Alfredo Castello Palomares
Posttranscriptional networks in infection
RNA is a central molecule in virus biology; in RNA viruses it functions not only as messenger (m)RNA, but also as genome. Because the complexity of the RNA life, the host cell dedicates more than 1,500 proteins to RNA metabolism, referred to as RNA-binding proteins (RBPs). In contrast, viral genomes typically encode few proteins, only a handful of which are RBPs. Hence, viruses rely on the repertoire of host RBPs to replicate, translate and package their RNA(s). On the other hand, host antiviral sensors are mostly RBPs that recognise intermediaries of viral replication, such as double stranded RNA, triggering the cell’s antiviral programmes. Therefore, cellular RBPs are key molecular partners of viruses, either helping or preventing viral replication. We thus hypothesise that host RBPs can be exploited as, still unexplored, antiviral targets.
My laboratory aims to identify the full complement of RBPs involved in virus infection employing system-wide approaches and viruses with distinct biological cycles. These include human immunodeficiency virus (HIV), sindbis virus (SINV) and influenza virus (FLU). We combine state-of-the-art proteomic (“RNA-interactome capture”; Castello et al., Cell 2012; Mol Cell 2016), transcriptomic (iCLIP; Koenig et al., NSMB, 2010), molecular and cellular biology techniques and super-resolution microscopy, to discover RBPs with important roles in virus infection and to characterise the molecular mechanisms underpinning their pro- or antiviral activities.
- 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