High-Resolution, High-Throughput Analysis of Hfq-Binding Sites Using UV Crosslinking and Analysis of cDNA (CRAC)

Abstract

Small regulatory nonprotein-coding RNAs (sRNAs) have emerged as ubiquitous and abundant regulators of gene expression in a diverse cross section of bacteria. They play key roles in most aspects of bacterial physiology, including central metabolism, nutrient acquisition, virulence, biofilm formation, and outer membrane composition. RNA sequencing technologies have accelerated the identification of bacterial regulatory RNAs and are now being employed to understand their functions. Many regulatory RNAs require protein partners for activity, or modulate the activity of interacting proteins. Understanding how and where proteins interact with the transcriptome is essential to elucidate the functions of the many sRNAs. Here, we describe the implementation in bacteria of a UV-crosslinking technique termed CRAC that allows stringent, transcriptome-wide recovery of bacterial RNA-protein interaction sites in vivo and at base-pair resolution. We have used CRAC to map protein-RNA interaction sites for the RNA chaperone Hfq and ribonuclease RNase E in pathogenic E. coli, and toxins from toxin-antitoxin systems in Mycobacterium smegmatis, demonstrating the broad applicability of this technique.

Defining the RNA interactome by total RNA ‐associated protein purification

“…We initially irradiated with 1.4 J cm −2 , since similar doses have previously been used in many publications mapping protein‐binding sites on RNA in yeast and E. coli (e.g. see Bohnsack et al , ; Sy et al , ). The workflow is outlined in Fig A.…”

Ribosome maturation by the endoribonuclease YbeY stabilizes a type 3 secretion system transcript required for virulence of enterohemorrhagic Escherichia coli

“…Linkers were ligated during purification, and cDNA libraries were synthesized from eluted RNA, PCR-amplified, and sequenced using the Illumina MiSeq platform. The YbeY binding profile was analyzed using the pyCRAC package ( 39 ), and statistically significant peaks were identified using pyCalculateFDR as described previously ( 40 ) ( Fig. 4 A ).…”

“…PCR-amplified cDNA libraries were sequenced using an Illumina MiSeq platform. Data analysis was performed using the pyCRAC software package ( 39 ) as described in Sy et al ( 40 ). To identify statistically significant YbeY-binding sites, pyCalculateFDR was used on mapped reads with the following settings: 100-nt flanking sequence for features (−r, 100), p value threshold of 0.05 (−m, 0.05), minimum coverage of 10 reads (−min, 10), and 500 iterations (−iterations, 500).…”

The Sponge RNAs of bacteria – How to find them and their role in regulating the post-transcriptional network

“…-Increased purification and stringency ensures the sample is not contaminated by other proteins or RNAs. -No information as to which RNAs interact with each other, so therefore like RIPseq computational and molecular work is required to determine [43,92,93] which RNAs in the list interact. CLASH (Crosslinking, ligation and sequencing of hybrids) This is a variation of CLIP and CRAC where not only is UV crosslinking used to pinpoint the exact point where the protein and RNA interact, but the interacting RNAs are ligated by proximity ligation to created chimeric RNAs.…”