Comparative genomics and interactomics of polyadenylation factors for the prediction of new parasite targets: Entamoeba histolytica as a working model.

Protein-protein interactions (PPI) play a key role in predicting the function of a target protein and drug ability to affect an entire biological system. Prediction of PPI networks greatly contributes to determine a target protein and signal pathways related to its function. Polyadenylation of mRNA 3'-end is essential for gene expression regulation and several polyadenylation factors have been shown as valuable targets for controlling protozoan parasites that affect human health. Here, by using a computational strategy based on sequence-based prediction approaches, phylogenetic analyses, and computational prediction of PPI networks, we compared interactomes of polyadenylation factors in relevant protozoan parasites and the human host, to identify key proteins and define potential targets for pathogen control. Then, we used Entamoeba histolytica as a working model to validate our computational results. RT-qPCR assays confirmed the coordinated modulation of connected proteins in the PPI network and evidenced that silencing of the bottleneck protein EhCFIm25 affects the expression of interacting proteins. In addition, molecular dynamics simulations and docking approaches allowed to characterize the relationships between EhCFIm25 and Ehnopp34, two connected bottleneck proteins. Interestingly, the experimental identification of EhCFIm25 interactome confirmed the close relationships among proteins involved in gene expression regulation and evidenced new links with moonlight proteins in E. histolytica, suggesting a connection between RNA biology and metabolism as described in other organisms. Altogether, our results strengthened the relevance of comparative genomics and interactomics of polyadenylation factors for the prediction of new targets for the control of these human pathogens.

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

We recently reported that silencing of the polyadenylation factor EhCFIm25 in Entamoeba histolytica, the protozoan which causes human amoebiasis, affects trophozoite proliferation, death, and virulence, suggesting that EhCFIm25 may have potential as a new biochemical target. Here, we performed a shotgun proteomic analysis to identify modulated proteins that could explain this phenotype. Data are available via ProteomeXchange with identifier PXD027784. Our results revealed changes in the abundance of 75 proteins. Interestingly, STRING analysis, functional GO-term annotations, KEGG analyses, and literature review showed that modulated proteins are mainly related to glycolysis and carbon metabolism, cytoskeleton dynamics, and parasite virulence, as well as gene expression and protein modifications. Further studies are needed to confirm the hypotheses emerging from this proteomic analysis, to thereby acquire a comprehensive view of the molecular mechanisms involved.

Expression of zebrafish cpsf6 in embryogenesis and role of protein domains on subcellular localization.

CPSF6 is a component of the CFIm complex, involved in mRNA 3'end processing. Despite increasing interest on this protein as a consequence of proposed roles in cancer and HIV infection, several aspects of CPSF6 biological function are poorly understood. In this work we studied the expression of the zebrafish ortholog cpsf6 in early stages of embryo development. Quantitative RT-PCR studies showed that zebrafish cpsf6 mRNA is maternally inherited and that its concentration markedly decreases during early development. We found a generalized distribution of cpsf6 mRNA in early stages through whole mount hybridization experiments. By performing Western blot, we also found a decrease in zebrafish Cpsf6 levels during development. Our analysis of the subcellular localization of this protein using a heterologous system showed a distinct pattern characterized by the presence of nuclear foci. We also studied the relevance of different protein domains on subcellular localization, showing that the C-terminal domain is critical for nuclear localization. Collectively, our results showed that cpsf6 expression changes during early development and that the subcellular localization of the protein is similar to that of the human ortholog.

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

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.

Targeting the polyadenylation factor EhCFIm25 with RNA aptamers controls survival in Entamoeba histolytica.

Messenger RNA 3'-end polyadenylation is an important regulator of gene expression in eukaryotic cells. In our search for new ways of treating parasitic infectious diseases, we looked at whether or not alterations in polyadenylation might control the survival of Entamoeba histolytica (the agent of amoebiasis in humans). We used molecular biology and computational tools to characterize the mRNA cleavage factor EhCFIm25, which is essential for polyadenylation in E. histolytica. By using a strategy based on the systematic evolution of ligands by exponential enrichment, we identified single-stranded RNA aptamers that target EhCFIm25. The results of RNA-protein binding assays showed that EhCFIm25 binds to the GUUG motif in vitro, which differs from the UGUA motif bound by the homologous human protein. Accordingly, docking experiments and molecular dynamic simulations confirmed that interaction with GUUG stabilizes EhCFIm25. Incubating E. histolytica trophozoites with selected aptamers inhibited parasite proliferation and rapidly led to cell death. Overall, our data indicate that targeting EhCFIm25 is an effective way of limiting the growth of E. histolytica in vitro. The present study is the first to have highlighted the potential value of RNA aptamers for controlling this human pathogen.

