Importance of amino acids Leu135 and Tyr236 for the interaction between EhCFIm25 and RNA: a molecular dynamics simulation study

Abstract

The CFIm25 subunit of the heterotetrameric cleavage factor Im (CFIm) is a critical factor in the formation of the poly(A) tail at mRNA 3' end, regulating the recruitment of polyadenylation factors, poly(A) site selection, and cleavage/polyadenylation reactions. We previously reported the homologous protein (EhCFIm25) in Entamoeba histolytica, the protozoan causing human amoebiasis, and showed the relevance of conserved Leu135 and Tyr236 residues for RNA binding. We also identified the GUUG sequence as the recognition site of EhCFIm25. To understand the interactions network that allows the EhCFIm25 to maintain its three-dimensional structure and function, here we performed molecular dynamics simulations of wild-type (WT) and mutant proteins, alone or interacting with the GUUG molecule. Our results indicated that in the presence of the GUUG sequence, WT converged more quickly to lower RMSD values in comparison with mutant proteins. However, RMSF values showed that movements of amino acids of WT and EhCFIm25*L135 T were almost identical, interacting or not with the GUUG molecule. Interestingly, EhCFIm25*L135 T, which is the only mutant with a slight RNA binding activity experimentally, presents the same stabilization of bend structures and alpha helices as WT, notably in the C-terminus. Moreover, WT and EhCFIm25*L135 T presented almost the same number of contacts that mainly involve lysine residues interacting with the G4 nucleotide. Overall, our data proposed a clear description of the structural and mechanistic data that govern the RNA binding capacity of EhCFIm25.

mRNA Polyadenylation Machineries in Intestinal Protozoan Parasites

“…Interestingly, the absence of certain conserved residues in the Nudix domain does not significantly affect the folding neither the RNA binding of CFIm25 in E. histolytica (Ospina‐Villa et al. ,b; Pezet‐Valdez et al. ), highlighting the relevance of the domain folding.…”

“…; Ospina‐Villa et al. ,b; Zamorano et al. ) and the conservation of the polyadenylation process through the evolutionary scale, we proposed a refined model in which the EhCPSF complex binds the consensus poly(A) signal UA(A/U)UU through the EhWDR33 polypeptide, while EhCstF complex recognizes the U‐rich motif downstream the poly(A) site, tethering other factors to RNA.…”

“…Moreover, we recently demonstrated that the 25 kDa subunit of cleavage factor Im (EhCFIm25) is necessary for trophozoites survival, indicating that polyadenylation factors are valuable targets for parasite control (Ospina‐Villa et al. , ,b) as it has been reported for other protozoan parasites (Hendriks et al. ; Palencia et al.…”

Advances on Aptamers against Protozoan Parasites

“…For several years, we have been investigating RNA sequences in 3′ UTR and proteins involved in this process in E. histolytica trophozoites. Particularly, we performed the molecular and functional characterization of the 25 kDa subunit of the Cleavage Factor Im (EhCFIm25) whose homologue participates in poly(A) site selection, polyadenylation factors recruitment, RNA cleavage and poly(A) tail synthesis in eukaryotic cells [68,69,70]. Notably, we showed that EhCFIm25 is essential for parasite survival and virulence, since its silencing by specific dsRNA reduced parasite mobility, and erythrophagocytosis capacity, and induced cell death [71].…”

Unraveling the relevance of the polyadenylation factor EhCFIm25 in Entamoeba histolytica through proteomic analysis

“…As the human protein, EhCFIm25 interacts with EhPAP [ 8 ], but also with the transcriptional coactivator EhPC4 (J. D. Ospina‐Villa, our unpublished data) related to virulence, DNA replication, and multinucleation in E. histolytica [ 9 , 10 ]. Although three‐dimensional structure prediction indicates the absence of a classical RNA‐binding domain, EhCFIm25 can bind the 3′ untranslated region (3′ UTR) of E. histolytica transcripts through the participation of the conserved Leu135 and Tyr236 residues [ 11 , 12 ]. By using the SELEX strategy, we isolated two RNA aptamers that contain the GUUG motif recognized by EhCFIm25.…”