"Cells have the beautiful capacity to assemble a great variety of dynamic structures performing very complex and specific tasks. Many of these macromolecular complexes form based on the self-organization of their constituting elements. The mitotic spindle is one of these wonderful cellular machineries, which is dedicated to the accurate segregation of the chromosomes during cell division."
RNA-dependent proteins during cell division
Mitotic spindle (microtubules are seen in magenta, chromosomes in blue, the green dots reveal protein-protein interactions as detected per proximity ligation assay). Confocal microscopy image (Jana Theiß).
We have created an atlas of RNA-dependent proteins (see concept below) which highlights a great number of mitotic factors as unconventional RNA-binding proteins: they seem to bind to RNA, in absence of known RNA-binding domains. This suggest an important role for RNA during cell division, in multiple parts of the cell division machinery (microtubules, condensed chromosomes, kinetochores, centrosomes etc ...), which we aim to understand.
The concept of RNA dependence
We developed a proteome-wide, specific, and quantitative analysis of RNA-dependent proteins based on density gradient ultracentrifugation. The R-DeeP enables the quantification of the RNA-dependent fraction of a protein and guided the discovery of unexpected RNA functions.
We recently developed the concept of RNA dependence, where we defined a protein as RNA dependent when its interactome depends on the presence of RNA. This concept has been translated into a comprehensive proteome-wide, unbiased and quantitative screening method called R-DeeP.
R-DeeP is based on the fractionation of cellular lysate – with and without prior RNase treatment – by sucrose density gradient ultracentrifugation and subsequent analysis by proteome-wide mass spectrometry or Western blotting of individual proteins.
R-DeeP determines the ability of a protein to form protein complexes exclusively in the presence of RNA by direct or indirect interaction with the RNA molecules. In the R-DeeP screen, we identified a significant enrichment for microtubule-related proteins. This indicates previously unknown potential functional implications of RNA-protein interactions, particularly in the context of cell division.
Please check our updated database at R-DeeP3.dkfz.de
The RBP2GO database
The analysis of RNA-protein complexes is central to elucidating the complex molecular networks that govern cellular processes. With the increasing recognition of their importance, proteins that interact directly with RNA have attracted growing interest. However, the comprehensive but specific identification of such RNA-binding proteins (RBPs) and the dissection of their functions in association with RNA remain major challenges in the field of RNA biology.
To further deepen our knowledge of RBPs, several proteome-wide strategies have been developed to identify RBPs in different species. Thus, a large number of studies have provided catalogs of experimentally identified as well as predicted RBP candidates. The rapid evolution of the field has led to an accumulation of isolated datasets, hampering both accessibility and comparability. Furthermore, tools for linking RBPs to cellular pathways and functions were lacking. Driven by our own research and as a service to the scientific community, we developed RBP2GO, a comprehensive database of all available proteome-wide datasets for RBPs across 13 species, providing a catalog of 22552 RBP candidates (RBP2GO.DKFZ.de). It not only includes data on RBP interaction partners but also provides information on related biological processes, molecular functions, and cellular components.
RBP2GO provides a user-friendly web interface with a scoring system that reflects the potential of RBP candidates to be bona fide RBPs. The RBP2GO advanced search provides users with options to navigate through the interconnected datasets to promote and stimulate new research directions.
Ongoing projects are currently refining the list of RBPs by incorporating information on RNA-binding domains (RBDs) and RNA-related protein families. These multi-purpose analyses aim to refine the extensive list of RBP candidates and to provide a list of high confidence RBPs, based on a scoring system.