@article{MichauxGerovacHansenetal.2023,
  author    = {Michaux, Charlotte and Gerovac, Milan and Hansen, Elisabeth E. and Barquist, Lars and Vogel, J{\"o}rg},
  title     = {Grad-seq analysis of Enterococcus faecalis and Enterococcus faecium provides a global view of RNA and protein complexes in these two opportunistic pathogens},
  series = {microLife},
  volume    = {4},
  journal   = {microLife},
  doi       = {10.1093/femsml/uqac027},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-313311},
  year      = {2023},
  abstract  = {Enterococcus faecalis and Enterococcus faecium are major nosocomial pathogens. Despite their relevance to public health and their role in the development of bacterial antibiotic resistance, relatively little is known about gene regulation in these species. RNA-protein complexes serve crucial functions in all cellular processes associated with gene expression, including post-transcriptional control mediated by small regulatory RNAs (sRNAs). Here, we present a new resource for the study of enterococcal RNA biology, employing the Grad-seq technique to comprehensively predict complexes formed by RNA and proteins in E. faecalis V583 and E. faecium AUS0004. Analysis of the generated global RNA and protein sedimentation profiles led to the identification of RNA-protein complexes and putative novel sRNAs. Validating our data sets, we observe well-established cellular RNA-protein complexes such as the 6S RNA-RNA polymerase complex, suggesting that 6S RNA-mediated global control of transcription is conserved in enterococci. Focusing on the largely uncharacterized RNA-binding protein KhpB, we use the RIP-seq technique to predict that KhpB interacts with sRNAs, tRNAs, and untranslated regions of mRNAs, and might be involved in the processing of specific tRNAs. Collectively, these datasets provide departure points for in-depth studies of the cellular interactome of enterococci that should facilitate functional discovery in these and related Gram-positive species. Our data are available to the community through a user-friendly Grad-seq browser that allows interactive searches of the sedimentation profiles (https://resources.helmholtz-hiri.de/gradseqef/).},
  language  = {en}
}
@phdthesis{Hoer2020,
  author    = {H{\"o}r, Jens},
  title     = {Discovery of RNA/protein complexes by Grad-seq},
  doi       = {10.25972/OPUS-21181},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-211811},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Complex formation between macromolecules constitutes the foundation of most cellular processes. Most known complexes are made up of two or more proteins interacting in order to build a functional entity and therefore enabling activities which the single proteins could otherwise not fulfill. With the increasing knowledge about noncoding RNAs (ncRNAs) it has become evident that, similar to proteins, many of them also need to form a complex to be functional. This functionalization is usually executed by specific or global RNA-binding proteins (RBPs) that are specialized binders of a certain class of ncRNAs. For instance, the enterobacterial global RBPs Hfq and ProQ together bind >80 \% of the known small regulatory RNAs (sRNAs), a class of ncRNAs involved in post-transcriptional regulation of gene expression. However, identification of RNA-protein interactions so far was performed individually by employing low-throughput biochemical methods and thereby hindered the discovery of such interactions, especially in less studied organisms such as Gram-positive bacteria. Using gradient profiling by sequencing (Grad-seq), the present thesis aimed to establish high-throughput, global RNA/protein complexome resources for Escherichia coli and Streptococcus pneumoniae in order to provide a new way to investigate RNA-protein as well as protein-protein interactions in these two important model organisms. In E. coli, Grad-seq revealed the sedimentation profiles of 4,095 (∼85 \% of total) transcripts and 2,145 (∼49 \% of total) proteins and with that reproduced its major ribonucleoprotein particles. Detailed analysis of the in-gradient distribution of the RNA and protein content uncovered two functionally unknown molecules—the ncRNA RyeG and the small protein YggL—to be ribosomeassociated. Characterization of RyeG revealed it to encode for a 48 aa long, toxic protein that drastically increases lag times when