The 25 kDa Subunit of Cleavage Factor Im Is a RNA-Binding Protein That Interacts with the Poly(A) Polymerase in Entamoeba histolytica

Marisol Pezet-Valdez,María Esther Ramírez-Moreno,Juan David Ospina-Villa,Jacqueline Soto-Sánchez,Guillermo Mendoza-Hernández,Laurence A Marchat,César López-Camarillo,Jorge Fernández-Retana,Esther Orozco,Mavil López-Casamicha,Socorro Charcas-López,Earl G Brown

doi:10.1371/journal.pone.0067977

Marisol Pezet-Valdez, María Esther Ramírez-Moreno + Show 10 more

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https://doi.org/10.1371/journal.pone.0067977

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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.

Highlights

RNA processing is an essential event for gene expression regulation
We focused on the study of the putative EhCFIm25 from E. histolytica
Bioinformatics analysis indicated that they possess the regions that have been shown to be important for RNA binding and poly(A) polymerase (PAP)/PAB binding in the human CFIm25 protein [24] as well as the Nudix domain that has been first identified in Mut T protein from E. coli [48] (Figure 1B)

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Introduction

RNA processing is an essential event for gene expression regulation. the poly(A) at the 3 ́ end of most eukaryotic mRNA influences mRNA stability, translation, and transport [1,2,3]. Polyadenylation is a two-step nuclear reaction that involves pre-mRNA 3 ́ end cleavage at the cleavage and polyadenylation site (poly(A) site) followed by the addition of a polyadenine tail [10,11,12,13]. Both reactions depend on trans-acting factors interacting in a coordinated way with specific motifs in pre-mRNA 3 ́ untranslated region (UTR). The 160 kDa subunit of the cleavage and polyadenylation specificity factor (CPSF) recognizes the PAS [17], while the Nterminal RNA recognition motif (RRM) from the 64 kDa subunit of the cleavage stimulatory factor (CstF) binds the DSE [18]. CPSF-73 is thought to perform pre-mRNA cleavage [22], before PAP catalyzes the poly(A) tail synthesis [21]

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