Posttranscriptional networks in infection
and cell-cycle progression
Co-workers: Marko Noerenberg, Manuel Garcia Moreno, Aino Jaervelin (Davis lab), Caroline Lenz, David Kulozik, Thomas Davis
Our main goal is to elucidate the role that RNA-binding proteins (RBPs) play in cell-fate decisions and infection with human viruses.
Viruses require host resources for replication. As an illustrative example, Human immunodeficiency virus (HIV) RNA genome is reverse transcribed into DNA, imported into the nucleus and integrated into the cellular chromosome. After integration, HIV-1 relies on host gene expression machinery to accomplish its biological cycle. Although system-wide approaches highlighted the fundamental role that host RBPs play in HIV infection, the identity of these proteins and the mechanisms by which they influence viral gene expression remain largely unsolved.
Cell cycle also represents an important biological process to study RBP function, since cells undergoing mitosis are subjected to significant biological alterations such as nucleus disassembly and release of nuclear RBPs into the cytoplasm.
Importantly, dysfunction of certain RBPs has been associated of uncontrolled cell proliferation and cancer. Both virus infection and cell division are indeed intimately related, since some virus replicate more efficiently in cells under a specific cell-cycle phase (e.g. HIV in G2/M).
We aim at determining the repertoire of RBPs involved in virus infection and cell-cycle progression applying the state-of-the-art proteomics and RNA sequencing. We expect to identify cell-fate master regulators that will be further characterized using molecular and cellular biology methods as well as animal models. Through understanding the biological role of these RBPs, we will unveil unexplored aspects of cell division and infection.
- Regulation of host translational machinery by african Swine Fever virus. Castello, A. , Quintas, A., Sanchez, E.G., Sabina, P., Nogal, M., Carrasco, L. & Revilla, Y. PLoS Pathogens. 2009 Aug;5(8):e1000562.
- Insights into RNA biology from a mammalian cell mRNA interactome. Castello, A. , Fischer, B., Schuschke, K., Horos, R., Beckmann, B.M., Strein, C., Humphreys, D.T., Preiss, T., Steinmetz, L.M., Krijgsveld, J. and Hentze, M.W. Cell. 2012 Jun 8;149(6):1393-406.
- System-wide identification and activity landscape mapping of RNA-binding proteins by interactome capture. Castello, A. , Strein, C., Horos, R., Beckmann, B., Hentze, M. Nature Protocols. 2013 Feb 14;8(3):491-500
- RNA-Binding Proteins in Mendelian Disease and Cancer. Castello, A. , Fischer, B., Hentze, M.W. and Preiss, T. Trends in Genetics 2013 Feb 14.
- A versatile assay for RNA-binding proteins in living cells. Strein, C., Allaeume, A.M., Hentze, M.W. and Castello, A. RNA. 2014 20 (5), 721-731.
- The new (dis)order in RNA regulation. Järvelin, A.I., Noerenberg, M., Davis, I., Castello, A. (2016) Cell Com. and Signaling.
- Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila. Sysoev, V.O., Fischer, B., Frese, C.K., Gupta, I., Krijgsveld, J., Hentze, M.W., Castello, A., and Ephrussi, A. Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila. (2016) Nature Communications
- The Cardiomyocyte mRNA-Binding Proteome: Links to Intermediary Metabolism and Heart Disease. Liao, Y., Castello, A., Fischer, B., Leicht, S., Föehr, S., Frese, C.K., Ragan, C., Kurscheid, S., Pagler, E., Yang, H. Krijgsveld, J., Hentze, M.W., Preiss, T. (2016) Cell Reports.
- Comprehensive Identification of RNA-Binding Domains in Human Cells. Castello, A., Fischer, B., Frese, C.K., Horos, R., Alleaume, A.M., Foehr, S., Curk, T., Krijgsveld, J., Hentze. M.W. (2016). Mol Cell.
Figure 1: Schematic representation of mRNA interactome capture
During my postdoc in the Hentze lab, I developed a new method for identification of RBPs in living cells called mRNA interactome capture. In brief, protein-RNA complexes are frozen by UV crosslinking, purified using oligo(dT) and identified by quantitative proteomics. Castello et al., Nature Prot.
Figure 2: mRNA interactome capture specifically isolates RBPs
Upon applying mRNA interactome capture to HeLa cells, eluates are analyzed either by silver staining (left panel) or using specific antibodies against well known RBPs (PTB and CELF1) or negative controls (β-actin, α-tubulin, histone 3 [H3] and H4). RBPs are exclusively identified in eluates when UV crosslinking (cCL or PAR-CL) is applied. Adapted from Castello et al., Cell, 2012
Figure 3: RBPs can be redistributed to replication foci upon virus infection
The eukaryotic initiation factor 4G, an RBP important for the first steps of protein synthesis, is recruited to African swine fever virus factories (localized by p72 viral protein and viral DNA labeled with To-Pro-3). Adapted from Castello et al., PLoS Pathogens, 2009