Research On Endogenous Nucleic Acids That Bind To Argonaute Protein In Natronobacterium Gregoryi SP2 Cells

The Argonaute(Ago)proteins are present in all domains of life.The eukaryotic Argonautes(e Agos)play a critic role in RNA interference(RNAi)and associated gene silence in eukaryotes.The e Agos lead to post-transcriptional gene silence by binding small guide RNA(g RNA)to target RNA which is complementary to g RNA.The properties and functions of Ago proteins in archaeal and bacterial species have striking diversity in comparison with e Agos.Many prokaryotic Argonautes(p Agos)contain divergent variants of the conserved domains involved in interactions with nucleic acids,while having extra domains that are absent in e Agos,suggesting that they might have unusual specificities in the nucleic acid recognition and cleavage.Many p Agos are associated with putative nucleases,helicases,and DNA binding proteins in the same gene or operon,suggesting that they are involved in target processing.The e Ago proteins and the RNAi pathways are widely used as a powerful tool in research and as potential therapeutics.In contrast,studying the diversity and functions of p Agos have great potential in discovering new cell pathways and new tools for genome manipulation.Existing data indicate that p Agos are involved in the protection of prokaryotic genomes,that is,host defense mechanisms against certain exogenous genetic elements,such as plasmids,transposons,and phages.At present,only few p Agos have been characterized by structural or biochemical approaches.Compared to e Agos,experimental data about p Agos functions are very scarce.Chunyu Han's research team proposed that Ng Ago is a DNA-guided endonuclease suitable for genome editing in human cells in 2016.However,this research result has caused great controversy worldwide because of it's unrepeatable experimental results.So far,Ng Ago-g DNA system was used for targeting exogenous DNA/RNA in almost all of Ng Ago-related researches,but the endogenous nucleic acids that bind to Ng Ago in Natronobacterium gregoryi SP2(N.gregoryi SP2)have not been characterized,which is crucial for research of Ng Ago.Up to now,there is no report on Ng Ago's preference for nucleic acid binding.This dissertation focuses on this issue.The purpose of the research is to explore whether Ng Ago targets DNA and/or RNA in vivo.Then characterize the nucleic acid targets and explore the most primitive functions and properties of Ng Ago.The main research content of this dissertation includes the following parts:1.The archaea Natronobacterium gregoryi SP2 strain is extremely halophilic and alkaliphilic.We purchased N.gregoryi SP2 strain from the China General Microbiological Culture Collection Center(CGMCC)under the accession number of1.1967 and cultivated it according to the standard protocol.The archaea N.gregoryi SP2 is red,aerobic and halophilic bacillus.The incubation period of N.gregoryi SP2 is about five days.The whole genome sequencing was performed and the draft genome sequence has been submitted to the Gen Bank database under accession number of PKKI00000000.Statistic analysis and comparison of the three genomes of N.gregoryi SP2 that have been submitted to NCBI(National Center for Biotechnology Information)were performed.The genes of N.gregoryi SP2 were analyzed for orthologous analysis,gene ontology(GO),and pathway classification analysis.What's more,we also identified and annotated three proteins in the N.gregoryi SP2 as type II secretion system protein E,Sir A family protein and uncharacterized membrane protein Yei H,which were previously annotated as hypothetical proteins.2.The PIWI domain of the Ng Ago gene was amplified and cloned into the p ET-28 a expression vector at the Bam HI and Xho I sites from the p BBR-Tac-Ng Ago plasmid.Recombinant p ET-28a-PIWI plasmid was constructed and PIWI domain was expressed in Rosetta Escherichia coli.PIWI domain was used as an immunogen to immunize New Zealand white rabbits,and the anti-PIWI antibodies were prepared by multiple subcutaneous injections.At the same time,the N.gregoryi SP2 strain(ATCC43098/NCIMB 2189/JCM 8860)was cultivated.The N.gregoryi SP2 strain was identified by 16 S r RNA sequencing.N.gregoryi SP2 strain and anti-PIWI antibody were used for subsequent immunoprecipitation,chromatin immunoprecipitation(Ch IP)and RNA immunoprecipitation(RIP)experiment.3.N.gregoryi SP2 lysate interacted with anti-PIWI antibody in the immunoprecipitation(IP)experiment.The nucleic acid was extracted from the Ng Ago protein complex for sequencing and analysis from IP assay.Only RNA was detected by Qubit fluorometer and Agilent 2100 Bioanalyzer in the complex.It is shown that Ng Ago in N.gregoryi SP2 bound significantly to nc RNA(ffs and rnp B),t RNA and the transcripts of non-histone chromosomal MC1 family protein,cold-shock protein,and twin-arginine translocation signal domain-containing protein by RNA-seq expression analysis.4.After the IP experiment,Ch IP and RIP experiments were performed separately to specifically enrich DNA and RNA for sequencing and analysis.The above experimental results show that Ng Ago directly binds RNA rather than DNA in vivo.The RNA bound to Ng Ago are mainly t RNA and transcripts of transcription regulator,RNA polymerase and RNA binding protein.Ng Ago mainly binds to the transcript of the internal or upstream sequence of the genes.The significantly enriched motif of peaks of RIP-Seq was the same as that of mi R-1289.GO enrichment analysis shown that peaks-related genes are enriched in the biological process of transmembrane transport.These results suggest that Ng Ago binds RNA endogenously and may play a post-transcriptional regulatory role in vivo.To sum up,this dissertation focuses on the endogenous nucleic acid targets that Ng Ago binds in the archaeal N.gregoryi SP2.It is proposed that Ng Ago itself directly binds to RNAs in N.gregoryi SP2,and RNAs is its natural target.This study laid the foundation for the follow-up research of Ng Ago.Ng Ago is an important family member of p Agos.It is extremely important to characterize Ng Ago protein,which has important scientific significance for the research of p Agos as a potential gene manipulation tool.