Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq.

PLoS Genetics
Alexandra SittkaJörg Vogel

Abstract

Recent advances in high-throughput pyrosequencing (HTPS) technology now allow a thorough analysis of RNA bound to cellular proteins, and, therefore, of post-transcriptional regulons. We used HTPS to discover the Salmonella RNAs that are targeted by the common bacterial Sm-like protein, Hfq. Initial transcriptomic analysis revealed that Hfq controls the expression of almost a fifth of all Salmonella genes, including several horizontally acquired pathogenicity islands (SPI-1, -2, -4, -5), two sigma factor regulons, and the flagellar gene cascade. Subsequent HTPS analysis of 350,000 cDNAs, derived from RNA co-immunoprecipitation (coIP) with epitope-tagged Hfq or control coIP, identified 727 mRNAs that are Hfq-bound in vivo. The cDNA analysis discovered new, small noncoding RNAs (sRNAs) and more than doubled the number of sRNAs known to be expressed in Salmonella to 64; about half of these are associated with Hfq. Our analysis explained aspects of the pleiotropic effects of Hfq loss-of-function. Specifically, we found that the mRNAs of hilD (master regulator of the SPI-1 invasion genes) and flhDC (flagellar master regulator) were bound by Hfq. We predicted that defective SPI-1 secretion and flagellar phenotypes of the hfq mutant wo...Continue Reading

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Datasets Mentioned

BETA
GSE8985
GSE10149

Methods Mentioned

BETA
co-immunoprecipitation
coIP
size-fractionation
immunoprecipitation
PCR
Assay
pull down

Software Mentioned

TargetRNA
Java Start
Affymetrix
BLAST
Integrated Genome Browser
SAM Statistical Analysis of Microarrays
WU

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