The heterogeneity of the RNA degradation exosome in Sulfolobus

Abstract

RNA is necessary for protein synthesis and for gene regulation in all living organisms. Most RNA molecules are transcribed as precursors which are then maturated by ribonucleases (RNases) and RNA modification enzymes. Often large multiprotein complexes are responsible for the maturation and for the degradation of various RNA molecules in the cell. The general mechanisms of RNA processing and degradation are highly conserved and include endonucleolytic cleavages, posttranscriptional modification at the 3'-end (RNA tailing) and exoribonucleolytic degradation or trimming in 3'-5' direction or in 5'-3' direction (for recent reviews see Refs (1-3). Controlled RNA degradation as a part of the posttranscriptional gene regulation is especially important for u nic ellular, prokaryotic microorganisms, which are exposed to changing environmental conditions. Prokaryotes are a highly heterogeneous group which include b a c te ria and Archaea. Archaea show morphological similarities to bacteria, but are closely related phylogenetically to eukarya than to bacteria (4). They also show a strong similarity to eukarya at the molecular level, for example in the mechanisms of replication, transcription and translation. Archaeal mRNA, however, is more similar to bacterial mRNA: it is generally intron-less and lacks a long stabilising poly (A)-tail at the 3'-end as well as a methylguanosine cap at the 5'-end. In accordance with this, the mechanisms of RNA degradation in Archaea show strong similarities to those operating in bacteria (2). For example, the archaeal protein complex named exosome is structurally and functionally similar to the bacterial polynucleotide phosphorylase (PNPase) (5-8). The bacterial PNPase and the archaeal exosome show structural similarities to the eukaryotic nine-subunit exosome (9), but there are important functional differences between the eukaryotic and prokaryotic exosome-like machineries. The activity of the eukaryotic nine subunit exosome is due to a tenth subunit with similarity to the bacterial RNase R, while its PNPase-like, nine-subunit core is catalytically inactive (10). The eukaryotic ten-subunit exosome that functions as a hydrolytic 3'-5' exoribonuclease and an endoribonuclease (11,12) is essential and participates in various RNA processing and RNA degrading pathways both in the nucleus and in the cytoplasm (13,14). Eukaryotic RNAs are intended for exoribonucleolytic 3'-5' degradation by the addition of short poly (A) tails. This destabilizing polyadenylation is performed by specialized protein complexes that are different from the eukaryotic exosome (15,16). In contrast, both the bacterial PNPase and the archaeal nine subunit exosome are phosphorolytic 3'-5' exoribonucleases, which also can use NDPs to synthesize destabilizing, heteropolymeric RNA tails, which are used as loading platforms for exo-ribonucleases (6,17,19). In Archaea lacking the exosome, RNA is not post-transcriptionally modified at the 3'-end (19). In these exosome-less Archaea, RNA is either exoribonucleolytically degraded in 3'-5' direction by a homologue of bacterial RNase R (in halophiles) or seems to be exclusively degraded in 5’-3’ direction by a homologue of the bacterial RNase J (in some methanogenic Archaea) (20,21). Like in Bacteria, in Archaea degradation in 5’-3’ direction is inhibited by a 5 ’ - triphosphate and is performed by RNase J homologues (22,23). The endoribonucleolytic mechanisms, which are of central importance for RNA degradation in Bacteria (3,24) are still not explored in Archaea. The recently described, endonucleolytically active RNase J homologues in methanogens are good c a n d id a te s fo r p r in c ip le endoribonucleases in the third domain of life (21). The existence of the archaeal exosome was proposed by bioinformatic analyses based on its similarity to the eukaryotic exosome (25) and was verified by co-immunoprecipitation from the thermoacidophilic archaeon Sulfolobus solfataricus (26). Later, its existence in vivo was verified for two further archaeal species, Methanotermobacter thermoautotrophicus and Thermococcus kodakarensis (27,28). Major components of the archaeal exosome are the orthologs of the eukaryotic exosomal subunits Rrp41, Rrp42, Rrp4 and Csl4 forming the ninesubunit form of the archaeal complex, and the archaeal DnaG protein, the function of which in respect to RNA is still unknown. The structure and the catalytic mechanism of the reconstituted archaeal nine-subunit exosome are well understood. It is built of a phosphorolytically active hexametric ring containing the subunits Rrp41 (harbouring the active centre) and Rrp42, to which a trimeric cap of the RNA-binding proteins Rrp4 and/or Csl4 is bound. It performs metal-dependent phosphorolysis of RNA in the presence of inorganic phosphate (Pi) and Mg2Cl, and synthesizes RNA using NDPs without a template (5-8,29-31).