Identification Of Non-Coding RNAs And It's Function In Mycobacterium Smegmatis

Mycobacterium tuberculosis is currently the world's high prevalence of tuberculosispathogens. It is estimated that one-third of worldwide population infected with tuberculosis,while those who infected with TB, about10percent of people develop to symptoms. Thegrowth of Mycobacterium tuberculosis is very slow, and it's highly pathogenic, relativelydifficult to manipulate, Mycobacterium smegmatis as a model organism, the first of itsstudy is very important for reseaching the Mycobacterium tuberculosis.The non-coding RNA is a class of RNA that is not translated into proteins, but playsan important role in a specific period. Non-coding RNA is emerging as a scientific hotspotlast decade, and more and more scientists focus on them, because a lot of literature hasreported that some non-coding RNAs play roles in the control of human disease and canhelp for designing drug targets. Due to the emergence of MDR or XDR Mycobacteriumtuberculosis, many drugs currently used for treatment of tuberculosis, such as isoniazid,now has not play a therapeutic role. So identification and characterization of thenon-coding RNA of Mycobacterium tuberculosis is imperative.Although the literature reports found that some of the non-coding RNA inMycobacterium tuberculosis, but the number was very little. As a model organism,Mycobacterium smegmatis can be a host agent as Mycobacterium tuberculosis forreseaching some Mycobacterium tuberculosis genes. So in this study, we constructedMycobacterium smegmatis50-500nt non-coding RNA library by using RNA genomics.Finally we sequenced a total of5085sequences, after bioinformatic analysis, we identified32non-coding RNAs. The genomic position of these non-coding RNAs, we found15ofthem belonging to the cis-encoded non-coding RNA, antisense in the open reading frame,and17belonging to the trans-encoded non-coding RNAs, located in the intergenic region.In this study, we randomly selected some of the newly acquired non-coding RNAs, andusing northern blot to further verify it's presence, and identification of seven trans-encodednon-coding RNAs in Mycobacterium smegmatis is truely expressed, five cis-encodednon-coding RNAs showed no hybridization signal that cis-encoded non-coding RNA may be expressed little than trans-encoded non-coding RNAs, because it is dilated base pairingto target. We also used RT-PCR method to strengthen the verification of the presence ofnon-coding RNA, and identified5of longer fragments of non-coding RNAs reallyexpressed. In this study, we blasted all non-coding RNAs with MTB complex,8genomes,found three homologous non-coding RNAs, cluster014, cluster017and cluster027, whichcluster027has been published in Mycobacterium tuberculosis. In order to study thefunction of non-coding RNAs, we randomly selected2non-coding RNAs that they havebeen verified by northern blot (cluster003, cluster021) for overexpression in the wild-typemycobacterium smegmatis and found no significant bacterial growth rate differences,indicating that these two non-coding RNAs do not affect the bacterial growth rate. In thisstudy, we provided some data for further identification and functional studies of non-codingRNAs of Mycobacterium tuberculosis.