Trypanosoma brucei EIF4E2 cap-binding protein binds a homolog of the histone-mRNA stem-loop-binding protein.

Trypanosomatids are parasitic protozoans characterized by several unique structural and metabolic processes that include exquisite mechanisms associated with gene expression and regulation. During the initiation of protein synthesis, for instance, mRNA selection for translation seems to be mediated by different eIF4F-like complexes, which may play a significant role in parasite adaptation to different hosts. In eukaryotes, the heterotrimeric eIF4F complex (formed by eIF4E, eIF4G, and eIF4A) mediates mRNA recognition and ribosome binding and participates in various translation regulatory events. Six eIF4Es and five eIF4Gs have been described in trypanosomatids with several of these forming different eIF4F-like complexes. This has raised questions about their role in differential mRNA translation. Here we have studied further TbEIF4E2, the least known eIF4E homologue from Trypanosoma brucei, and found that it is not associated with an eIF4G homolog. It is, however, associated with mature mRNAs and binds to a histone mRNA stem-loop-binding protein (SLBP), one of two Trypanosoma SLBP homologs (TbSLBP1 and TbSLBP2). TbSLBP1 is more similar to the mammalian counterpart while TbSLBP2 is exclusive to trypanosomatids and related organisms. TbSLBP2 binds to TbEIF4E2 through a conserved central region missing in other SLBP homologs. Both SLBPs, as well as TbEIF4E2, were found to localize to the cytoplasm. TbEIF4E2 and TbSLBP2 are differentially expressed during cell culture, being more abundant in early-log phase, with TbSLBP2 also showing cell-cycle dependent expression. The new data reinforce unique aspects of the trypanosomatid eIF4Es, with the TbEIF4E2-TbSLBP complex possibly having a role in differential selection of mRNAs containing stem-loop structures.

Silencing the cleavage factor CFIm25 as a new strategy to control Entamoeba histolytica parasite.

The 25 kDa subunit of the Clevage Factor Im (CFIm25) is an essential factor for messenger RNA polyadenylation in human cells. Therefore, here we investigated whether the homologous protein of Entamoeba histolytica, the protozoan responsible for human amoebiasis, might be considered as a biochemical target for parasite control. Trophozoites were cultured with bacterial double-stranded RNA molecules targeting the EhCFIm25 gene, and inhibition of mRNA and protein expression was confirmed by RT-PCR and Western blot assays, respectively. EhCFIm25 silencing was associated with a significant acceleration of cell proliferation and cell death. Moreover, trophozoites appeared as larger and multinucleated cells. These morphological changes were accompanied by a reduced mobility, and erythrophagocytosis was significantly diminished. Lastly, the knockdown of EhCFIm25 affected the poly(A) site selection in two reporter genes and revealed that EhCFIm25 stimulates the utilization of downstream poly(A) sites in E. histolytica mRNA. Overall, our data confirm that targeting the polyadenylation process represents an interesting strategy for controlling parasites, including E. histolytica. To our best knowledge, the present study is the first to have revealed the relevance of the cleavage factor CFIm25 as a biochemical target in parasites.

Guía de práctica clínica para la prevención y manejo del cáncer de cuello uterino. Guía técnica / Guide to clinical practice for the prevention and management of cervical cancer. Technical guide

La guía incluye estrategias innovadoras, como un modelo de tamizaje y tratamiento de casos que orienta a los profesionales de la salud para el manejo adecuado del cáncer de cuello uterino a nivel nacional

Pilocarpine-induced seizures trigger differential regulation of microRNA-stability related genes in rat hippocampal neurons.

