Expression of EhRAD54, EhRAD51, and EhBLM proteins during DNA repair by homologous recombination in

Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan

Expression of EhRAD54, EhRAD51, and EhBLM proteins during DNA repair by homologous recombination in Entamoeba histolytica

Entamoeba histolytica, the protozoan responsible for human amoebiasis, exhibits a great genome plasticity that is probably related to homologous recombination events. It contains the RAD52 epistasis group genes, including Ehrad51 and Ehrad54, and the Ehblm gene, which are key homologous recombination factors in other organisms. Ehrad51 and Ehrad54 genes are differentially transcribed in trophozoites when DNA double-strand breaks are induced by ultraviolet-C irradiation. Moreover, the EhRAD51 recombinase is overexpressed at 30 min in the nucleus. Here, we extend our analysis of the homologous recombination mechanism in E. histolytica by studying EhRAD51, EhRAD54, and EhBLM expression in response to DNA damage. Bioinformatic analyses show that EhRAD54 has the molecular features of homologous proteins, indicating that it may have similar functions. Western blot assays evidence the differential expression of EhRAD51, EhRAD54, and EhBLM at different times after DNA damage, suggesting their potential roles in the different steps of homologous recombination in this protozoan

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

<div><p>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 <i>Entamoeba histolytica</i>, 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 <u>X</u>) 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 <i>EhPgp5</i> 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 <i>E. histolytica</i>. 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.</p> </div

Molecular characteristics of predicted EhCFIm25.

<p>A) Comparative molecular organization of CFIm25 proteins from <i>E. histolytica</i> and human. Upper panel, Schematic representation. The scale at the top indicates the size in aa. Numbers at the right are relative to the initial methionine in each protein. Lower panel, ClustalW sequence alignment of Nudix box. Black box, identical aa; grey box, similar aa. X, any residue; U, hydrophobic residue. D) Three dimensional organization of CFIm25 proteins from <i>E. histolytica</i> (left) and human (right). 3D modeling of EhCFIm25 was obtained using crystal data from human CFIm25 as template. The Nudix box is in black color. The UniProt KnowledgeBase database accession number for each predicted protein is indicated.</p

Expression of the putative EhCFIm25 in <i>E. histolytica</i> trophozoites.

<p>A and B) Western blot assays. A) Cytoplasmic (lanes 1, 3, 5 and 7) and nuclear (lanes 2, 4, 6 and 8) extracts of <i>E. histolytica</i> trophozoites were separated through 10% SDS-PAGE, electrotransferred to a nitrocellulose membrane and incubated with antibodies. Lanes 1 and 2, anti-EhCFIm25 antibodies; lanes 3 and 4, pre-immune serum; lanes 5 and 6, anti-EhPAP antibodies; lanes 7 and 8, anti-EhPC4 antibodies. B) Cytoplasmic (lanes 1, 3, 5 and 7) and nuclear (lanes 2, 4, 6 and 8) extracts were treated with 5% β-mercaptoethanol (lanes 1 to 4) or 8% β-mercaptoethanol (lanes 5 to 8) and separated through 10% SDS-PAGE in the presence of 8 M urea, and electrotransferred to a nitrocellulose membrane before being incubated with antibodies. Lanes 1, 2, 5 and 6: anti-EhCFIm25 antibodies; lanes 3, 4, 7 and 8: pre-immune serum. Arrowhead, endogenous EhCFIm25. C) Primers to evidence mRNA expression of genes at locus EHI_077110 (up) and EHI_077000 bottom) in RT-PCR assays. D) RT-PCR assays. <i>EhCFIm25</i> transcript was PCR amplified using cDNA synthesized from total RNA and products were analyzed through ethidium bromide stained polyacrylamide gels. Lane 1, molecular size markers; lane 2, cDNA; lane 3, control using genomic DNA from <i>E. histolytica</i>; lane 4, control using oligonucleotides for <i>actin</i> gene; lane 5, control using pRSET<i>-EhCFIm25</i>; lane 6, control without cDNA.</p

Expression of the recombinant EhCFIm25.

