Proteomic analysis identifies endoribouclease EhL-PSP and EhRRP41 exosome protein as novel interactors of EhCAFl deadenylase

摘要

In higher eukaryotic cells mRNA degradation initiates by poly(A) tail shortening catalyzed by deadenylases CAF1 and CCR4. In spite of the key role of mRNA turnover in gene expression regulation, the underlying mechanisms remain poorly understood in parasites. Here, we aimed to study the function of EhCAFl and identify associated proteins in Entamoeba histolytica. By biochemical assays, we evidenced that EhCAFl has both RNA binding and deadenylase activities in vitro. EhCAFl was located in cytoplasmic P-bodies that increased in number and size after cellular stress induced by DNA damage, heat shock, ?and nitric oxide. Using pull-down assays and ESI-MS/MS mass spectrometry, we identified 15 potential EhCAFl-interacting proteins, including the endoribonuclease EhL-PSP. Remarkably, EhCAFl colocalized with EhL-PSP in cytoplasmic P-bodies in trophozoites. Bioinformatic analysis of EhL-PSP network proteins predicts a potential interaction with EhRRP41 exosome protein. Consistently, we evidenced that EhL-PSP colocalizes and physically interacts with EhRRP41. Strikingly, EhRRP41 did not coimmunoprecipitate EhCAFl, suggesting the existence of two EhL-PSP-containing complexes. In conclusion, our results showed novel interactions between mRNA degradation proteins and evidenced for the first time that EhCAFl is a functional deadenylase that interacts with EhL-PSP endoribonuclease in P-bodies, while EhL-PSP interacts with EhRRP41 exosome protein in this early-branched eukaryote.