TY - JOUR
T1 - Comprehensive analysis reveals how single nucleotides contribute to noncoding RNA function in bacterial quorum sensing
AU - Rutherford, Steven T.
AU - Valastyan, Julie S.
AU - Taillefumier, Thibaud
AU - Wingreen, Ned S.
AU - Bassler, Bonnie Lynn
PY - 2015/11/3
Y1 - 2015/11/3
N2 - Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544 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, fluorescenceactivated 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.
AB - Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544 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, fluorescenceactivated 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.
KW - Qrr
KW - Quorum sensing
KW - RSort-Seq
KW - Regulation
KW - Regulatory sRNA
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U2 - 10.1073/pnas.1518958112
DO - 10.1073/pnas.1518958112
M3 - Article
C2 - 26483489
AN - SCOPUS:84946220577
SN - 0027-8424
VL - 112
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 44
M1 - E6038
ER -