Mycobacterium Non-coding RNA Discovery And Identification Systems

Tuberculosis (TB) presents a major threat to global health, and Mycobacteriumtuberculosis, the causative agent of TB, is one of the world’s most ancient andsuccessful pathogens. During the course of infection, in order to adapt to survival incomplex host microenvironments, M. tuberculosis has evolved and established specialmechanisms to quickly regulate gene expression and switch between differentphysiological states, including periods of active cell division and persistence in anon-replicating state. While transcriptional regulation in response to environmentalchange has been extensively analyzed in M. tuberculosis, less attention has beengiven to the regulatory processes that occur subsequent to the initiation oftranscription. Non-coding RNAs (ncRNAs), a class of RNAs that are not translatedinto proteins, play an important role in the post-transcriptional regulation. It isbecoming increasingly evident that ncRNAs are widespread in all kinds of species,and play important roles in nearly all physiological and pathological processes.Non-coding RNAs can help bacteria to cope with different stresses and also regulatethe expression of virulence genes and affect their pathogenicity to human beings.However, at present, little is known about the classes, quantity, and regulatorynetwork of ncRNAs in M. tuberculosis. The most urgent thing we need to do is todiscover and identify ncRNA molecules of M. tuberculosis systematically. Recentadvances in high-throughput DNA sequencing have opened the way forcomprehensive analysis of ncRNAs.In the present study, high-throughput complememtary DNA sequencing(RNA-seq) was used to sequence four exponential phase M. tuberculosis cDNAlibraries with different sizes of inserted fragments. A set of2837candidate ncRNAswas identified, including2269antisense(AS) ncRNAs and568intergenic(IGR) ncRNAs. The length of candidate molecules ranged from16to428nt, and most ofthem were concentrated in the region of16to80nt, including a large number of16to30nt candidates.Using Northern blotting and reverse transcription PCR (RT-PCR) followed bysequencing, the expression of30ncRNAs including14IGR-ncRNAs and16AS-ncRNAs of different lengths, expression levels and genomic positions wasconfirmed, validating their existence in exponential phase cultures of M. tuberculosis.Genes located on the complementary strands of AS-ncRNAs coded for16S rRNA,proteins involved in information pathways, lipid metabolism, cell wall and cellprocesses, and also hypothetical proteins with unknown functions.Quantitative reverse transcription PCR (qRT-PCR) was performed to investigatethe expression levels of M. tuberculosis ncRNAs during different growth phases.Results indicated that while some ncRNAs were expressed stably in different growthphases, many ncRNAs were apparently differentially expressed. Increased expressionlevels during the stationary phase were of particular note.Phylogenetic conservation of the30verified ncRNAs was examined usingnucleotide BLAST. All30ncRNAs are highly conserved in the three members of theM. tuberculosis complex. Thirteen ncRNAs were highly conserved in the slowgrowing M. tuberculosis, M. africanum, M. bovis, M. marinum, M. ulcerans, M.avium and M. intracellular, and7ncRNAs in the fast growing M. abscessus and M.smegmatis. In addition, five ncRNAs (Number:0149,0130,0041,0205and0190)were highly.conserved in M. leprae and many slow and fast growing mycobacteria.This genome-wide study identified ncRNAs in M. tuberculosis H37Rv byRNA-seq, the expression level and phylogenetic conservation of some of which werefurther verified and analyzed. This work will lay the foundation for future detailedstudies on their regulatory function in virulence, persistence, interaction with the host and drug resistance. This finding may also open the way for new research onprevention and treatment of TB.