In vivo and in vitro studies of RNA degrading activities in Archaea.
Controlled degradation of RNA is important for the regulation of gene expression in Bacteria and Eukarya, but information about these processes is limited in the domain of Archaea. To address this, we studied the half-life of different mRNAs in halophilic Archaea after blocking transcription with actinomycin D. We found that the stability of mRNAs of the gvp operons in Haloferax mediterranei varies under different growth conditions. To understand regulated mRNA decay in Archaea, we need to identify stability determinants within mRNAs and proteins, mainly ribonucleases (RNases), which recognize these determinants. First, we wanted to identify archaeal RNases independently of their sequence similarity to known RNases from Bacteria and Eukarya. To this end we performed fractionation of proteins from Halobacterium salinarum and tested the fractions for RNase activity with an internally labeled in vitro-synthesized mRNA. After three purification steps, we isolated an endoribonucleolytically active protein with similarities to the eukaryotic initiation factor 5A. Further characterization was performed with recombinant halobacterial IF-5A, which was purified from H. salinarum or Escherichia coli. Mutational analysis confirmed unambiguously its RNase activity. In another study, we aimed to purify a double-strand-specific endoribonuclease from Sulfolobus solfataricus. Seven purification steps led to the isolation of two different dehydrogenases with RNase properties. Interestingly, their RNase activity resembled that of aIF-5A and of highly diluted RNase A. RNA was cleaved preferentially between C and A nucleotides in single-stranded regions, and the activity was inhibited at MgCl(2) concentrations >5 mM and at KCl concentrations >200 mM. However, it was possible to distinguish the activity of the archaeal proteins from the activity of RNase A. In a different approach, we used a bioinformatics prediction of the archaeal exosome to purify this protein complex from S. solfataricus. Isolation by coimmunoprecipitation revealed the presence of four orthologs of eukaryotic exosomal subunits and at least one archaea-specific subunit. We characterized the S. solfataricus exosome as a major enzyme involved in phosphorolytic RNA degradation and in RNA polyadenylation. Here we describe in detail the techniques used to achieve these results.
Evguenieva-Hackenberg
Elena
E
Institut für Mikrobiologie und Molekularbiologie, University of Giessen, Giessen, Germany.
Wagner
Steffen
S
Klug
Gabriele
G
D001105:Archaea / Q000235:genetics / Q000378:metabolism*; D015152:Blotting, Northern; D003609:Dactinomycin / Q000494:pharmacology; D047468:Immunoprecipitation; D008970:Molecular Weight; D019642:RNA, Archaeal / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011995:Recombination, Genetic; D048229:Sulfolobus solfataricus / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects
D019642:RNA, Archaeal; D003609:Dactinomycin