GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation

Han, Kyu‐Sung; Tjaden, Brian; Lory, Stephen

doi:10.1038/nmicrobiol.2016.239

Cited by 87 publications

(107 citation statements)

References 50 publications

Supporting

Mentioning

107

Contrasting

Order By: Relevance

“…A related method is GRIL-seq (Global small noncoding RNA target identification by ligation and sequencing), which is based on the in vivo coexpression of an RNA ligase that fuses the sRNA to its targets. The generated chimeric RNAs can be analyzed by RNA-seq and the method comes with the advantage that the sRNA of choice does not require genetic manipulation, e.g., by adding an aptamer tag (Han, Tjaden, & Lory, 2016).…”

Section: Discussionmentioning

confidence: 99%

Transcriptomic Approaches for Studying Quorum Sensing in Vibrio cholerae

Herzog

Papenfort

2018

Methods in Enzymology

View full text Add to dashboard Cite

Transcriptome analysis using RNA-sequencing (RNA-seq) has now become the standard approach to determine the transcriptional output of an organism. Various modifications to this technology have been developed over the years, usually aiming to improve the annotation of transcript borders, or to identify novel classes of RNAs, such as small regulatory RNAs (sRNAs) and antisense transcripts. RNA-seq has also led to the identification of dozens of new sRNAs in the major human pathogen, Vibrio cholerae. Several of these sRNAs function in the context of a cell-to-cell communication process, called quorum sensing (QS). QS is key for pathogenicity and biofilm formation of V. cholerae and the

show abstract

Section: Discussionmentioning

confidence: 99%

Transcriptomic Approaches for Studying Quorum Sensing in Vibrio cholerae

Herzog

Papenfort

2018

Methods in Enzymology

View full text Add to dashboard Cite

show abstract

“…Experimental techniques to identify bacterial sRNA targets have also expanded. Many of these use affinity purification or co-immunoprecipitation approaches, with or without crosslinking (15, 17, 20, 58, 59, 60). To help streamline the process of sRNA target identification, the SPOT pipeline was constructed to be used in conjunction with other identification methods.…”

Section: Discussionmentioning

confidence: 99%

sRNA-target Prediction Organizing Tool (SPOT) integrates computational and experimental data to facilitate functional characterization of bacterial small RNAs

King

Vanderpool²,

Degnan³

2018

Preprint

View full text Add to dashboard Cite

Small RNAs (sRNAs) post-transcriptionally regulate mRNA targets, typically under conditions of environmental stress. Although hundreds of sRNAs have been discovered in diverse bacterial genomes, most sRNAs remain uncharacterized, even in model organisms. Identification of mRNA targets directly regulated by sRNAs is rate-limiting for sRNA functional characterization. To address this, we developed a computational pipeline that we named SPOT for sRNA-targetPredictionOrganizingTool. SPOT incorporates existing computational tools to search for sRNA binding sites, allows filtering based on experimental data, and organizes the results into a standardized report. SPOT sensitivity (Correctly Predicted Targets/Total Known Targets) was equal to or exceeded any individual method when used on 12 characterized sRNAs. Using SPOT, we generated a set of target predictions for the sRNA RydC, which was previously shown to positively regulatecfamRNA, encoding cyclopropane fatty acid synthase. SPOT identifiedcfaalong with additional putative mRNA targets, which we then tested experimentally. Our results demonstrated that in addition tocfamRNA, RydC also regulatestrpEandpheAmRNAs, which encode aromatic amino acid biosynthesis enzymes. Our results suggest that SPOT can facilitate elucidation of sRNA target regulons to expand our understanding of the many regulatory roles played by bacterial sRNAs.IMPORTANCESmall RNAs (sRNAs) regulate gene expression in diverse bacteria by interacting with mRNAs to change their structure, stability or translation. Hundreds of sRNAs have been identified in bacteria, but characterization of their regulatory functions is limited by difficulty with sensitive and accurate identification of mRNA targets. Thus, new robust methods of bacterial sRNA target identification are in demand. Here, we describe ourSmall RNA-targetPredictionOrganizingTool, which streamlines the process of sRNA target prediction by providing a single pipeline that combines available computational prediction tools with customizable results filtering based on experimental data. SPOT allows the user to rapidly produce a prioritized list of predicted sRNA-target mRNA interactions that serves as a basis for further experimental characterization. This tool will facilitate elucidation of sRNA regulons in bacteria, allowing new discoveries regarding the roles of sRNAs in bacterial stress responses and metabolic regulation.

show abstract

“…A comparative analysis of small RNA transcriptome of Neisseria gonorrhoeae lacking RppH with the transcriptome of wild-type cells showed that RppH influences the quality and quantity of small RNAs originating from different chromosomal locations (Wachter and Hill, 2015). It was also shown that RNA pyrophosphpohydrolase activity is required for the novel GRIL-seq technique, which was developed to identify noncoding RNA targets in bacterial cells (Han et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Nudix‐type RNA pyrophosphohydrolase provides homeostasis of virulence factor pyocyanin and functions as a global regulator in Pseudomonas aeruginosa

Kujawa

Lirski

Ziecina

et al. 2017

Molecular Microbiology

View full text Add to dashboard Cite

The PA0336 protein from Pseudomonas aeruginosa belongs to the family of widely distributed Nudix pyrophosphohydrolases, which catalyze the hydrolysis of pyrophosphate bonds in a variety of nucleoside diphosphate derivatives. The amino acid sequence of the PA0336 protein is highly similar to that of the RppH Nudix RNA pyrophosphohydrolase from Escherichia coli, which removes pyrophosphate from 5'-end of triphosphorylated RNA transcripts. Trans-complementation experiments showed that the P. aeruginosa enzyme can functionally substitute for RppH in E. coli cells indicating that, similar to RppH, the Pseudomonas hydrolase mediates RNA turnover in vivo. In order to elucidate the biological significance of the PA0336 protein in Pseudomonas cells, a PA0336 mutant strain was constructed. The mutated strain considerably increased level of the virulence factor pyocyanin compared to wild type, suggesting that PA0336 could be involved in downregulation of P. aeruginosa pathogenicity. This phenotype was reversed by complementation with the wild type but not catalytically inactive PA0336, indicating that the catalytic activity was indispensable for its biological function. Pathogenesis tests in Caenorhabditis elegans showed that the PA0336 mutant of P. aeruginosa was significantly more virulent than the parental strain, confirming further that the P. aeruginosa RNA pyrophosphohydrolase PA0336 modulates bacterial pathogenesis by down-regulating production of virulence-associated factors. To study the role of PA0336 further, transcriptomes of the PA0336 mutant and the wild-type strain were compared using RNA sequencing. The level of 537 transcripts coding for proteins involved in a variety of cellular processes such as replication, transcription, translation, central metabolism and pathogenesis, was affected by the lack of PA0336. These results indicate that the PA0336 RNA pyrophosphohydrolase functions as a global regulator that influences many of transcripts including those involved in P. aeruginosa virulence.

show abstract

GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation

Cited by 87 publications

References 50 publications

Transcriptomic Approaches for Studying Quorum Sensing in Vibrio cholerae

Transcriptomic Approaches for Studying Quorum Sensing in Vibrio cholerae

sRNA-target Prediction Organizing Tool (SPOT) integrates computational and experimental data to facilitate functional characterization of bacterial small RNAs

Nudix‐type RNA pyrophosphohydrolase provides homeostasis of virulence factor pyocyanin and functions as a global regulator in Pseudomonas aeruginosa

Contact Info

Product

Resources

About