescence-activated cell sorting, high-throughput sequencing, and mutual information theory to explore the role that every nucleotide in Qrr4 plays in regulation of two mRNA targets, luxR and luxO. Companion biochemical assays allowed us to assign specific regulatory functions/underlying molecular mechanisms to each important base. This strategy yielded a regional map of nucleotides in Qrr4 vital for stability, Hfq interaction, stem-loop formation, and base pairing to both luxR and luxO, to luxR only, and to luxO only. In terms of nucleotides critical for sRNA function, the RSort-Seq analysis provided strikingly different results from those predicted by commonly used regulatory RNA-folding algorithms. This approach is applicable to any RNA-RNA interaction, including sRNAs in other bacteria and regulatory RNAs in higher organisms.</p>

DOI10.1073/pnas.1518958112
Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID26483489
PubMed Central IDPMC4640798
Grant List5R01GM065859 / GM / NIGMS NIH HHS / United States
F32AI085922 / AI / NIAID NIH HHS / United States
R37 GM065859 / GM / NIGMS NIH HHS / United States
/ / Howard Hughes Medical Institute / United States