| Eimeria spp. as an intracellular parasite, causes a severe form of an enteric disease,coccidiosis, which has an enormous impact on worldwide poultry industry. Theapplication ofanticoccidial drugs and vaccines is the main control strategy against coccidiosis in poultry.However, due to little knowledge of cellular and molecular biology of coccidian, there is no idealdrugs and vaccines to control coccidiosis. Telomerase and related proteins facilitate further studyof cellular and molecular biology of coccidian, and may be potentially good targets forcoccidiosis control.Telomerase is a ribonucleoprotein enzyme consisting of RNA component which contains atemplate region complementary to the telomeric repeat sequence, telomerase reversetranscriptase (TERT), and associated proteins. The role of telomerase is to maintain the length oftelomeres by adding telomeric repeats onto the end of chromosomes by the reverse transcriptaseusing the telomerase RNA as a template. TERT, as the catalytic subunit of telomerase, plays acritical role in telomerase activity. With the in-depth study of telomerase, a variety oftelomerase-associated proteins have been reported, and these proteins play an important role inregulating telomerase activity. p23, identifed in a yeast two hybrid screen using hTERT as thebait, along with Hsp90, associated with an active telomerase following assembly. PinX1bindsthe RNA binding domain of hTERT (amino acids326~620) and the telomerase RNA. The yeasthomolog of human PinX1may negatively regulate telomerase function. p65and p45ofTetrahymena have essential roles in the maintenance of telomere length as part of telomeraseholoenzyme. Previously, E. tenella telomerase gene had been identified and characterized, andtelomere-like sequences had been predicted. These indicating that E. tenella may utilize aclassical telomerase-based mechanism for its telomeric DNA maintenance. Notelomerase-associated proteins have been described in coccidian. In the present study, the yeasttwo hybrid system, pull-down and co-immunoprecipitation assays were applied to identify a14-3-3protein as TERT-interacting partner in E. tenella. Further, we investigate the function of14-3-3in telomerase regulation by a virus-mediated hammerhead ribozyme. Construction of a yeast two-hybrid cDNA library of E. tenella. Sporulated oocysts of E.tenella purified were collected and purified. Total RNA was extracted by using Trizol reagent,then was reverse transcribed into cDNA. A yeast two-hybrid cDNA library was established bythe SMART technology. The titer and capacity of the library were5×107cfu/mL and2×1010cfu,respectively. The inserted cDNA fragments of48clones were between0.8kb and2kb, and thelibrary contained100%recombinant clones.Construction of a bait plasmid pGBKT7-TRBD and screening its interaction proteinsby yeast two-hybrid. The RNA binding domain of TERT (TRBD) in E. tenella was amplified byPCR and ligated to pGBKT7to construct the recombinant bait plasimd pGBKT7-TRBD.pGBKT7-TRBD was transformed into yeast competent cells. The results showed that the baitprotein TRBD can be expressed in yeast and is not toxic.-galactosidase assay indicated thatTRBD has no transcriptional activation effect. In order to screen the interaction proteins withTRBD, the pGBKT7-TRBD/Y187and yeast two-hybrid cDNA library were co-cultured.Nucleolar phosphoprotein nucleolin,14-3-3, and uridine phosphorylase were obtained.14-3-3was found to associate with TRBD based on-galactosidase assay.Identification the interaction of TRBD and14-3-3protein. The TRBD and14-3-3werecloned into prokaryotic expression vector pET-32a and pGEX-4T-1, respectively. Thenexpression and purification of E.tenella TRBD and14-3-3were performed. SDS-PAGE showedthat both recombinant proteins could be expressed in the soluble form. GST-14-3-3andHis-TRBD purified were incubated and applied to pull-down assay to verify the interaction.Results showed His-TRBD bound directly to GST-14-3-3in vitro.The recombinant plasmids pcDNA-3.1-Myc-TRBD and pcDNA-3.1-HA-14-3-3wereconstructed, then co-transfected into293T cell. Protein extracts were subjected to Western blot,and expressions of TRBD and14-3-3were detected using anti-Myc and anti-HA antibodies,respectively. Immunoprecipitation showed that there was an interaction between Myc-TRBD andHA-14-3-3in eukaryotic cell.Function studies of14-3-3protein. Hammerhead ribozyme synthesized for cleavage14-3-3were cloned into a viral vector derived from the genome of E. tenella virus, andpEtV-RFP-Ham-14-3-3was obtained. In vitro, the cleavage activity of pEtV-RFP-Ham-14-3-3transcripts on14-3-3mRNA was85.24%. pEtV-RFP-Ham-14-3-3transcripts were introduced into E. tenella by electroporation. Western blot indicated that the expression level of14-3-3wasdown-regulated11.24%, comparing with RFP-GFP strain. When the expression of14-3-3wasinhibited, the telomerase activity was up-relugated. The results indicated that14-3-3maynegatively regulate telomerase activity.In summary, the studies successfully established a yeast two-hybrid cDNA library of E.tenella, and TERT-interacting protein14-3-3was screened. Pull-down and immunoprecipitationconfirmed that14-3-3is a binding partner for TERT. E. tenella virus-mediated hammerheadribozyme down-regulated the expression of14-3-3in vivo, then the results suggested14-3-3maynegatively regulate telomerase activity. This study make the foundation for further elucidating themechanism of14-3-3regulating telomerase activity, and benefit to develop more efficientmolecule-based drugs and vaccines for coccidiosis control. |
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