Epileptogenesis in the temporal lobe elicits regulation of gene expression and protein translation, leading to reorganization of neuronal networks. In this process, miRNAs were described as being regulated in a cell-specific manner, although mechanistics of miRNAs activity are poorly understood. The specificity of miRNAs on their target genes depends on their intracellular concentration, reflecting the balance of biosynthesis and degradation. Herein, we confirmed that pilocarpine application promptly (<30 min) induces status epilepticus (SE) as revealed by changes in rat electrocorticogram particularly in fast-beta range (21-30 Hz). SE simultaneously upregulated XRN2 and downregulated PAPD4 gene expression in the hippocampus, two genes related to miRNA degradation and stability, respectively. Moreover, SE decreased the number of XRN2-positive cells in the hilus, while reduced the number of PAPD4-positive cells in CA1. XRN2 and PAPD4 levels did not change in calretinin- and CamKII-positive cells, although it was possible to determine that PAPD4, but not XRN2, was upregulated in parvalbumin-positive cells, revealing that SE induction unbalances the accumulation of these functional-opposed proteins in inhibitory interneurons that directly innervate distinct domains of pyramidal cells. Therefore, we were able to disclose a possible mechanism underlying the differential regulation of miRNAs in specific neurons during epileptogenesis.

Interference on cytoplasmic polyadenylation element-binding proteins affects the invasion ability of glioma stem cells.

Glioma stem cells derived from primary cultures were divided into an experiment group, a control group, and a blank group and subjected to cytoplasmic polyadenilation element-binding protein (CPEBs) interference, transfection with empty vector, and normal culture, respectively, to compare their invasion abilities. Western blotting showed that siRNA-3 had the strongest interfering effect on CPEBs. CPEBs were expressed in the experiment group with green fluorescence at an expression rate of over 70%. Significantly lower CPEB expression was observed in the experiment group compared to in the control and blank groups (P < 0.05). After 48-h treatment, the apoptotic rate in the experiment group was 21.43%, which was significantly higher than that in the blank (0.51%) and control (1.43%) groups (P < 0.05). After 3 days of treatment, the experiment group grew significantly more slowly than did the control and blank groups (P < 0.05). The transwell invasion assay showed that significantly fewer cells in the experiment group penetrated the membrane than did cells in the control and blank groups (P < 0.05). After CPEB interference, the growth, proliferation, and invasion of glioma stem cells were substantially inhibited, providing support for targeted therapy of glioma and for improving prognosis.

The 25 kDa subunit of cleavage factor Im Is a RNA-binding protein that interacts with the poly(A) polymerase in Entamoeba histolytica.

In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability and translation. Taking advantage of the knowledge of genomic sequences of Entamoeba histolytica, the protozoan responsible for human amoebiasis, we previously reported the putative polyadenylation machinery of this parasite. Here, we focused on the predicted protein that has the molecular features of the 25 kDa subunit of the Cleavage Factor Im (CFIm25) from other organisms, including the Nudix (nucleoside diphosphate linked to another moiety X) domain, as well as the RNA binding domain and the PAP/PAB interacting region. The recombinant EhCFIm25 protein (rEhCFIm25) was expressed in bacteria and used to generate specific antibodies in rabbit. Subcellular localization assays showed the presence of the endogenous protein in nuclear and cytoplasmic fractions. In RNA electrophoretic mobility shift assays, rEhCFIm25 was able to form specific RNA-protein complexes with the EhPgp5 mRNA 3´ UTR used as probe. In addition, Pull-Down and LC/ESI-MS/MS tandem mass spectrometry assays evidenced that the putative EhCFIm25 was able to interact with the poly(A) polymerase (EhPAP) that is responsible for the synthesis of the poly(A) tail in other eukaryotic cells. By Far-Western experiments, we confirmed the interaction between the putative EhCFIm25 and EhPAP in E. histolytica. Taken altogether, our results showed that the putative EhCFIm25 is a conserved RNA binding protein that interacts with the poly(A) polymerase, another member of the pre-mRNA 3' end processing machinery in this protozoan parasite.

The FIP-1 like polyadenylation factor in trypanosomes and the structural basis for its interaction with CPSF30.

In trypanosomes transcription is polycistronic and individual mRNAs are generated by a trans-splicing/polyadenylation coupled reaction. We identified a divergent trypanosome FIP1-like, a factor required for mRNA 3' end formation from yeasts to human. Here we showed that it is a nuclear protein with a speckled distribution essential for trypanosome viability. A strong interaction was found between TcFIP1-like and TcCPSF30, a component of the polyadenylation complex. We determined the specific amino acids in each protein involved in the interaction. Significant differences were found between the trypanosome interaction surface and its human counterpart. Although CPSF30/FIP1 interaction is known in other organisms, this is the first report mapping the interaction surface at the amino acid level.