HTRIR (C19orf43) Is A Novel RNase

Ribonucleases(RNase)play an important role in RNA metabolism which is critical for RNA biogenesis,localization,accumulation,stability and degradation.Following identification and characterization of the bovine pancreatic RNase A in 1920,more and more ribonucleases and homologs were identified in different organisms with the explosion of genomic sequences in recent years along with the development of biochemistry,molecular biology and biochemical detection methods.In our study of non-coding RNA h TR biogenesis through in vitro transcription and processing assay,we identified an uncharacterized protein,C19orf43,which could interact with h TR with unknown function.We then named the C19orf43 as h TRIR(h TR interacting proteins).To analyze the function of h TRIR,bioinformatics software was used to analyze the C19orf43 protein,we found that h TRIR was widely expressed in eukaryotic cells and conserved in genetic analysis.The C part(aa: 111-176)was highly conserved.7 proteins were searched interacted with h TRIR in string database and 3 of them involved in RNA metabolism.The other 3 proteins were functional unknown.The prokaryotic expressed and purified h TRIR protein digested ?-32 P marked T7 transcribed structured h TR and non-structured Amp(nt1-300)RNA efficiently.h TRIR could also digest total cellular RNA and chemically synthesized small RNA.h TRIR could digest different forms of RNA,but not plasmid DNA,linearized DNA and single-stranded DNA.The results suggested that h TRIR play critical role in RNA metabolism.To optimize the digestion conditions of h TRIR,we incubated T7 transcribed RNA with h TRIR at different time,temperature and ions.h TRIR actively digest RNA between 37 to 75 oC,p H 6.5 to 12 when incubated for 30 min.h TRIR had high efficiency in RNA digestion without need of ions as co-factors and ATP as energy.In order to find out the RNase active domain,the truncate of h TRIR were constructed,expressed,purified and incubated with RNA in in vitro digestion assay.The C part which termed h TRIRD held excellent RNA digestion ability while the N part(aa: 1-110)had not.To clarify the molecular mechanism of h TRIR in RNA digestion,we designed and chemically synthesized poly(N)RNA with both methylated ends.h TRIR could digest all the bases but had high efficiency in purine bases A/G digestion which could surpass 90%.h TRIR cleaves RNA from both ends and preferentially purine bases which proved that h TRIR was a novel RNase which belongs to exoribonuclease.In conclusion,h TRIR which possessed exoribonuclease activity could digest RNA under normal conditions without need of ions and ATP.The RNase domain of h TRIR was located at the C part of protein and it could digest RNA from 5' and 3' ends.