The development of exosomes pre-dates the divergence of eukaryotes and archaea. Exosomes appear to have developed from bacterial proteins responsible for RNA degradation, as their overall structures display striking similarities, and the proteins involved carry homologous domains.
PNPases (polynucleotide phosphorylases) are exoribonucleases that act as degradosomes, and are responsible for the 3’-5’ degradation of unstructured RNA in bacteria, mitochondria and chloroplasts. Bacterial PNPases contain three copies of a polypeptide that has two RNase-like PH domains, a KH domain and an S1 domain arranged in a single polypeptide. The homotrimer forms a ring of six RNase PH domains, with three S1 and KH domains arranged on one side of the ring. The PNPase PH (phosphorolytic) domain acts as the exoribonuclease to cleave nucleotides from the RNA substrates, while the KH and S1 are protein-nucleic acid recognition motifs. The KH domain (K homology) possesses a nucleic acid-recognising helix-turn-helix motif, and was originally identified in hnRNP-K, from which it takes its name. The S1 domain belongs to the olilgonucleotide/oligosaccharide-binding (OB) fold proteins, and was originally identified in ribosomal protein S1.
Exosomes display remarkable similarity in structure to PNPases. Archaeal exosomes have a simpler subunit composition than eukaryotic exosomes, but structurally they are very similar. Archaeal exosomes have four subunits: Rrp41, Rrp42, Rrp4 and Csl4. The Rrp41 and Rrp42 subunits contain the RNase PH domains, and are organised as a hexameric PH-ring consisting of a trimer of Rrp41/Rrp42 pairs that form a central degradation chamber, similar to that of PNPases. There are three exonuclease active sites within the Rrp41 subunit, which face inside the degradation chamber opposite the cap; Rrp42 is inactive, but contributes to the positioning of the Rrp41 active sites. The cap sits on one end of the hexameric PH-ring, and consists of a homotrimer of Rrp4 or Csl4 subunits, which contain S1, KH and zinc-ribbon domains used to bind the RNA substrate and feed it into the degradation chamber formed by the hexameric PH-ring.
Eukaryotic exosomes diverged from archaeal ones by increasing in complexity, but the core structure of a hexameric PH-ring with a cap of RNA-binding domains remains the same. Eukaryotic exosomes have 9-11 different subunits. The hexameric PH-ring forming the degradation chamber is usually composed of six different subunits, alternating Rrp41-like subunits (Rrp41, Rrp46 and Mtr3) with Rrp42-like subunits (Rrp42, Rrp43 and Rrp45). These subunits make specific pairs: Rrp41/45, Rrp46/43 and Mtr3/Rrp42. Some of these subunits appear to have lost enzymatic activity (in humans, only the Rrp41/45 dimer displays activity), and may play more of a regulatory role within the degradation chamber. The cap is formed by S1/KH domain-containing proteins (Csl4, Rrp4 and Rrp40).
In addition, eukaryotic exosomes often have ancillary proteins, such as Rrp6 or Rrp44. Rrp6 is a hydrolytic exonuclease (homologous to E. coli RNase-D) that is associated with nuclear exosomes. Rrp44 is another hydrolytic exonuclease (homologous to E. coli RNase-R and RNase-II) found in yeast exosomes. Both Rrp6 and Rrp44 interact with the bottom of the hexameric PH-ring opposite the cap.
The number of active sites can vary between species. Human exosomes appear to have only one active site/complex. Yeast have one active site/complex within the hexameric PH-ring, and another provided by Rrp44.
Eukaryotes possess both nuclear and cytoplasmic exosomes, which share the same core subunit composition. Nuclear exosomes process rRNA, snRNA and snoRNA, as well as unspliced pre-mRNAs, such as nonsense transcripts. Cytoplasmic exosomes are involved in the 3’-5’ pathway of mRNA degradation. Eukaryotes also have a separate 5’-3’ degradative pathway carried out by Xrn1 exonuclease within P bodies.