Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF

Kayley H Schulmeyer; Manisha R Diaz; Thomas B Bair; Wes Sanders; Cindy J Gode; Alain Laederach; Matthew C Wolfgang; Timothy L Yahr

doi:10.1128/jb.00343-16

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Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF

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Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF

Kayley H Schulmeyer, Manisha R Diaz, Thomas B Bair, Wes Sanders, Cindy J Gode, Alain Laederach, Matthew C Wolfgang and Timothy L Yahr

Journal of bacteriology, Vol.198(18), pp.2458-2469

09/15/2016

DOI: 10.1128/jb.00343-16

PMCID: PMC4999922

PMID: 27381913

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Abstract

CsrA family RNA-binding proteins are widely distributed in bacteria and regulate gene expression at the posttranscriptional level. Pseudomonas aeruginosa has a canonical member of the CsrA family (RsmA) and a novel, structurally distinct variant (RsmF). To better understand RsmF binding properties, we performed parallel systematic evolution of ligands by exponential enrichment (SELEX) experiments for RsmA and RsmF. The initial target library consisted of 62-nucleotide (nt) RNA transcripts with central cores randomized at 15 sequential positions. Most targets selected by RsmA and RsmF were the expected size and shared a common consensus sequence (CANGGAYG) that was positioned in a hexaloop region of the stem-loop structure. RsmA and RsmF also selected for longer targets (≥96 nt) that were likely generated by rare PCR errors. Most of the long targets contained two consensus-binding sites. Representative short (single consensus site) and long (two consensus sites) targets were tested for RsmA and RsmF binding. Whereas RsmA bound the short targets with high affinity, RsmF was unable to bind the same targets. RsmA and RsmF both bound the long targets. Mutation of either consensus GGA site in the long targets reduced or eliminated RsmF binding, suggesting a requirement for two tandem binding sites. Conversely, RsmA bound long targets containing only a single GGA site with unaltered affinity. The RsmF requirement for two binding sites was confirmed with tssA1, an in vivo regulatory target of RsmA and RsmF. Our findings suggest that RsmF binding requires two GGA-containing sites, while RsmA binding requirements are less stringent. The CsrA family of RNA-binding proteins is widely conserved in bacteria and plays important roles in the posttranscriptional regulation of protein synthesis. P. aeruginosa has two CsrA proteins, RsmA and RsmF. Although RsmA and RsmF share a few RNA targets, RsmF is unable to bind to other targets recognized by RsmA. The goal of the present study was to better understand the basis for differential binding by RsmF. Our data indicate that RsmF binding requires target RNAs with two consensus-binding sites, while RsmA recognizes targets with just a single binding site. This information should prove useful to future efforts to define the RsmF regulon and its contribution to P. aeruginosa physiology and virulence.

Pseudomonas aeruginosa - genetics

Pseudomonas aeruginosa - metabolism

Bacterial Proteins - genetics

Protein Binding

RNA, Bacterial - genetics

Bacterial Proteins - metabolism

Nucleic Acid Conformation

Binding Sites

RNA, Bacterial - metabolism

Gene Expression Regulation, Bacterial - physiology

DNA, Bacterial

Details

Title: Subtitle: Primary and Secondary Sequence Structure Requirements for Recognition and Discrimination of Target RNAs by Pseudomonas aeruginosa RsmA and RsmF
Creators: Kayley H Schulmeyer - Department of Microbiology, University of Iowa, Iowa City, Iowa, USA
Manisha R Diaz - Department of Microbiology, University of Iowa, Iowa City, Iowa, USA
Thomas B Bair - Iowa Institute of Human Genetics, University of Iowa, Iowa City, Iowa, USA
Wes Sanders - Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
Cindy J Gode - Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
Alain Laederach - Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Caroline, USA
Matthew C Wolfgang - Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
Timothy L Yahr - Department of Microbiology, University of Iowa, Iowa City, Iowa, USA tim-yahr@uiowa.edu
Resource Type: Journal article
Publication Details: Journal of bacteriology, Vol.198(18), pp.2458-2469
Publisher: United States
DOI: 10.1128/jb.00343-16
PMID: 27381913
PMCID: PMC4999922
ISSN: 1098-5530
eISSN: 1098-5530
Grant note: R01 HL111527 / NHLBI NIH HHS T32 AI007511 / NIAID NIH HHS T32 GM082729 / NIGMS NIH HHS R01 GM101237 / NIGMS NIH HHS R01 HG008133 / NHGRI NIH HHS R01 AI097264 / NIAID NIH HHS P30 DK054759 / NIDDK NIH HHS
Language: English
Date published: 09/15/2016
Academic Unit: Microbiology and Immunology
Record Identifier: 9984001146002771

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