The RNA Biology group is interested in gene regulation by noncoding RNA molecules in bacterial pathogens and eukaryotic host cells. We use a wide range of biochemical, genetic, biocomputational and RNA deep sequencing approaches to discover new regulatory RNA molecules and their functions. Our work is aided by many fruitful collaborations with laboratories in Germany, Europe and overseas.
Small regulatory RNAs in bacteria
A major focus of our research is in small regulatory RNAs (sRNAs) that associate with the conserved RNA-binding protein Hfq in the model pathogen Salmonella Typhimurium. Hfq-dependent sRNAs constitute the largest post-transcriptional network presently known in bacteria, rivaling the complex regulations by eukaryotic microRNAs. Salmonella expresses ~100 sRNAs from both core genomic regions that are conserved in closely related Escherichia coli and from Salmonella-specific, pathogenicity islands. The Hfq-dependent sRNAs typically modulate protein synthesis by using short imperfect base-pairing with target mRNAs, thus altering translation and stability of the mRNA. We now understand that a single sRNA can regulate many target mRNAs using a highly-conserved short (â‰¥7 nucleotide) seed sequence, yet how sRNAs act select with high specificity their targets in the background of thousands of other cellular transcripts is not understood. Equally, do proteins other than Hfq help mediate sRNA activity? Other fundamental questions which we are addressing are what are the benefits of using an RNA regulator versus a transcriptional factor in complex regulatory networks; how are the sRNAs themselves regulated; and how does this relate to virulence.
Massively parallel sequencing of cellular transcripts has been revolutionizing the discovery of coding and noncoding RNAs in virtually any organism. We were one of the first groups to use RNA deep sequencing in bacteria, and developed generic methods such as differential RNA sequencing (dRNA-seq) to report the primary transcriptomes of the major human pathogen, Helicobacter pylori, and many other species. We also pioneered the use of deep sequencing to identify the interaction partners of bacterial RNA binding proteins, for example, the small noncoding RNAs and mRNA targets of Hfq, for which we combined chromosomal epitope tagging of the protein with sequencing the co-immunoprecipitated RNA. Current projects use Illumina sequencing to discover new RNA-binding proteins and the landscape of post-transcriptional regulations in bacteria and host. Furthermore, we want to develop RNA deep sequencing as a robust tool to studyâ€•in parallelâ€•the transcriptomes of bacterial pathogens and eukaryotic host over the course of infection, ideally at the single-cell level.
Whereas there has been much progress on base pairing RNAs, the abundance and mechanisms of RNA molecules that target proteins to modulate their activity is little understood. For example, may RNA molecules serve to tether virulence proteins until they are needed, or how many enzymes are targeted by regulatory RNAs to fine-tune metabolism? We are using in vivo cross-linking and RNA deep sequencing (CLIP-seq) to discover new RNA-binding proteins and map RNA-protein contacts in pathogenic bacteria. The ultimate goal is to understand how many proteins a regulatory RNA or even mRNA “seesâ€ from its birth to death, how many RNA-protein interactions there are in the cell, how many of these are productive versus non-productive, and what the productive ones look like at the molecular level.
Contrary to previous beliefs, the bacterial cell contains complex structures with many proteins, and indeed mRNAs, localized in distinct foci within the cell. An emerging focus of our research is to investigate the sub-cellular localization of sRNAs and other major components of the post-transcriptional regulon in an attempt to decipher whether sRNAs are localized in distinct regions within the cell and determine how this localization may relate to the function of each sRNA.
MicroRNAs and Long Noncoding RNAs in infected eukaryotic hosts
Research over the last decade has implicated microRNAs in a plethora of eukaryotic disease-related pathways, including the mammalian immune response, but surprisingly little remains known as to the microRNA response to bacterial infections. Likewise, it is estimated that the human and mouse genomes express several hundred long noncoding RNA (lncRNA) molecules, with transcript lengths in the range of less than 1 to more than 100 kilobases. These lncRNA seem to play important roles in the epigenetic control of gene expression and in organizing RNA-protein particles. We are investigating which microRNAs and lncRNAs play a role in the response to infections by Salmonella and other bacterial pathogens, again using systematic screening approaches followed by in-depth characterization of differentially expressed candidate molecule. For example, we recently showed that the conserved Let-7 microRNA family is down-regulated by bacterial LPS in mouse and human cells, and that this removes a post-transcriptional break on the IL-6 and IL-10 cytokine mRNAs upon infection with Salmonella.