RNomics in Escherichia coli detects new sRNA species and indicates parallel transcriptional output in bacteria
Jo rg Vogel
1
2
Verena Bartels
0
Thean Hock Tang
4
Gennady Churakov
0
Jacoba G. Slagter-Ja ger
2
Alexander Hu ttenhofer
0
3
E. Gerhart H. Wagner
2
0
Institut fu r Experimentelle Pathologie/Molekulare Neurobiologie, Universita t Mu nster
, Von-Esmarch-Str. 56, D-48149 Mu nster,
Germany
1
Department of Molecular Genetics and Biotechnology, The Hebrew University-Hadassah Medical School
, PO Box 12272,
91120 Jerusalem, Israel
2
Institute of Cell and Molecular Biology, Biomedical Center, Uppsala University
, Box 596, 75124 Uppsala,
Sweden
3
Institut fu r Molekularbiologie
, Abt. Funktionelle Genomik,
Universita t Innsbruck
, Peter-Mayr-Str. 4b, 6020 Innsbruck,
Austria
4
Department of Medical Microbiology and Parasitology, School of Medical Sciences
, 16150 Kubang Kerian, Kelantan,
Malaysia
Recent bioinformatics-aided searches have identified many new small RNAs (sRNAs) in the intergenic regions of the bacterium Escherichia coli. Here, a shot-gun cloning approach (RNomics) was used to generate cDNA libraries of small sized RNAs. Besides many of the known sRNAs, we found new species that were not predicted previously. The present work brings the number of sRNAs in E.coli to 62. Experimental transcription start site mapping showed that some sRNAs were encoded from independent genes, while others were processed from mRNA leaders or trailers, indicative of a parallel transcriptional output generating sRNAs coexpressed with mRNAs. Two of these RNAs (SroA and SroG) consist of known (THI and RFN) riboswitch elements. We also show that two recently identified sRNAs (RyeB and SraC/RyeA) interact, resulting in RNase III-dependent cleavage. To the best of our knowledge, this represents the first case of two non-coding RNAs interacting by a putative antisense mechanism. In addition, intracellular metabolic stabilities of sRNAs were determined, including ones from previous screens. The wide range of half-lives (<2 to >32 min) indicates that sRNAs cannot generally be assumed to be metabolically stable. The experimental characterization of sRNAs analyzed here suggests that the definition of an sRNA is more complex than previously assumed.
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In the traditional view, the transcriptional output of a genome
comprises three major classes of RNAs, which function either
in genetic information transfer (mRNA) or in protein synthesis
(rRNA and tRNA). A minor addition were a few small,
untranslated RNAs with housekeeping or regulatory roles
[reviewed in Wagner and Vogel (1) and Wassarman et al. (2)].
Recent systematic searches for such small non-coding RNAs
(sRNAs) have significantly changed our view about their
prevalence, since numerous representatives of these molecules
are now known to be encoded by bacterial, archaeal and
eukaryal genomes (3). In 2001, three groups searched the
empty spaces between known protein-coding regions, i.e.
intergenic regions (IGRs), of Escherichia coli and discovered
31 new sRNAs (46). Bioinformatic analysis of sequence
conservation among closely related bacteria and/or structural
conservation at the RNA level proved to be powerful tools for
identification of such sRNA genes. Additional strategies used
were analyses of transcription initiation and termination
features, or transcript detection on microarrays specifically
in IGRs. Other recent approaches to predict sRNAs in E.coli
employed neural networks to extract common features among
known RNAs (7), relied on transcription features alone (8) or
used whole genome arrays (9). A recent compilation sets the
present number of E.coli sRNA genes at 55 and, furthermore,
about 1000 non-redundant sRNA candidates have been
proposed but are as yet unconfirmed (10).
Though sRNAs comprise a significant fraction of the
overall transcriptional output in E.coli, the screens for these
molecules have not been saturated. Experimental verification
of sRNA candidates might have been limited by the growth
conditions tested, and stable secondary structures of sRNAs
could have hampered detection on oligoribonucleotide arrays
[e.g. Wassarman et al. (6)]. Moreover, most screens (4,6,8)
deliberately searched for sRNAs encoded by independent
genes, although sRNAs can be generated by processing of
longer transcripts, as in the case of E.coli 6S RNA (11).
In contrast to screens that rely on predictions, cDNA
cloning of RNAs in the size range of 50500 nt aims at
identifying those sRNAs that are expressed in a given genome
*To whom correspondence should be addressed Tel: +46 18 471 4579; Fax: +46 18 530 396; Email:
Correspondence may also be addressed to Alexander Huttenhofer. Tel: +43 512 507 3630; Fax: +43 512 507 9880; Email:
under a given set of conditions, irrespective of whether they
are encoded independently or generated by processing. This
approach, experimental RNomics, has been successfully
employed to discover a vast number of non-coding RNAs in
several eukaryotic organisms and in the archaeon
Archaeoglobus fulgidus (1217).
To complement previous studies of the overa (...truncated)