The N-terminal region of Rrp44 has ribonuclease activity. (a) The yeast exosome contains three layers. The exosome contains a PH-ring of 6 proteins with an RNase PH domain. Assembly and/or stability of the PH-ring requires three additional proteins that form a cap on one side of the ring. The exoribonuclease activity of the exosome is provided by a tenth subunit (Rrp44) that associates with the bottom of the ring. RNA substrates are thought to associate with the cap proteins, pass through the central channel in the PH-ring, and get degraded by Rrp44 after it emerges from the bottom of the ring. (b) Schematic representation of Rrp44 domains. (c) The N-terminal region of Rrp44, which contains a CR3 domain and a PIN domain, has ribonuclease activity in vitro. The N-terminal part of Rrp44 was purified as a GST-fusion recombinant protein from E. coli and incubated with 5′ end labeled U₃₀ RNA. (d) GST by itself, purified and incubated under identical conditions does not have ribonuclease activity. (e) The N-terminal region of Rrp44 degrades both 5′ end labeled and 3′ end labeled substrates to smaller labeled oligoribonucleotides, suggesting it acts as an endonuclease. (f) The N-terminal region of Rrp44 degrades an internally labeled RNA corresponding to 5′ETS in vitro. (g) The N-terminal region of Rrp44 prefers substrates with a 5′ phosphate (left), over those with a 5′ hydroxyl (right). The apparent slight change in mobility in the right panel is an artifact of this particular gel, and does not reflect a slow degradation of the 5′ OH substrate. (h) Mutation the conserved acidic amino acid residue D171 to A in the N-terminal region of Rrp44 abolishes ribonuclease activity. The asterisk in panels C to G indicates the position of the P³² label.

The RNB domain of Rrp44 is required for both RNA processing and RNA degradation activities of the exosome. (a) The indicated Rrp44 truncations were expressed in a GAL∷rrp44 yeast strain. RNA was isolated from cultures grown in dextrose (to inhibit expression of the GAL∷rrp44 gene), and analyzed by northern blotting with probes that hybridize to the 7S precursor of 5.8S rRNA (top panel), the 5′ ETS of the rRNA (middle panel), and the RNA subunit of the signal recognition particle (bottom panel). The relative levels of the 7S pre-rRNA and the 5′ETS were normalized for loading using the SRP signal and are indicated under the top and middle panels. Shown is a representative experiment. (b) RRP44 alleles containing the D171A, the D551N, or the double mutant D171A D551N where introduced into a GAL∷rrp44 yeast strain. Cultures where first grown in media containing galactose, and then incubated in media containing glucose for the indicated time (in hours), and analyzed by northern blotting with probes that hybridize to the 5.8S rRNA (top panel), the 5′ ETS of the rRNA (middle panel), and the RNA subunit of the signal recognition particle (bottom panel). Shown is a representative experiment.

A model of how RNA might access the 5′ end stimulated endonuclease activity of Rrp44

The PIN active site is important for exosome function. (a) The N-terminal region of Rrp44 is required and sufficient for viability. An rrp44Δ strain complemented by full length RRP44 on a plasmid with a URA3 marker was transformed with LEU2 plasmids encoding each of the depicted truncations. Growth on 5FOA indicates that truncated Rrp44 can carry out the essential function of the exosome. The smallest complementing Rrp44 allele includes amino acids 1-235. Similar results were obtained with a GAL∷rrp44 strain (Supplemental Fig. 2 online) (b) Mutation of the three conserved cysteine residues in the CR3 domain to serine reduces growth. (c) Mutations in the PIN domain active site residues of Rrp44 do not have a large affect on growth, but are lethal in combination with a mutation of the active site residue of the RNB domain. Similar results were obtained for the D91A and E120A mutations (Supplemental Fig. 3 online) (d) Mutations of the PIN domain active site residues are lethal in combination with a truncation that removes the RNB domain. CCDEDD indicates Cys47, Cys52, Cys55, Asp91, Glu120 Asp171, and Asp198, which where mutated in the last 5 rows as indicated.

The essential Csl4 does not contain any essential domains, but its zinc-ribbon domain is required for cytoplasmic exosome-mediated mRNA decay. (a) Rrp4 and Rrp40 make extensive contacts with two neighboring subunits of the PH-ring. Csl4 makes extensive contacts with Mtr3p, but relatively small contacts with Rrp43p. The width of the double headed arrows is proportional to the extent of the buried surface calculated using Pymol. (b) The essential Csl4 does not contain any essential domains. Csl4 contains a RPL27-like domain (red), a 38 amino acid linker, not present in other eukaryotes (white), an S1 domain (Orange), and a zinc-ribbon like domain that is missing the zinc-coordinating amino acid residues (yellow). A csl4Δ strain complemented by full length CSL4 on a plasmid with a URA3 marker was transformed with LEU2 plasmids encoding the depicted truncations. Growth on 5FOA indicates that truncated Csl4 can carry out the essential function of the exosome. Similar results were obtained with a GAL∷csl4 strain (Supplemental Fig. 4 online). (c) The zinc-ribbon domain of Csl4 is required for exosome-mediated mRNA decay. The decay rate of PGK1pG-nonstop mRNA was measured in a wild-type strain (half life = 2 minutes), a strain lacking the zinc-ribbon domain of Csl4 (half life = 10 minutes), and a ski7Δ strain (half life = 10 minutes). Shown is the average value from two independent experiments. The conclusion that the zinc-ribbon domain of Csl4 is required for exosome-mediated mRNA decay was confirmed using two other approaches (Supplemental Fig. 7 online).