RNase PH, PNPase, archaeal and eukaryotic exosome cores are composed of evolutionarily related domains () that oligomerize to form rings with central pores large enough to accommodate single stranded RNA (). RNase PH achieves this architecture through oligomerization of six RNase PH proteins (), resulting in a pseudo-hexameric ring with three-fold symmetry ()12–14
. The RNase PH ring includes six phosphorolytic active sites that are located in the interface between respective RNase PH proteins. The head to tail arrangement of RNase PH proteins around the ring generates a molecular two fold axis that situates three active sites on the bottom of the ring, another three active sites on the top of the ring, and RNA binding surfaces situated within the pore ().
‘Exosomes’ from bacteria, archaea, and eukaryotes have a similar architecture
Similar to RNase PH, PNPase forms a related pseudo-hexameric ring through oligomerization of three PNPase molecules that contain an N-terminal RNase PH-like domain which we term RNase PH 1, an alpha domain, a second RNase PH-like domain which we term RNase PH 2 which is then followed by a KH domain and an S1 domain (). The RNase PH 2 domain contains residues responsible for phosphorolytic activity, while the amino terminal RNase PH 1 domain is catalytically inactive2
. The phosphorolytic active site and RNA binding surfaces are formed at the interface between the RNase PH 2 and RNase PH 1 domains (). Because only one RNA PH-like domain in PNPase contains residues that form the phosphorolytic active site, only three active sites are formed in PNPase. In addition, the RNase PH 2 domain of PNPase is partially occluded from solvent by the alpha domain (bottom orientation) while additional putative RNA binding surfaces are formed by the KH and S1 domains (top orientation, & ).
Crystal structures from the hyper-thermophiles Sulfolobus solfataricus
, Archaeoglobus fulgidus
, and Pyrococcus abyssi
revealed that archaeal exosomes are composed of trimers of Rrp41-Rrp42 heterodimers which oligomerize to form pseudo-hexameric rings (). Rrp41 contains residues that comprise the phosphorolytic active site that share sequence similarity with both the RNase PH 2 domain of PNPase and RNase PH (). Rrp42 shares sequence similarity with the RNase PH 1 domain and is catalytically inert ( & ). Analogous to bacterial PNPase, the RNA binding surfaces and active sites are located in a composite surface formed between the Rrp41 and Rrp42 heterodimer (). The six-subunit rings are capped by three copies of Rrp4, Csl4, or combinations therein5–7, 15
. Rrp4 and Csl4 both contain putative sites for RNA interaction via their S1 and KH domains or S1 domain, respectively. The phosphorolytic active sites are exposed to solvent and visible at the bottom of the ring while Rrp4 or Csl4 cap the top of the ring to presumably restrict access or guide substrates into the pore for degradation (, top view).
The human exosome core features a pseudo-hexameric six-component ring, three-component cap, and a central pore, an architecture common to bacterial PNPase and archaeal exosomes ( & )9
. With that said, the human exosome architecture differs somewhat from archaeal exosomes because the nine-subunit core is formed through oligomerization of nine individually encoded subunits that form the ring (Rrp41, Rrp45, Rrp42, Rrp43, Mtr3 and Rrp46) or the three-component cap (Rrp4, Rrp40, and Csl4). While it is likely that the general architecture observed for the human exosome core is predictive of other eukaryotic exosomes, subtle distinctions between protozoa and metazoa are expected; for instance, metazoan Rrp45 subunits include a large (~150 amino acid) C-terminal extension that is absent in lower eukaryotes (). Interestingly in human Rrp45, this extension contains a phosphorylation-dependent SUMO interaction motif suggesting that this region of Rrp45 may be important for regulation of exosome activities or assembly16
Subunits that comprise the six-component ring of eukaryotic exosomes share higher sequence and structural similarities to either archaeal Rrp41 or PNPase RNase PH 2-like proteins (Rrp41, Mtr3, and Rrp46) or archaeal Rrp42 or PNPase RNase PH 1-like proteins (Rrp42, Rrp43, and Rrp45). The six-component ring is formed by oligomerization of three distinct RNase PH 2-like and RNase PH 1-like heterodimers: Rrp41-Rrp45, Rrp43-Rrp46, and Mtr3-Rrp42. However, unlike the archaeal exosome or PNPase, both classes of eukaryotic RNase PH-like domains are devoid of catalytic activity and do not contain key catalytic residues that are conserved in PNPase or archaeal exosome phosphorolytic active sites ( & )5, 9–11, 17
Csl4, Rrp4, and Rrp40 contain N-terminal Domains (NTD) and putative RNA binding S1 domains, but they differ with respect to inclusion of either a KH domain (as observed in Rrp4 and Rrp40) or a C-Terminal Domain (CTD), as observed for Csl4 (). In addition, subunits of the cap are required to stabilize interactions between the different RNase PH-like heterodimers. Specifically in the human exosome, Rrp4 bridges interactions between Rrp41 and Rrp42, Rrp40 bridges the Rrp45 and Rrp46 interface, and Csl4 interacts with Mtr3 and to a lesser extent with Rrp43 (). This phenomenon of the three-component cap stabilizing the hexameric core is unique to eukaryotes, insofar as the archaeal exosome forms stable six-component rings in the absence of the three-component cap5
. A feature unique to eukaryotic and archaeal exosomes is that the S1 domains of the three-component cap face the central pore surface, while in bacterial PNPase the KH domains face the central pore. The significance of the orientations for the S1 and KH putative RNA binding domains with respect to the central pore has not been ascertained.