Mycobacterium tuberculosis: I have always been fascinated by this one of the most successful human pathogens. Whenever I happen to see any research work on this pathogen, I try to read it. While doing some random search on pubmed, I happened to come across this very beautiful article. This article is about identification of small RNAs in M. tuberculosis and the authors are Dr. Kristine B. Arnving and Dr. Douglas B. Young, Division of Mycobacterial Research, London UK.
In many bacterial species, it has been shown that the mechanisms of transcription control are complemented by a post transcriptional regulatory network dependent on small regulatory RNA (sRNA) molecules. These sRNA molecules can enhance or suppress translation of mRNA targets by base pairing with the 5’ end of mRNA molecules at different locations relative to the ribosome binding site and start codon. They can also alter stability of mRNA by generating duplex molecules which act as substrate for RNase III or RNase E. It is observed that these sRNAs play important roles in regulation of stress response, bacterial cell cycle and also in bacterial pathogenesis.
The present study was conducted to investigate the occurrence of sRNAs in M. tuberculosis.
Two general approaches have been used for identification of sRNAs in bacteria. The first is bioinformatics prediction by sequence alignment of intergenic regions with known sRNAs, together with identification of appropriately positioned signals for transcriptional initiation and termination. Other approach is direct analysis of low molecular weight RNA molecules from culture of bacteria. In this paper authors used the second approach due to relatively poor definition of transcriptional signals in Mycobacteria. The authors have described a set of nine putative sRNAs identified by this approach and characterized by Northern Blotting and transcript mapping by 5’ and 3’ RACE analysis.
The major results of the study are as follows:
1. Cloning of small RNAs from M. tuberculosis- The initial experiment was aimed for determining the actual presence and abundance of small transcripts, regarded as putative sRNAs. For this, M. tuberculosis was cultured and total RNA was extracted from exponential and stationary growth phases. This total RNA was depleted of rRNA (16SrRNA and 23SrRNA) by microbeExpress kit. After the depletion, it was labeled with 32P-pCp and RNA ligase. The RNA was separated on a denaturing acrylamide gel and visualized by phosphorimaging. Multiple abundant and well defined small transcripts were observed. The pattern of expression was different between the two growth phases. Now, two independent cDNA libraries were constructed from stationary and exponential phase cultures. For preparation of cDNA library, total RNA was size fractionated and transcripts between 20-75 nucleotides were eluted from the gel. The eluted RNA was tailed with CTP and an RNA linker was attached to the 5’end. The RNA was then converted to cDNA, PCR amplified, cloned and sequenced. A total of 192 clones were sequenced and they fell into 6 categories. They were:
a. mRNA fragments with open reading frames (ORF): 11 clones
b. rRNA spacer fragments (5 clones)
c. tRNA fragments (1 clone)
d. unknown RNA encoded in intergenic regions (trans candidates, 23 clones representing six unique regions and 19 repeat clones).
e. Unknown RNA encoded antisense to annotated ORFs
f. Fragments that could not be assigned to a single region due to small size (<17bp) or chimeric sequences.
In order to determine the likelihood of each unknown to be a genuine sRNA or to be a part of an adjacent gene, the genomic positions and context of unknown RNAs were identified. If an intergenic cDNA clone was encoded on the opposite strand of an adjacent gene or more than 100 base pairs from an adjacent gene on the same strand, it was considered to be a valid sRNA candidate. When this criterion was applied, most of the intergenic clones appeared to be independent transcripts and not 5’ or 3’ UTRs.
2. Northern Blots verify the presence of sRNAs in M. tuberculosis- The cDNA libraries identified nine sRNA candidate. To further verify them, northern blotting with riboprobes complementary to the original cDNA clones was performed. The results of Northern blotting demonstrated signals corresponding to small transcripts from each of the candidates. Judging from the signals on the Northern blots, trans encoded sRNAs were expressed at significantly higher levels than the cis encoded sRNAs. All transcripts were larger than the cloned fragments, suggesting that the cloned fragments were probably degradation products.
3. Mapping of transcripts: sRNA transcripts were further characterized by RNA ligase mediated rapid amplification of 5’ and 3’ complementary DNA ends (RLM-RACE). The 5′ ends were mapped by comparing the RACE products obtained with and without prior treatment with tobacco acid pyrophosphatase (TAP), which facilitates the differentiation of transcription start sites from processed 5′ ends. The results suggested several putative transcription start sites. The authors also performed 5’ RACE on RNA from stationary phase.
4. Transcriptional coupling and sequence conservation: Excluding G2, all of the trans encoded sRNA candidates were encoded on the same strand as one of the adjacent protein-encoding genes. To test whether these sRNAs were co-transcribed with the particular upstream or downstream gene, RT PCR was performed by using primers that spanned the sRNA and the adjacent gene. It was observed that B55 was co-transcribed with the upstream Rv0609A. This suggested the possibility that B55 is part of 3’ UTR of the Rv0609A mRNA rather than being an sRNA. C8 was found to be co-transcribed with Rv3722c. On searching the Rfam database with the C8 sequence revealed that this RNA was in fact 4.5SRNA. F6 was co-transcribed with upstream fadA2. No RT PCR product was obtained using primers spanning B11 and Rv3660c. This indicated that B11 is a bonafide sRNA.
5. Prediction of secondary structure of sRNAs: All sRNA candidates except B55 and C8 had a C:G ratio>1.
6. Expression of M. tuberculosis sRNAs during stress: To study this, cultures of M. tuberculosis were subjected to oxidative stress (induced by H2O2), DNA damage (induced by Mitomycin C) and acid stress. It was observed that stress induced expression varied significantly between sRNAs.
7. Overexpression of M. tuberculosis sRNAs: Trans encoded sRNAs, B11, F6 and G2 were cloned in plasmid vectors under the control of the strong rrnB promoter of M. smegmatis and transformed into M. tuberculosis and M. smegmatis mc2155. The constructs expressing B11 and G2 both proved lethal in M. tuberculosis, while the expression of F6 resulted in extremely slow growth with pin prick colonies visible after 3-4 weeks.
In summary, the present study provides first evidence that M. tuberculosis expresses sRNA molecules and that these play important role in bacterial physiology. The authors anticipate that further functional studies in combination with sequence-based RNomics will provide novel insights into the fundamental biology of tuberculosis with the potential to inform development of improved strategies for disease control.
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