dRNA-seq-based analysis of the Thermococcus kodakarensis KOD1 transcriptional landscape to identify growth-regulated small regulatory RNAs
Final Report Abstract
Prokaryotes have relatively small genomes, densely-packed and apparently dominated by proteinencoding sequences. However, data now generated by high throughput RNA sequencing (RNA-seq) reveal surprisingly more complex transcriptomes with many previously unrecognized and unanticipated non-coding small and antisense transcripts. To date, such studies have investigated primarily Bacteria. Here, we report the transcripts present in Thermococcus kodakarensis, a model hyperthermophilic Archaeon, synthesized under different physiological conditions. Sequencing cDNA libraries generated from RNA preparations isolated from growing and stationary phase cells has identified >2,700 sites of transcription initiation, established a genome-wide map of transcripts in T. kodakarensis, and consensus sequences for transcription initiation and translation regulatory elements. Primary transcription start sites (TSS) have been identified upstream of 1,254 annotated genes, including ~78 % of those previously predicted, with an additional 644 primary TSS and their promoters identified within genes. The results identify 1,018 antisense transcripts, most complementary to either the 5'- or 3'-region of a sense mRNA. They confirm the presence of 69 predicted snoRNAs, transcripts from the three CRISPR loci and transcripts predicted to be riboswitches. The T. kodakarensis genome is small (~2.1 Mbp) and tightly packed with proteinencoding genes, but the results reveal the presence of many non-coding RNAs and predict extensive RNA-based regulation in T. kodakarensis. Transcripts have been documented from many regions of the T. kodakarensis genome that were not previously recognized as encoding small RNAs, some of which are ≤ 50% GC with sequences that do not fold readily into base paired secondary structures, contrary to the classical expectation for non-coding RNAs in a hyperthermophile. In a second project, a novel fluorescent reporter-systems for T. kodakarensis was developed using TK1971 as the foundation, which encodes an O6-methylguanine-DNA methyltransferase (MGMT) and was proven to be involved in the DNA-repair upon exposure of alkylating agents in other species. Several genetically engineered constructs have been generated, including a markerless deletion of TK1971 gene and none of the studied constructs showed a negative impact in cell culture experiments. An experimental protocol was developed to study the TK1971 reporter by using confocal laser scanning microscopy. In further in vitro an in vivo experiments the concept of this reporter-system was proven and an reporter assay with readily detectable fluorescent activity was established. The further development of the described reporter will make the system an indispensible tool for future studies in T. kodakarensis and might be easily applied to other (hyperthermophilic) Archaea.
Publications
- Primary transcriptome map of the hyperthermophilic archaeon Thermococcus kodakarensis. BMC Genomics. 2014 Aug 16;15:684
Jäger D, Förstner KU, Sharma CM, Santangelo TJ, Reeve JN
(See online at https://doi.org/10.1186/1471-2164-15-684) - Small regulatory RNAs in Archaea. RNA Biol. 2014;11(5):484-93
Babski J, Maier LK, Heyer R, Jaschinski K, Prasse D, Jäger D, Randau L, Schmitz RA, Marchfelder A, Soppa J
(See online at https://doi.org/10.4161/rna.28452)
