Controlled cell death may occur, or not, depending on which isoform of the Fas protein is expressed—one isoform includes a molecular anchor, so that Fas fastens to the cell membrane; another isoform lacks the anchor, so that Fas floats into the cytoplasm. When it is anchored, Fas promotes cell death, or apoptosis; when it is adrift, Fas may spare the cell, even if it is dysfunctional, cancerous.
Details on the state of Fas expression—shipshape or otherwise—emerged from a study conducted by an international team of researchers. The team, which included scientists from Germany (the Helmholtz Zentrum München and the Technical University of Munich) and Spain (Centre de Regulació Genòmica), report that Fas expression depends on a protein called RNA-binding motif 5 (RBM5), which often exhibits mutations in lung tumors. RBM5, it turns out, has structural secrets that are relevant to the molecular mechanisms of tumor diseases.
The scientists presented their findings November 29 in the journal eLife, in an article entitled, “Structural Basis for the Recognition of Spliceosomal SmN/B/B’ Proteins by the RBM5 OCRE Domain in Splicing Regulation.” The article notes that the so-called OCRE (octamer repeat of aromatic residues) domain of the protein RBM5 binds to the carboxyl terminus (C-terminus) of the spliceosomal protein SmN. The OCRE domain, the article’s authors explain, determines whether the spliceosome reaches the pre-mRNA of the Fas protein, and thus decides which isoform of Fas is expressed. Essentially, it controls the balance between the two different isoforms.
“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,” said Professor Michael Sattler, senior author of the eLife paper and director of the Institute of Structural Biology (STB) at Helmholtz Zentrum München.
“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 the complex works,” explained Professor Sattler, who directed the study.
“We show that the RBM5 OCRE domain adopts a unique β–sheet fold,” wrote the authors of the eLife article. “NMR and biochemical experiments demonstrate that the OCRE domain directly binds to the proline-rich C-terminal tail of the essential snRNP core proteins SmN/B/B’.”
To confirm their findings, the scientists mutated the corresponding interaction residues of the proteins and observed the results: “Mutation of conserved aromatic residues impairs binding to the Sm proteins in vitro and compromises RBM5-mediated alternative splicing regulation of FAS/CD95.” The OCRE domain’s interaction no longer took place in the test tube. The splicing activities of RBM5 in cell culture were 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,” emphasized Professor Sattler. 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.