Research at a glance

In recent years, there has been increased awareness of the importance of post-transcriptional regulation of gene expression by regulatory RNAs in all three domains of life: bacteria, archaea, and eukaryotes. The major group of regulatory RNAs in bacteria is comprised of short, 50-400 nt long, RNA molecules denoted small RNAs (sRNAs). sRNAs affect many aspects of cell physiology, including pathogenicity.

Most of the sRNAs act in trans by base-pairing with their RNA targets upon binding the RNA chaperone Hfq and affect the target stability/and or translation. For a long time, a major unresolved challenge was the identification of the RNA pairs on Hfq. For this reason, we developed a broadly applicable method, named RIL-seq (RNA Interaction by Ligation and sequencing), for a transcriptome-wide identification of mRNA targets of sRNAs. In RIL-seq, RNAs are UV-crosslinked to an RNA-binding protein, and the protein is purified with its bound RNAs. Proximal RNA ends are ligated, yielding RNA chimeras. These chimeras are sequenced and filtered for statistically significant over-represented chimeras, generating a reliable dataset of RNA pairs.

Application of RIL-seq to Escherichia coli Hfq revealed thousands of novel interactions, significant re-wiring of the network upon changes in cellular conditions, and the fact that sRNAs are encoded in all regions of the genome. Application of RIL-seq to another RNA chaperone in E. coli, the understudied ProQ, demonstrated that the Hfq and ProQ interactomes overlap, resulting in competition for some RNA pairs.

Goal

The goal of the Melamed lab is to understand the roles played by sRNAs during infection and in the responses to the environment, the host, bacteriophage (phage) and other bacteria. Understanding the role sRNAs play in these relationships can be valuable for coping with different pathogens.