Final Report Summary - POSTTRANS (An interdisciplinary genome-wide study of posttranscriptional regulation by small RNAs: from individual interactions to networks and evolution)
Gene expression is tightly regulated at various levels. A main regulation mechanism operates before the gene is transcribed from DNA to RNA, and involves transcription factors that bind the DNA and influence the transcript level. In recent years it has been acknowledged that post-transcriptional regulation by non-coding small RNAs (sRNAs in bacteria and microRNAs in higher organisms) plays also a major role in gene expression regulation. Many sRNAs and microRNAs were identified in many organisms, suggesting that this level of regulation might be as prominent as transcription regulation. Yet, while for transcription factors many of their targets were revealed, little was known about the targets of the non-coding RNAs. Such knowledge is essential for understanding the physiological roles of the various sRNAs. A major focus of our study regarded large-scale experimental-computational determination of targets of sRNAs in bacteria. This involved development of an experimental protocol using a variation of deep sequencing to discover targets of non-coding RNAs, as well as a computational pipeline to analyze the deep sequencing results and filter spurious targets. By applying our methodology to the model organism Escherichia coli grown under three different conditions, we managed to increase substantially the number of deciphered sRNA-target interactions. Our methodology can be applied to other bacteria, including pathogenic bacteria, towards understanding the role of sRNAs in pathogenicity. Having sufficient data of transcription factor-target and small RNA-target interactions enables the description of the transcriptional and post-transcriptional regulatory networks. We apply network analysis tools to these networks to reveal regulatory circuits involving the two layers of regulation and study their functionality and dynamics.
Additional important players in the post-transcriptional regulation of gene expression are endoribonucleases, which are enzymes that cleave RNA and play important roles in the processing and/or degradation of RNA transcripts. A major goal is to identify their cleavage targets and cleavage specificities. In the current study we focused on bacterial RNase III, an endoribonuclease that is known to cleave double stranded RNA, however the full scope of its targets is yet to be found. We applied to E. coli cells a tailored RNA-seq-based technology together with state of the art computational analysis, which have allowed transcriptome-wide in-vivo mapping of RNase III cleavage sites at a nucleotide resolution. We generated a comprehensive map of the cleavage sites in both intra-molecular and inter-molecular duplex substrates, providing novel insights into its in-vivo cleavage rules and setting the framework for the study of interweaved sRNA and RNase III post-transcriptional regulation.
Additional important players in the post-transcriptional regulation of gene expression are endoribonucleases, which are enzymes that cleave RNA and play important roles in the processing and/or degradation of RNA transcripts. A major goal is to identify their cleavage targets and cleavage specificities. In the current study we focused on bacterial RNase III, an endoribonuclease that is known to cleave double stranded RNA, however the full scope of its targets is yet to be found. We applied to E. coli cells a tailored RNA-seq-based technology together with state of the art computational analysis, which have allowed transcriptome-wide in-vivo mapping of RNase III cleavage sites at a nucleotide resolution. We generated a comprehensive map of the cleavage sites in both intra-molecular and inter-molecular duplex substrates, providing novel insights into its in-vivo cleavage rules and setting the framework for the study of interweaved sRNA and RNase III post-transcriptional regulation.