TY  - JOUR
A1  - Jäger, Dominik
A1  - Förstner, Konrad U.
A1  - Sharma, Cynthia M.
A1  - Santangelo, Thomas J.
A1  - Reeve, John N.
T1  - Primary transcriptome map of the hyperthermophilic archaeon Thermococcus kodakarensis
JF  - BMC Genomics
N2  - Background
Prokaryotes have relatively small genomes, densely-packed with protein-encoding sequences. RNA sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in the model hyperthermophilic Archaeon, Thermococcus kodakarensis, under different physiological conditions.

Results
Sequencing cDNA libraries, generated from RNA isolated from cells under different growth and metabolic conditions has identified >2,700 sites of transcription initiation, established a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements. The primary transcription start sites (TSS) upstream of 1,254 annotated genes, plus 644 primary TSS and their promoters within genes, are identified. Most mRNAs have a 5'-untranslated region (5'-UTR) 10 to 50 nt long (median = 16 nt), but ~20% have 5'-UTRs from 50 to 300 nt long and ~14% are leaderless. Approximately 50% of mRNAs contain a consensus ribosome binding sequence. The results identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA, and confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 predicted snoRNAs. Two putative riboswitch RNAs were present in growing but not in stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the results also provide a semi-quantitative documentation of the differences in T. kodakarensis genome expression under different growth conditions and confirm previous observations of substrate-dependent specific gene expression. Many previously unanticipated small RNAs have been identified, some with relative low GC contents (≤50%) and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding RNAs in a hyperthermophile.

Conclusion
The results identify >2,700 TSS, including almost all of the primary sites of transcription initiation upstream of annotated genes, plus many secondary sites, sites within genes and sites resulting in antisense transcripts. The T. kodakarensis genome is small (~2.1 Mbp) and tightly packed with protein-encoding genes, but the transcriptomes established also contain many non-coding RNAs and predict extensive RNA-based regulation in this model Archaeon.
KW  - riboswitch
KW  - hyperthermophile
KW  - hydrogen regulation
KW  - transcriptome
KW  - archaea
KW  - promoters
KW  - antisense RNAs
KW  - small non-coding RNAs
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-120966
SN  - 1471-2164
VL  - 15
IS  - 684
ER  - 
TY  - JOUR
A1  - Jäger, Dominik
A1  - Pernitzsch, Sandy R.
A1  - Richter, Andreas S.
A1  - Backofen, Rolf
A1  - Sharma, Cynthia M.
A1  - Schmitz, Ruth A.
T1  - An archaeal sRNA targeting cis- and trans-encoded mRNAs via two distinct domains
JF  - Nucleic Acids Research
N2  - We report on the characterization and target analysis of the small (s) RNA\(_{162}\) in the methanoarchaeon Methanosarcina mazei. Using a combination of genetic approaches, transcriptome analysis and computational predictions, the bicistronic MM2441-MM2440 mRNA encoding the transcription factor MM2441 and a protein of unknown function was identified as a potential target of this sRNA, which due to processing accumulates as three stabile 5' fragments in late exponential growth. Mobility shift assays using various mutants verified that the non-structured single-stranded linker region of sRNA\(_{162}\) (SLR) base-pairs with the MM2440-MM2441 mRNA internally, thereby masking the predicted ribosome binding site of MM2441. This most likely leads to translational repression of the second cistron resulting in dis-coordinated operon expression. Analysis of mutant RNAs in vivo confirmed that the SLR of sRNA\(_{162}\) is crucial for target interactions. Furthermore, our results indicate that sRNA\(_{162}\)-controlled MM2441 is involved in regulating the metabolic switch between the carbon sources methanol and methylamine. Moreover, biochemical studies demonstrated that the 50 end of sRNA\(_{162}\) targets the 5'-untranslated region of the cis-encoded MM2442 mRNA. Overall, this first study of archaeal sRNA/mRNA-target interactions unraveled that sRNA\(_{162}\) acts as an antisense (as) RNA on cis- and trans-encoded mRNAs via two distinct domains, indicating that cis-encoded asRNAs can have larger target regulons than previously anticipated.
KW  - strain
KW  - escherichia coli
KW  - methanosarcina mazei GO1
KW  - methanol methyltransferase isozymes
KW  - small nucleolar RNAs
KW  - acetivorans C2A
KW  - antisense RNAs
KW  - GO1
KW  - transcriptional regulator
KW  - translational initiation
KW  - pyrococcus furiosus
Y1  - 2012
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134972
VL  - 40
IS  - 21
ER  -