The Duplex Unwinding Coupled To Hydrolysis Mechanism And Functional Analysis Of Conserved Nuclease RNase J In Methanogenic Archaea

Background: The quality control of intracellular RNA includes the degradation,processing and circulation of RNA.There have been many studies showing that abnormal RNA quality control in human body is related to tumor development,neurodegenerative diseases and various pathogenic viral infections.For eukaryotes,the composition of nucleases related to RNA quality control in vivo is comprehensive,and research is not easy.Archaea,considered to be the evolutionary origin of eukaryotes,have genetic mechanisms similar to eukaryotes such as DNA replication and repair,RNA transcription,degradation,and translation,which can provide simple models for the study of eukaryotes;at the same time,Archeara,as the third life evolution form juxtaposed with bacteria and eukaryotes,has a unique and indispensable role in life evolution,genetic metabolism,biogeochemical cycle and functional applications,and co-parasitic with the human body,Related to the development of human diseases.Methanogenic archaea are one of the most studied archaeal representative groups with the most abundant genetic metabolism.They are strictly anaerobic microorganisms and can be used as model species to analyze basic life processes.The processing and degradation of RNA are the core steps of intracellular RNA metabolism,contributing to the correct formation of functional RNA,efficient translation of proteins,and rapid regulation required for environmental responses.The processing and degradation of RNA is usually performed by a series of ribonucleases,such as 5′-3′ or 3′-5′ exonucleases and endonucleases.At present,researches on RNA processing and degradation are mainly concentrated in eukaryotes and bacteria,but little research in archaea.In the early stage of our laboratory,we found that β-CASP family nucleases-RNase J are widely distributed and evolved conservatively in archaea through biochemical analysis;and through biochemical experiments,we found that the model methanogensRNase J(mpy-RNase J)of Methanolobus psychrophilus R15)Has 5′-3′ exonuclease activity,and its molecular mechanism such as RNA recognition,catalysis,and efficient exo-continuity are analyzed through crystal structure;and it is found that it may have newduplex RNA unwinding activity,but its solution The chain mechanism is unknown.Objective: To expound the unwinding mechanism of duplex RNA of β-CASP nuclease RNase J of Methanolobus psychrophilus R15 and perceive its function about RNA turnover.Methods: Methanolobus psychrophilus R15 RNase J(mpy-RNase J)was used as a representative,and its doubled-strand RNA unwinding activity was confirmed through its biochemical experiments,by analyzing the co-crystal structure of mpy-RNase J and 6nt poly(A)RNA,and combining mutations and biochemical analysis to analyze the molecular mechanism of itsduplex RNA unwinding activity and analyze its function in RNA metabolism.Results: through different types of duplex RNA substrates(Gx & Hx),confirming that mpy-RNase J hasduplex RNA unwinding activity,and can untie duplex RNA up to 40 bp,by mutating its hydrolytic active center and key residues of the 5 monophosphate binding pocket,confirming its solution The chain activity is coupled to its hydrolytic activity,driven by the energy released by RNA hydrolysis;By analyzing the co-crystal structure of mpy-RNase J-S247 A and 6 nt poly(A),and combining key residue mutations and biochemistry In the activity analysis,we analyzed the molecular mechanism of its duplex RNA unwinding activity: the rapid conversion of RNA driven by the energy released by the exohydrolysis of the single-stranded RNA substrate at the 5′ end of its singlestranded RNA binding channel In the position process,every 1 nt of the exolytic hydrolysis cycle can promote the duplex RNA at the entrance of the RNA channel by the three-dimensional exclusion effect formed by the archaeal Loops Ⅰ,Ⅱ and α5,α9 to open the complementary pairing of theduplex RNA.Realize its unwinding and degradation;We found that the double stranded RNA unwinding activity of mpy-RNase J can assist Help it to degrade RNA with complex secondary structure in cells,including r RNA,t RNA and m RNA 5′UTR region,suggesting that mpy-RNase J should play an important role in the processing,degradation and functional supervision of these functional RNAs in the body;We found that the bacterial homolog Sco-RNase J also hasduplex RNA unwinding activity,suggesting that this activity should be conserved in RNase J family proteins,the duplex unwinding mechanism of mpy-RNase J— Hydrolysis promotes and relies on the exclusion effect at the entrance of the RNA channel,and has evolving strategic similarities with eukaryotic 5′-3 ′ exonuclease Xrn1 and RNase II family proteins,eukaryotic Rrp44 / Dis3 and bacterial RNase R It explains the evolutionary principle of "different proteins and similar strategies".Conclusions: We confirmed that mpy-RNase J has duplex RNA unwinding activity;We expounded the mechanisms of duplex RNA unwinding coupled exohydrolysis based on the steric clamp in the entrance of archae;We revealedmpy-RNase J plays an important role in the RNA metabolisms by it degrades the lined RNA or structured RNA in vivo.