Reconstitution, Activities, and Structure of the Eukaryotic RNA Exosome

Abstract

The RNA exosome is a multisubunit 3' to 5' exoribonuclease complex that participates in degradation and processing of cellular RNA. To determine the activities and structure of the eukaryotic exosome, we report the reconstitution of 9-subunit exosomes from yeast and human and reconstitution of 10- and 11-subunit exosomes from yeast. Comparative biochemical analysis between purified subunits and reconstituted exosomes using AU-rich, polyadenylated (poly[A]), generic, and structured RNA substrates reveals processive phosphorolytic activities for human Rrp41/Rrp45 and the 9-subunit human exosome, processive hydrolytic activities for yeast Rrp44 and the yeast 10-subunit exosome, distributive hydrolytic activities for Rrp6, and processive and distributive hydrolytic activities for the yeast 11-subunit exosome. To elucidate the architecture of a eukaryotic exosome, its conserved surfaces, and the structural basis for RNA decay, we report the X-ray structure determination for the 286 kDa nine-subunit human exosome at 3.35 A.

The PNPase, exosome and RNA helicases as the building components of evolutionarily-conserved RNA degradation machines

“…Nonetheless, alignment of the proteins of this complex clearly points to a great similarity between the number and characteristics of the different domains in the archaeal and eukaryotic exosomes and the PNPases (Figure 2; Table 1). The same core structure is formed: a ring-shaped structure created by six different PHlike polypeptides, where three different subunits each contribute a KH and an S1 domain ( Figure 3) [31]. In addition to RNase PH-like polypeptides, the yeast exosome core contains a novel subunit with hydrolytic 3¢ exonuclease activity (Rrp44 or Dis3) that belongs to the family of RNase R and RNase II in bacteria (Table 1) [38,49].…”

“…Similarities in the structure of the RNase PH and 3¢ fi 5¢ RNA degradation machines: the PNPase and the exosome. The structure of the RNase PH [22,23], the bacterial PNPase [27], archaeal [29,30] and eukaryotic [31] exosomes, as well as the predicted structure of the chloroplast PNPase [24], are shown in order to compare the ring shape of these complexes. The molecular surfaces of these structures are represented in the same view and colored as in Figure 2.…”

“…The structures were generated using Pymol (see The PyMOL Molecular Graphics System in Further Information). Rrp41 [29,30] Rrp44 [31,49] Rrp6 and Rrp44 [31] hRrp41/Rrp45…”

The evolutionarily conserved subunits Rrp4 and Csl4 confer different substrate specificities to the archaeal exosome

“…This nine-subunit complex recruits substrates in a similar way like PNPase and the archaeal exosome, and channels the single stranded 3 0 -end to the catalytically active, 10th core subunit of the exosome, Rrp44, which interacts with the bottom of the hexameric ring ( Supplementary Fig. 1 [12][13][14][15]). …”

Poring over exosome structure

“…the crystal structures of overlapping parts of the eukaryotic exosome (Liu et al, 2006;Bonneau et al, 2009) and the related bacterial pNpase (Symmons et al, 2000) and archaeal exosome (Lorentzen et al, 2007) have been solved, and show that these rNa degrading machines from the three domains of life have a similar structure (Fig 1). they are all composed of a ring of six rNase pH domains, one side of which has a cap that contains putative rNa binding domains.…”