MassIVE MSV000090547

Complete Public PXD037523

Implementation of PAR-CLIP to characterize RNA-protein interactions in prokaryotes at nucleotide resolution

Description

The identification of RNAs that are recognized by RNA-binding proteins (RNA-BPs) using techniques such as Crosslinking and Immunoprecipitation (CLIP) has revolutionized the genome-wide discovery of RNA-BP RNA targets. Among the different versions of CLIP that have been developed, the use of photoactivable nucleoside analogs has resulted in high efficiency photoactivable ribonucleoside-enhanced CLIP (PAR-CLIP) in vivo. Nonetheless, PAR-CLIP has not yet been applied in prokaryotes. To determine if PAR-CLIP can be used in prokaryotes, we determined suitable conditions for the incorporation of 4-thiouridine (4SU), a photoactivable nucleoside, into E. coli RNA and for the isolation of RNA crosslinked to RNA-BPs of interest. Applying this technique to Hfq, a well-characterized regulator of small RNA (sRNA)-messenger RNA (mRNA) interactions, we showed that PAR-CLIP identified most of the known sRNA targets of Hfq, as well as functionally relevant sites of Hfq-mRNA interactions at nucleotide resolution. Based on our findings, PAR-CLIP represents an improved method to identify both the RNAs and the specific regulatory sites that are recognized by RNA-BPs in prokaryotes. [doi:10.25345/C5B853P0G] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: RNA-protein interactions ; TMT ; CLIP ; Hfq ; RNA-Seq

Contact

Principal Investigators:
(in alphabetical order)
Chaitanya Jain, University of Miami, United States
Submitting User: gcrynen
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