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

• Published in: PloS one Vol. 8; no. 6; p. e67977
• Main Authors: Pezet-Valdez, Marisol, Fernández-Retana, Jorge, Ospina-Villa, Juan David, Ramírez-Moreno, María Esther, Orozco, Esther, Charcas-López, Socorro, Soto-Sánchez, Jacqueline, Mendoza-Hernández, Guillermo, López-Casamicha, Mavil, López-Camarillo, César, Marchat, Laurence A
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 28.06.2013; Public Library of Science (PLoS)
• Subjects: 3' Untranslated regions; Amino Acid Sequence; Antibodies; Assaying; Bacteria; Biochemistry; Cell Nucleus - genetics; Cell Nucleus - metabolism; Chemical synthesis; Cleavage; Cytoplasm - genetics; Cytoplasm - metabolism; Electrophoretic mobility; Entamoeba histolytica; Entamoeba histolytica - genetics; Entamoeba histolytica - metabolism; Eukaryotes; Exoribonucleases - genetics; Exoribonucleases - metabolism; Gene expression; Genomes; Human behavior; Localization; Machinery; Machinery and equipment; Mass spectrometry; Mass spectroscopy; Molecular Sequence Data; mRNA Cleavage and Polyadenylation Factors - genetics; mRNA Cleavage and Polyadenylation Factors - metabolism; mRNA stability; Poly A - genetics; Poly A - metabolism; Poly(A); Polyadenine; Polyadenylation; Polymerase; Polynucleotide adenylyltransferase; Protein Subunits - genetics; Protein Subunits - metabolism; Proteins; Protozoa; Ribonucleic acid; RNA; RNA polymerase; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA-binding protein; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Mexico
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0067977

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.