<p>A) Expression of the 6x-His-labeled EhCFIm25 protein. Bacteria <i>E. coli</i> were transformed with pRSET<i>-EhCFIm25</i> plasmid and protein expression was induced by the addition of 1 mM IPTG for 3 h. Proteins extracts (30 µg) were separated through 10% SDS-PAGE and gels were stained with Coomassie blue. Lane 1, molecular weight; lane 2, non-induced bacterial extract; lane 3, IPTG-induced bacterial extract. B) Immunodetection of rEhCFIm25 polypeptide by Western blot assays using anti-6x His tag antibodies. Lane 1, non-induced bacterial extract (30 μg); lane 2, IPTG-induced bacterial extract (30 μg). C) Purification of the 6x-His-labeled EhCFIm25 protein through affinity chromatography using a Ni-NTA column. Lane 1, molecular weight; lane 2, IPTG-induced bacterial extract; lane 3, unbound proteins; lane 4, wash using 150 mM imidazole; lanes 5-8, elution with 250 mM imidazole. D) Immunodetection of rEhCFIm25 polypeptide by Western blot assays using specific rabbit antibodies anti-EhCFm25Im. Lane 1, non-induced bacterial extract; lane 2, IPTG-induced bacterial extract; lane 3, IPTG-induced bacterial extract and anti-6x His tag antibodies used as control. E) Immunodetection of purified rEhCFIm25 polypeptide by Western blot assays. Lane 1, anti-6x His tag antibodies; lane 2, specific rabbit antibodies anti-EhCFIm25; lane 3, pre-immune serum; lane 4, control without primary antibody. Arrowhead, EhCFIm25.</p

RNA binding activity of rEhCFIm25.

<p>A) REMSA. rEhCFIm25 was incubated with [α-³²P] UTP-labeled PSIII¹⁵⁶ RNA probe (5×10⁵ cpm) at 4 °C for 15 min. Complexes were resolved through 6% non-denaturing PAGE and detected in a Phosphor Imager apparatus. Lane 1, free probe; lane 2, RNA probe plus 5 nM rEhCFIm25; lane 3, as in lane 2 plus specific competitor (Sc) (350-fold molar excess of unlabeled probe); lane 4, as in lane 2 plus unspecific competitor (Uc) (350-fold molar excess of unlabeled tRNA); lane 5, RNA probe plus 10 nM rEhCFIm25; lane 6, as in lane 2 plus proteinase K; lane 7, as in lane 2 plus RNAse; lanes 8 and 9, as in lane 2 plus antibody anti-EhCFIm25. Arrowhead, RNA-protein complex. B) Densitometry analysis of complexes detected in A. Pixels corresponding to C_I in lane 2 was taken as 100% and used to normalize data.</p

<i>In vitro</i> protein-protein interaction assays.

<p>A) Pull-Down assay. The rEhCFIm25 immobilized on Ni²⁺-NTA column was incubated with NE. After washing, proteins were eluted, separated though 10% SDS-PAGE and stained with Coomassie Brilliant blue. Lane 1, Molecular weight markers; lane 2, proteins not retained on the column; lanes 3 and 4, washing fraction; lane 5, elution fraction. B) Far-Western assay. Purified rEhCFIm25 (lanes 1 to 4) and rEhPAP (lanes 5 to 8) were subjected to 10% SDS-PAGE and electrotransferred to a nitrocellulose membrane that was incubated with rEhPAP (lane 1) or rEhCFIm25 (lane 5). Proteins were immunodetected by specific antibody anti-EhPAP (lanes 1, 2 and 7) or anti-EhCFIm25 (lanes 3, 5 and 6) and revealed by the ECL Plus Western blotting system (Amersham). Anti-His antibody was used as control (lanes 4 and 8).</p

Crystal structure of human poly(A) polymerase gamma reveals a conserved catalytic core for canonical poly(A) polymerases

In eukaryotes, the poly(A) tail added at the 3′ end of an mRNA precursor is essential for the regulation ofmRNA stability and the initiation of translation. Poly(A) polymerase (PAP) is the enzyme that catalyzes thepoly(A) addition reaction. Multiple isoforms of PAP have been identified in vertebrates, which originate fromgene duplication, alternative splicing or post-translational modifications. The complexity of PAP isoformssuggests that they might play different roles in the cell. Phylogenetic studies indicate that vertebrate PAPs aregrouped into three clades termed α, β and γ, which originated from two gene duplication events. To date, allthe available PAP structures are from the PAPα clade. Here, we present the crystal structure of the firstrepresentative of the PAPγ clade, human PAPγ bound to cordycepin triphosphate (3′dATP) and Ca2+. Thestructure revealed that PAPγ closely resembles its PAPα ortholog. An analysis of residue conservationreveals a conserved catalytic binding pocket, whereas residues at the surface of the polymerase are moredivergent