Proteomic analysis identifies endoribouclease EhL-PSP and EhRRP41 exosome protein as novel interactors of EhCAF1 deadenylase.

UNLABELLED

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 EhCAF1 and identify associated proteins in Entamoeba histolytica. By biochemical assays, we evidenced that EhCAF1 has both RNA binding and deadenylase activities in vitro. EhCAF1 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 EhCAF1-interacting proteins, including the endoribonuclease EhL-PSP. Remarkably, EhCAF1 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 EhCAF1, 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 EhCAF1 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.

BIOLOGICAL SIGNIFICANCE

This study provides evidences for the functional deadenylase activity of EhCAF1 and shows a link between different mRNA degradation proteins in E. histolytica. By proteomic tools and pull down assays, we evidenced that EhCAF1 interacts with the putative endoribonuclease EhL-PSP, which in turn interacts with exosome EhRRP41 protein. Our data suggest for the first time the presence of two complexes, one containing the endoribonuclease EhL-PSP and the deadenylase EhCAF1 in P-bodies; and another containing the endoribonuclease EhL-PSP and the exosome EhRRP41 exoribonuclease. Overall, these results provide novel data that may help to understand mRNA decay mechanisms in this parasite.