The Fas protein can either inhibit or promote the controlled cell death (apoptosis), depending on the isoform in which it occurs. Researchers from the Helmholtz Zentrum München, the Technical University of Munich, and the Centre for Genomic Regulation in Barcelona, in collaboration with the Institute of Molecular Genetics of Montpeller, have elucidated how this decision is guided. These results provide new insights into the molecular mechanisms of tumor diseases and have now been published in 'eLife'.
We know the problem: When assembling the parts and pieces of furniture purchased at a store, everyone uses the same blueprint. Nevertheless, the end product can differ greatly in the course of assembling the whole product over several intermediate steps. Something quite similar can happen during the production of proteins from genes. The genome (the blueprint) is first transcribed into a messenger molecule, the mRNA, and then translated into proteins (furniture). However, the mRNA can be altered and trimmed during intermediate steps in a process called alternative splicing, so that ultimately different proteins are produced from the same blueprint.
An interesting example of alternative splicing is the mRNA of the Fas gene*. Depending on which intermediate steps take place, the finished protein can either prevent or promote controlled cell death (apoptosis). "The right balance between these opposing results is dependent on the cell type and can also lead to uncontrolled cell growth and cancer when alternative splicing is dysregulated," explains Professor Michael Sattler, Director of the Institute of Structural Biology (STB) at Helmholtz Zentrum München. In collaboration with Professor Juan Valcárcel Juárez of the Centre de Regulació Genòmica (CRG) in Barcelona, he and his team have now gained insight into which intermediate steps are taken and how these lead to different isoforms of the Fas protein.
"The focus of our interest was the protein RBM5, which often exhibits mutations in lung tumors," says Dr. André Mourão of the STB. "RBM5 helps to bring the spliceosome to the mRNA by binding to a spliceosomal protein", explains coauthor Dr. Sophie Bonnal of the CRG Barcelona. In this central position, RBM5 decides which isoform of Fas is expressed and thus controls the balance between the two different isoforms.**
"By employing nuclear magnetic resonance (NMR) spectroscopy at the Bavarian NMR Center in Garching, we were able to elucidate the spatial structure of RBM5-OCRE in complex with SmN (a protein present in the spliceosome) and to understand exactly how these interaction occurs," states Sattler, who directed the study.*** To confirm their findings, the scientists mutated the corresponding interaction residues of the proteins and observed that the interactions no longer took place in the test tube and that the splicing activities of RBM5 in cell culture was impaired.
"The process of alternative splicing affects numerous essential functions and processes in an organism, and dysregulation can trigger cancer. That is why it is very important to precisely understand the mechanisms that regulate these processes,” explains Sattler, summarizing the results. According to the authors, only a few protein interactions that influence alternative splicing by binding to spliceosomal proteins have been analyzed in such structural depth. In the future, the researchers want to determine exactly how RBM5 binds to the mRNA and whether there are additional interactions with the spliceosome, which consists of numerous other components.
* Fas is also known as CD95 or APO-1. Depending on whether a specific region (exon 6) is contained in the mRNA or not, a membrane-bound pro-apoptotic protein or a soluble isoform arises in the cell interior, which counteracts apoptosis. As a pro-apoptotic protein, Fas prevents errant cells from multiplying uncontrolled, whereas the anti-apoptotic isoform leads to the proliferation of such cells.
** The name is an acronym for RNA Binding Motif 5. RBM5 is a protein, which is demonstrably dysregulated in different cancers (especially in the lungs).
*** A so-called OCRE (octamer repeat of aromatic residues) domain of the protein RBM5 binds to the C-terminus of the spliceosomal protein SmN and is thus important for the regulation of the alternative splicing.
Publication: Mourão, A. & Bonnal, S. & Soni, K. & Warner, L. et al. (2016): Structural basis for the recognition of spliceosomal SmN/B/B’ proteins by the RBM5 OCRE domain in splicing regulation. eLife, doi: 10.7554/eLife.14707