Study Of The Novel Programmed Ribosomal Frameshifting Gene ARF1 In Euplotes Octocarinatus

It is becoming increasingly apparent that PRF is much more widespreadand likely exists in all branches of life from bacteria to higher eukaryotes.There are two main mechanisms that produce out of frame peptides: changes in the genome sequence that result in insertions or deletions(indels)and programmed ribosomal frameshifting.PRF is a recoding event by which the translating ribosome switches from the initial(0)reading frame to the-1 or +1 reading frame at a specific position,and then continues its translation.-1 PRF was identified as important mechanisms of regulating virus gene and cellular gene expression.Furthermore,frameshifting is not limited to the-1 frame,it is becoming increasingly apparent thatmuch more bacteria to higher eukaryotes.Presentstudies confirmed that the synthesis of partial proteinproducts in Euplotes needed +1 PRF.As a typical ciliate,Euplotes octocarinatus exhibits nuclear dimorphism [micronucleus(MIC)and macronucleus(MAC)].Weconducted a genome-wide investigation of+1 PRF in E.octocarinatus through genome andtranscriptome sequencing previously.An approximately 11.4% putative +1 PRF genes were identified in E.octocarinatus.In addition to the 3,489 +1 PRF genes with the classical ‘Euplotes frameshift motif'(5?-AAA-TAR-3?),we also identified 211 novel +1 PRF genes with different types of slippery sequences.Among these novel +1 PRF genes,the most abundant slippery sequence motif was the 5?-TTT-TAR-3? motif with 54 genes?To further investigate the gene secquence,frameshift mechanism and gene function of predicted novel +1 PFR.We choosedthe ARF1 gene from E.octocarinatuswith a novel +1 frameshift motif 5?-TTT-TAA-C-3? for further analysis.Below is a summary of key results.1.Gene cloning and sequence analysis of ARF1 from Euplotes octocarinatusThe EoARF1 genewas obtained by PCR.Including telomeres the macronuclear Eo-ARF1 is 984 bp long.The coding region contains two ORFs.The first ORF encodes the first 109 amino acids and the second ORF encodes the remained 78 amino acids of ARF.The slippery site has the heptameric motif TTTTAAC.Compared the amino acid sequence of EoARF1 with that fromother organisms,it contains only two conservative motifs GXXXXGKTand DLGG before frameshifting site.Other two motifs involved in GTP binding are encoded by the sequence in +1 frame.These data suggested that the polypeptide produced by EoARF1 gene belong to the group of ARF1.However,ribosomal frameshifting is likely required for expression of the Eo-ARF1 gene.2.Analysis of the frameshifting gene EoARF1To verify the +1 PRF gene EoARF1,it was inserted into a dual luciferase reporter vector.The recombinant plasmid pDB722-ARF1 was constructed for testing its expression in yeast.Plasmids pDB722-ARF1-T and pDB722-ARF1-S were constructed by the sited mutation.Dual-Luciferase assay was employed from a single construct to measure the frameshifting efficiency of EoARF1,the result demonstrated thatthe Frameshift efficiency of EoARF1 is 5.86%,suggesting that +1 PRF is required for the translation of EoARF1.In addition,the heptanucleotide slippery sequence TTT TAA C was mutated,the results indicated that the frameshift efficiency was reduced after the slippery sequence was abolished,and it proved that frameshift is occurred in the region.3.Expression and purification of recombinant ARF1 protein from Euplotes octocarinatusThe recombinant plasmid pET28a-EoARF1 was constructed and expressed in E.coli BL21.Recombinant protein of EoARF1 was purified by Ni-NTA affinity chromatography and gel chromatography.The purified protein was detected by SDS-PAGE and confirmed by Western blotting 4.Assay of interaction between EoARF1 and purine nucleotidesThe interactions between EoARF1 and purine nucleotides were investigated by using fluorescence spectroscopy.The results showed that the purified protein EoARF1 and purine nucleotides have interactions,and theaffect strong-to-weak sequence isGTP>GDP>GMP.Furthermore,the bonding strength of Eo ARF1 with GDP was exceeded ADP.Therefore,the EoARF1 gene was cloned to verify that +1 PRF gene ARF1 is actual occurred in Euplotes octocarinatusand produced transcription products.We used the dual luciferase report system to demonstrate that +1 PRF is required for the translation of EoARF1.Recombinant protein of EoARF1 was expressed and purified in E.coli BL21 and the purified protein EoARF1 and purine nucleotides have interactions.These results provided experimental data for further studies of the mechanisms of programmed ribosomal frameshifting in Euplotes.