12-12-2012, 11:44 AM
Identification of small RNAs in Mycobacterium tuberculosis
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Summary
In spite of being one of our most prominent bacterial
pathogens, the presence of small regulatory RNAs
(sRNAs) has not previously been investigated in
Mycobacterium tuberculosis. Post-transcriptional
regulation of gene expression by sRNA molecules
has been demonstrated in a wide range of pathogenic
bacteria and has been shown to play a significant role
in the control of virulence. By screening cDNA libraries
prepared from low-molecular weight RNA from
M. tuberculosis we have identified nine putative sRNA
molecules, including cis-encoded antisense transcripts
from within open reading frames and transencoded
transcripts from intergenic regions. sRNAs
displayed differential expression between exponential
and stationary phase, and during a variety of
stress conditions. Two of the cis-encoded sRNAs
were associated with genes encoding enzymes
involved in lipid metabolism, desA1 and pks12.
These sRNAs showed complementarity to multiple
M. tuberculosis genes, suggesting the potential to act
as both cis-encoded and trans-encoded sRNAs. Overexpression
of selected trans-encoded sRNAs had
profound impact on growth of M. tuberculosis and
M. smegmatis. This is the first experimental evidence
of sRNAs in M. tuberculosis and it will be important to
consider the potential influence of sRNA regulation
when studying the transcriptome and the proteome of
M. tuberculosis during infection.
Introduction
Mycobacterium tuberculosis, the causative agent of tuberculosis,
is one of the most successful human pathogens. It
is estimated that a third of the world’s population has
been infected, with new infections occurring at a rate of
approximately one per second. Less than 10% of infected
individuals go on to develop active disease, resulting in
around 9million new cases and 1.6 million deaths annually.
The remaining 90% will not develop any symptoms but the
bacteria may persist in the form of an asymptomatic latent
infection with the potential to reactivate at any time.
Results
Cloning of small RNAs from M. tuberculosis
Previous reports have identified the two small structural
RNAs, tmRNA and RNaseP RNA from M. tuberculosis
(Svard et al., 1996; Mignard and Flandrois, 2007);
however, no regulatory RNAs have been identified to
date. Therefore our initial experiment was aimed at determining
the actual presence and abundance of small transcripts,
regarded as putative sRNAs. Total RNA from
exponential and stationary growth phases was depleted
of rRNA, and labelled 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, with a difference
in the pattern of expression between the two
growth phases (Fig. S1). This prompted us to proceed
with further experiments.
Discussion
We have shown here for the first time experimental
evidence of sRNAs in M. tuberculosis. By screening
cDNA libraries prepared from low-molecular-weight
M. tuberculosis RNA, we cloned nine sRNAs which were
all readily visualized by Northern blotting and were further
characterized by mapping of 5′ and 3′ ends. All of the
cloned transcripts appeared to be degradation products of
the native sRNAs, judging from size differences detected
by Northern blot. All of the M. tuberculosis sRNAs display
stable predicted secondary structures and in most cases
a C:G ratio > 1. They include transcripts from intergenic
regions (trans-encoded sRNAs) as well as short antisense
transcripts encoded within ORFs (cis-encoded
sRNAs). One of the trans-encoded sRNAs was identified
as the M. tuberculosis homologue of the 4.5S RNA molecule
that forms part of the signal recognition particle
involved in protein secretion (Driessen and Nouwen,
2008).