Euryarchaeal β-CASP Proteins with Homology to Bacterial RNase J Have 5′- to 3′-Exoribonuclease Activity

Abstract

In the Archaea only a handful of ribonucleases involved in RNA processing and degradation have been characterized. One potential group of archaeal ribonucleases are homologues of the bacterial RNase J family, which have a ␤-CASP metallo-␤-lactamase fold. Here we show that ␤-CASP proteins encoded in the genomes of the hyperthermophilic Euryarchaeota Pyrococcus abyssi and Thermococcus kodakaraensis are processive exoribonucleases with a 5 end dependence and a 5 to 3 directionality. We named these enzymes Pab-RNase J and Tk-RNase J, respectively. RNAs with 5-monophosphate or 5-hydroxyl ends are preferred substrates of Pab-RNase J, whereas circularized RNA is resistant to Pab-RNase J activity. Degradation of a 3 endlabeled synthetic RNA in which an internal nucleoside is substituted by three ethylene glycol units generates intermediates demonstrating 5 to 3 directionality. The substitution of conserved residues in Pab-RNase J predicted to be involved in the coordination of metal ions demonstrates their importance for ribonuclease activity, although the detailed geometry of the catalytic site is likely to differ from bacterial RNase J. This is the first identification of a 5-exoribonuclease encoded in the genomes of the Archaea. Phylogenetic analysis shows that euryarchaeal RNase J has been inherited vertically, suggesting an ancient origin predating the separation of the Bacteria and the Archaea.The study of RNA metabolism in Archaea is in its infancy (for a recent review, see Ref. 1). The control of RNA processing and mRNA stability are important steps of gene expression. As in all domains of life, processing of tRNA in Archaea is performed by the ribonucleoprotein RNase P (2) and the endoribonuclease RNase Z (3, 4). The RNA-splicing endoribonuclease removes introns from archaeal pre-tRNA and participates in rRNA processing (5, 6). The coupling of transcription and translation in Archaea (7) suggests that mRNA degradation in the Archaea might be more closely related to processes in bacteria than in eukaryotes. To our knowledge, archaeal mRNA is not capped, nor have homologues of eukaryotic enzymes involved in mRNA capping, decapping, and 5Ј-to 3Ј-exoribonucleolytic degradation been identified in archaeal genomes (1, 8, 9). However, a eukaryotic-like exosome with a 3Ј-to 5Ј-exoribonucleolytic activity is present in most Archaea (10, 11) with the exception of the halophilic archaeon Haloferax where this activity is carried out by RNase R (12). Overall, our comprehension of mRNA degradation in Archaea is very limited, whereas it is quite advanced in Eukarya and bacteria.In bacteria as well as in Eukarya, the nature of the 5Ј end of mRNA is an important determinant for stability. In the model Gram-negative bacterium Escherichia coli, RNase E, a key enzyme in mRNA degradation, is a 5Ј end-dependent endoribonuclease (13,14). Indeed substrates with 5Ј-monophosphate ends stimulate RNase E activity (15-17). In the model Grampositive bacterium Bacillus subtilis, the recently identified and characterized RNase J was discovere...

Identification of an RNase J ortholog in Sulfolobus solfataricus: Implications for 5′-to-3′ directional decay and 5′-end protection of mRNA in Crenarchaeota

“…59-End protection of mRNAs is occurring in all three kingdoms of life ( Fig. 1; Tourriere et al 2002;Mathy et al 2007;Clouet-d'Orval et al 2010). Interestingly, in the three kingdoms, different means are exploited to protect the tri-phosphate group and consequently the body of the mRNA from 59-to-39 directional decay.…”

“…Furthermore, no RNase E orthologs have been found in Archaea. RNase J othologs were predicted for Euryarchaeota by bioinformatics (Even et al 2005) and were recently experimentally confirmed in the hyperthermophilic Euryarchaeota Pyrococcus abyssi and Thermococcus kodakaraensis (Clouet-d'Orval et al 2010).…”

mRNA degradation and maturation in prokaryotes: the global players

“…At present, it is not clear whether it is the dimerization of the enzyme, the C-terminal domain itself, or both, that is important for enzyme function. By comparison, archeal RNase J orthologs, which show only 59 exonuclease activity (31,32) have no equivalent C-terminal domain, but their oligomerization status is unknown.…”

“…However, new crystallographic data, discussed further on, provide a rational insight into why the endonucleolytic activity of RNase J might be restricted in vivo (47,48). In the archea, recently identified RNase J orthologs have a strong 59 exonucleolytic activity but no significant endonuclease activity (31,32). In contrast, mycobacterial RNase J has both 59 exo-and endonucleolytic activity (Taverniti and Putzer, unpublished results).…”

“…RNase J orthologs have recently also been characterized as 59 exoribonucleases in the eury-and crenarcheal clades of the archea (31,32). Their importance in global mRNA turnover is not yet well established.…”