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Series GSE77463 Query DataSets for GSE77463
Status Public on Feb 14, 2017
Title Small RNA interactome revealed through crosslinking of the degradosome
Organism Escherichia coli O157:H7 str. Sakai
Experiment type Other
Summary UV-crosslining of protein-RNA complexes was employed to capture sRNA-mRNA interactions occuring on the RNA degradosome protein, RNase E, in enterohaemorhaggic E. coli. Abstract from associated mansucript: In many organisms small regulatory RNAs (sRNA) play important roles in the regulation of gene expression by base-pairing to specific target mRNAs. In enterohaemorrhagic E. coli (EHEC), sRNAs are encoded by both the “core” genome and in numerous horizontally acquired pathogenicity islands. To identify functionally important sRNA-target RNA interactions we applied crosslinking and sequencing of hybrids (CLASH) to the core degradosome component RNase E in EHEC. RNase E was shown to bind to many classes of RNA, confirming the wide distribution of degradosome targets. These included several hundred sRNA-mRNA duplexes, and the distribution of non-templated oligo(A) tails indicated that the sRNA target RNase E-mediated cleavage at these interaction sites. Functional repression of target mRNAs was confirmed for the core sRNA RyhB, and the pathogenicity-associated sRNA Esr41. In the case of Esr41, three confirmed target mRNAs participate in iron accumulation and the ∆esr41 strain showed increased growth under conditions of iron limitation. We conclude that CLASH can be used to identify functional targets for bacterial sRNAs.
 
Overall design The endoribonuclease, RNase E, was affiity tagged with a His-FLAG tag. Cultures were UV irradiated with 1800 mJ of UV-C and harvested. RNA-protein complexes were purified using M2-anti-FLAG resin and eluted but incubating with TEV protease to release the FLAG tag. RNAs were trimmed using RNase T1/A and bound to Ni-NTA resin under denaturing conditions. Complexes were washed, 5' and 3' ends phophorylated and dephophorylated respectively and RNA linkers ligated. Complexes were eluted and cDNA libraries prepared from recovered RNAs. cDNA were PCR amplified and gel extracted before Illumina HiSeq2500 sequencing. Sequence files were analysed using the hyb package and pyCRAC software.
 
Contributor(s) Tree JJ, Kudla G, Gally DP, Tollervey D
Citation(s) 27836995, 33585289
Submission date Feb 02, 2016
Last update date Feb 16, 2021
Contact name Jai Justin Tree
E-mail(s) [email protected]
Phone +61 2 938 59142
Organization name University of New South Wales
Department School of Biotechnology and Biomolecular Sciences
Lab Tree lab
Street address Rm s110 Bldg F25, UNSW, Gate 11 Botany St
City Sydney
State/province NSW
ZIP/Postal code 2033
Country Australia
 
Platforms (1)
GPL21393 Illumina HiSeq 2500 (Escherichia coli O157:H7 str. Sakai)
Samples (2)
GSM2052447 RNaseE_HTF_1
GSM2052448 RNaseE_HTF_2
Relations
BioProject PRJNA310426
SRA SRP069179

Download family Format
SOFT formatted family file(s) SOFTHelp
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Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE77463_RAW.tar 33.9 Mb (http)(custom) TAR (of GTF, HYB)
GSE77463_RNaseE_peaks_threshold100_FDR0_05.gtf.gz 18.4 Kb (ftp)(http) GTF
GSE77463_RNaseE_peaks_threshold50_FDR0_05.gtf.gz 38.9 Kb (ftp)(http) GTF
GSE77463_RNaseE_peaks_threshold50_FDR0_05_1kb_of_Hfq.gtf.gz 10.6 Kb (ftp)(http) GTF
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Raw data are available in SRA
Processed data provided as supplementary file

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