overexpressed. YggL was shown to be bound by the 50S subunit of the 70S ribosome, possibly indicating involvement of YggL in ribosome biogenesis or translation of specific mRNAs. S. pneumoniae Grad-seq detected 2,240 (∼88 \% of total) transcripts and 1,301 (∼62 \% of total) proteins, whose gradient migration patterns were successfully reconstructed, and thereby represents the first RNA/protein complexome resource of a Gram-positive organism. The dataset readily verified many conserved major complexes for the first time in S. pneumoniae and led to the discovery of a specific interaction between the 3'!5' exonuclease Cbf1 and the competence-regulating ciadependent sRNAs (csRNAs). Unexpectedly, trimming of the csRNAs by Cbf1 stabilized the former, thereby promoting their inhibitory function. cbf1 was further shown to be part of the late competence genes and as such to act as a negative regulator of competence.},
  subject      = {Multiproteinkomplex},
  language  = {en}
}
@article{GerovaWickeChiharaetal.2021,
  author    = {Gerova, Milan and Wicke, Laura and Chihara, Kotaro and Schneider, Cornelius and Lavigne, Rob and Vogel, J{\"o}rg},
  title     = {A grad-seq view of RNA and protein complexes in Pseudomonas aeruginosa under standard and bacteriophage predation conditions},
  series = {mbio},
  volume    = {12},
  journal   = {mbio},
  number    = {1},
  doi       = {10.1128/mBio.03454-20},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259054},
  pages     = {e03454-20},
  year      = {2021},
  abstract  = {The Gram-negative rod-shaped bacterium Pseudomonas aeruginosa is not only a major cause of nosocomial infections but also serves as a model species of bacterial RNA biology. While its transcriptome architecture and posttranscriptional regulation through the RNA-binding proteins Hfq, RsmA, and RsmN have been studied in detail, global information about stable RNA-protein complexes in this human pathogen is currently lacking. Here, we implement gradient profiling by sequencing (Grad-seq) in exponentially growing P. aeruginosa cells to comprehensively predict RNA and protein complexes, based on glycerol gradient sedimentation profiles of >73\% of all transcripts and ∼40\% of all proteins. As to benchmarking, our global profiles readily reported complexes of stable RNAs of P. aeruginosa, including 6S RNA with RNA polymerase and associated product RNAs (pRNAs). We observe specific clusters of noncoding RNAs, which correlate with Hfq and RsmA/N, and provide a first hint that P. aeruginosa expresses a ProQ-like FinO domain-containing RNA-binding protein. To understand how biological stress may perturb cellular RNA/protein complexes, we performed Grad-seq after infection by the bacteriophage ΦKZ. This model phage, which has a well-defined transcription profile during host takeover, displayed efficient translational utilization of phage mRNAs and tRNAs, as evident from their increased cosedimentation with ribosomal subunits. Additionally, Grad-seq experimentally determines previously overlooked phage-encoded noncoding RNAs. Taken together, the Pseudomonas protein and RNA complex data provided here will pave the way to a better understanding of RNA-protein interactions during viral predation of the bacterial cell. IMPORTANCE Stable complexes by cellular proteins and RNA molecules lie at the heart of gene regulation and physiology in any bacterium of interest. It is therefore crucial to globally determine these complexes in order to identify and characterize new molecular players and regulation mechanisms. Pseudomonads harbor some of the largest genomes known in bacteria, encoding ∼5,500 different proteins. Here, we provide a first glimpse on which proteins and cellular transcripts form stable complexes in the human pathogen Pseudomonas aeruginosa. We additionally performed this analysis with bacteria subjected to the important and frequently encountered biological stress of a bacteriophage infection. We identified several molecules with established roles in a variety of cellular pathways, which were affected by the phage and can now be explored for their role during phage infection. Most importantly, we observed strong colocalization of phage transcripts and host ribosomes, indicating the existence of specialized translation mechanisms during phage infection. All data are publicly available in an interactive and easy to use browser.},
  language  = {en}
}