Central Project Z2: “Ribosome Profiling and Bioinformatics”
University of Würzburg (Germany), Institute of Molecular Infection Biology
The recent development of a variety of global approaches and new methodologies, combining bioinformatics, peptidomics, genomics and molecular biology, has greatly facilitated the genome-wide identification of small ORFs (sORFs) and their translation products, so-called small proteins of less than 50 aa. In this central project, we will provide a platform for ribosome profiling (Ribo-seq) and bioinformatics support for the SPP 2002. The Z2-project Ribosome profiling and Bioinformatics aims to identify and characterize small proteins, and to ensure that all methods will be available to all members of the SPP 2002 with high standards and reproducibility. The Z2-project will focus on the development and establishment of novel analytical tools and optimization of existing technologies (both experimentally and computationally) to detect small proteins and translation of sORFs. The recently developed Ribo-seq technology allows for a global translatome analysis based on deep sequencing of ribosome-protected fragments of mRNAs. To validate predicted sORFs and also to identify novel sORFs, we aim to establish Ribo-seq for global cataloging of translated mRNAs for a selected number of bacteria and archaea, which are studied in the individual projects within the SPP 2002. Therefore, we will adapt and modify Ribo-seq protocols for the use in the different prokaryotes. The set-up of conditions for Ribo-seq in the individual organisms will be performed in close collaboration with researchers from the individual projects. Furthermore, we will employ Ribo-seq to investigate changes in translation rates of sORFs under selected growth or stress conditions to get insights into their physiological roles. In addition, we aim to further develop prokaryotic Ribo-seq protocols, e.g., for translatome analysis of membrane-associated ribosomes or in the context of host-pathogen or symbiotic interactions. Moreover, a strong bioinformatics input is necessary for the analysis of Ribo-seq data as well as integration of genomics, transcriptomics, translatomics and peptidomics data to link the genome with the proteome level. We will provide a comprehensive analysis of Ribo-seq as well as existing or new RNA-seq data for the prediction, detection and validation of sORFs. This will provide the experimental groups with a level of bioinformatics analysis of their data that would not be achieved by the individual groups alone, and fosters a common standard within the SPP for this type of analysis. Furthermore, we will help the groups with the genome-wide search for new coding regions on the basis of mass spectrometry data and provide them with a comprehensive and extensively tested toolbox for the analysis of Ribo-seq data.
Exploring small proteins in the food-borne pathogen Campylobacter jejuni
Defining the entire gene complement of a bacterial pathogen is essential to understand how it survives and causes disease. Deep sequencing technologies have revolutionized genome sequencing and revealed an unexpected complexity in bacterial genomes and transcriptomes. The recently established ribosome profiling technique (Ribo-seq) for global translatome analysis, based on deep sequencing of ribosome-protected fragments, is revealing a wealth of novel open reading frames (ORFs) encoding potential small proteins (<50 amino acids) in diverse organisms. This class of cellular macromolecules is still understudied. The small number of characterized small proteins in bacteria is involved in diverse physiological processes, including modulation of virulence and antibiotic resistance.
The food-borne pathogen Campylobacter jejuni is currently the leading cause of bacterial gastroenteritis worldwide. However, since its annotated genome lacks homologues of key virulence factors used by other enteric pathogens, little is known about how it causes disease. Our comparative RNA-seq-based transcriptome analysis of multiple C. jejuni strains revealed conserved and strain-specific transcriptional output, including many novel transcripts, and suggests much remains to be learned about how its expressed genome contributes to virulence. In this project we aim to define the C. jejuni small proteome and functionally characterize selected small proteins. Translatome analysis under different growth conditions using Ribo-seq combined with our transcriptome maps and comparative genomics will identify expressed C. jejuni small ORFs. Translation will be further validated using mass spectrometry and epitope tagging. Our first Ribo-seq of C. jejuni grown in broth culture has already identified several potentially translated small proteins, including Cj0878 (48 codons), for which we have validated translation using a GFP reporter. Cj0878 is highly conserved in Campylobacter and its amino acid sequence suggests it is basic, amphipathic, and might associate with the cell envelope. Cj0878 also appears to be regulated post-transcriptionally by a flagellar co-regulated sRNA, and is transcriptionally induced under iron limitation. Subcellular localization studies as well as phenotypic analyses, infection studies in 3D intestinal tissue models, and gene expression analyses of wild-type and Cj0878 mutant strains will be used to study its role and mechanisms in C. jejuni physiology. To identify potential biochemical and genetic interaction partners of Cj0878, we will employ co-immunoprecipitation and transposon-sequencing, respectively. We also aim to develop a protocol for selective capture of bacterial ribosomes for Ribo-seq studies in infection samples. Overall, this project will fill in gaps in the genome map of C. jejuni. Moreover, it might reveal novel paradigms of small protein activity, which could be used as novel targets for antimicrobial strategies in diverse pathogens.