Prof. Dr. Rolf Backofen

Central Project Z2: “Ribosome Profiling and Bioinformatics”

University of Freiburg, Informatics

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.