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In vitro tRNA Methylation Assay with the Entamoeba histolytica DNA and tRNA Methyltransferase Dnmt2 (Ehmeth) Enzyme
Published on: October 19, 2010
Entamoeba histolytica: cloning and expression of the poly(A) polymerase EhPAP
Jessica García-Vivas1, César López-Camarillo, Elisa Azuara-Liceaga
1Programa Institucional de Biomedicina Molecular, Escuela Nacional de Medicina y Homeopatía del IPN, Guillermo Massieu Helguera #239, Fracc. La Escalera, Ticomán, Mexico D.F. 07300, Mexico.
Abstract:
In eukaryotes, polyadenylation of pre-mRNA 3' end is essential for mRNA export, stability, and translation. Here we identified and cloned a gene codifying for a putative nuclear poly(A) polymerase (EhPAP) in Entamoeba histolytica. Protein sequence alignments with eukaryotic PAPs showed that EhPAP has the RNA-binding region and the PAP central domain with the catalytic nucleotidyl transferase domain described for other nuclear PAPs. Recombinant EhPAP expressed in bacteria was used to generate specific antibodies, which recognized two EhPAP isoforms of 60 and 63kDa in nuclear and cytoplasmic extracts by Western blot assays. RT-PCR assays showed that EhPap mRNA expression varies in multidrug-resistant trophozoites growing in different emetine concentrations. Moreover, EhPap mRNA expression is about 10- and 7-fold increased in G1 and S phase, respectively, through cell cycle progression. These results suggest the existence of a link between EhPAP expression and MDR and cell cycle regulation, respectively.
Insights
Researchers identified a nuclear poly(A) polymerase (EhPAP) in Entamoeba histolytica, finding its expression links to multidrug resistance and cell cycle regulation.
Area of Science:
- Molecular Biology
- Biochemistry
- Parasitology
Background:
- Polyadenylation is crucial for eukaryotic mRNA processing, affecting export, stability, and translation.
- Entamoeba histolytica is an important human pathogen where mRNA regulation mechanisms are less understood.
Purpose of the Study:
- To identify and characterize a nuclear poly(A) polymerase (EhPAP) in Entamoeba histolytica.
- To investigate the role of EhPAP in multidrug resistance (MDR) and cell cycle regulation.
Main Methods:
- Gene cloning and protein expression of EhPAP.
- Antibody generation and Western blot analysis to detect EhPAP isoforms.
- RT-PCR to analyze EhPap mRNA expression levels.
Main Results:
- Identified and cloned the EhPAP gene, confirming conserved domains with other eukaryotic PAPs.
- Detected two EhPAP isoforms (60 and 63kDa) in nuclear and cytoplasmic extracts.
- EhPap mRNA expression increased in multidrug-resistant trophozoites and during specific cell cycle phases (G1 and S).
Conclusions:
- EhPAP is a functional nuclear poly(A) polymerase in Entamoeba histolytica.
- EhPAP expression is potentially linked to multidrug resistance and cell cycle progression in this parasite.
