The Enzyme Activity Identification Of Nanornase From Aeropyrum Pernix

RNA degradation is common in three domains of life.Although RNA degradation pathway is highly complex,RNA metabolism among bacteria,archaea and eukaryote is essentially similar.In cell there are three main types of ribonucleases(RNases)that can degrade RNA: endonuclease,5'-exonuclease and 3'-exonuclease.In bacteria,RNase E,RNase Y and RNase III are the main endonuclease,in addition,there are also 5'-exonuclease(RNaseJ)and 3'-exonuclease(PNPase and RNase II/R),but all these RNases are not able to degrade oligoribonucleotide shorter than five nucleotides.Oligonucleotide with a length of 2-5nt is usually called nanoRNA,and oligoribonucleases that can degrade nanoRNA had been found in bacteria,including Orn in E.coli,Nrn A/B in Bacillus subtilis and Nrn C in Bartonella.Archaea can be divided into two main phylums(Euryarchaeota and Crenarchaeota),and the members in Crenarchaeota are mainly hyperthermophilic archaea.Aeropyrum pernix is a strictly aerobic crenarchaeota,and its optimal growth temperature is 95?.During cell growth process,DNA can suffer spontaneous damages,for example,hydrolytic deamination of cytosine and adenine,hydrolytic depurination or oxidation of guanine.These spontaneous processes can accelerate in high temperature environment and threaten the survival of hyperthermophiles.Under high temperature and alkaline conditions,RNA also can be spontaneously hydrolyzed;therefore the extreme environment can cause potential RNA degradation for hyperthermophiles.At present,there is no Orn homologous protein found in archaea.Besides,little is known about nano RNase in archaea,especially the specific nanoRNA degradation pathway is not clear.Recently we have found three hypothetical nano RNase candidate proteins(Ape0124,Ape1437 and Ape2190)in Aeropyrum pernix,and the biochemical result demonstrated that only Ape0124 possessed nuclease activity.Through protein multiple sequence alignment and protein homology comparison we find that Ape0124 belongs to the DHH/DHHA1 family with highly conserved motif III and motif VI,and is homologous with Bacillus subtilis Nrn B.In addition to nano RNase and nano DNase activities,Ape0124 can also degrade long ss DNA and ss RNA,but prefers short ss DNA and long ss RNA.As a 3'-exonuclease,Ape0124 has no preference for 3'-terminal bases and substrate sequences,however,3'-P can inhibit exonuclease activity of Appe0124.This study is the first report on the enzyme activity of nano RNase in archaea,and deepens the understanding of complete RNA degradation pathway in archaea and knowledge about nanoRNA degradation enzymology.