The experimental results reported here shed light on the question of whether the activity of the exoribonuclease Rrp6p requires physical association with the core exosome. Our findings indicate that Rrp6p functions in a core exosome-independent manner for some activities in vivo
. Previous studies that analyzed polypeptides associated with affinity purified core exosomes showed that Rrp6p co-purifies with these complexes (48
). Likewise, affinity purification of yeast Rrp6p in some of these studies co-purified the core exosome components (25
). However, these studies did not address the relative stoichiometries of the proteins or the possibility that some of these proteins might exist in sub-complexes or free pools of individual subunits. More recent analyses in human cells, plants and flies suggested that functionally important sub-complexes of core exosome subunits may exist (67–69
). Initial evidence for functional independence of Rrp6p from the core exosome comes from the fact that the core exosome functions prior to Rrp6p in the 3′-end processing of 5.8S rRNA and many snRNAs and snoRNAs (20
). Indeed, our results show that deletion of the C-terminal domain of Rrp6p significantly impairs it ability to interact with the core exosome, but has no effect on these 3′-end formation activities. Moreover, Dis3p, the active component of the core exosome, and Rrp6p exhibit significant differences in their exoribonuclease activities. Dis3p appears to degrade RNA processively, while Rrp6p uses a more distributive mode (15
). Rrp6p also showed better activity than Dis3p in the removal of poly(A) tails from model substrates in vivo
). Interestingly, the interaction of Dis3p with the rest of the core proteins significantly inhibited its activity relative to free Dis3p, while Rrp6p showed similar activity in the presence or absence of the core exosome.
The findings reported here suggest that Rrp6p may function in vivo
without direct interaction with the core exosome. Deletion of the C-terminal domain of Rrp6p disrupts its ability to co-purify with the core exosome. Nevertheless, the mutant enzyme retains its exoribonuclease activity as shown by the fact that it carries out Rrp6p-specific 3′-end processing of 5.8S rRNA and snoRNAs. In contrast, the ΔC2 mutant fails to efficiently degrade some rRNA intermediates that appear to require the action of Rrp6p and the core exosome. For example, the 5′-ETS by-product of rRNA processing accumulates in rrp6
- mutants and in strains depleted of core exosome components, and combination of these defects causes a synergistic increase in 5′-ETS levels (, and ) (20
). The ΔC2 mutant fails to degrade this RNA suggesting that efficient degradation requires physical interaction between Rrp6p and the core exosome, or that the C-terminal domain plays a specific role in the recognition of this RNA. The 5′-ETS RNAs also accumulate as poly(A)+ forms in Rrp6p- and core exosome-deficient cells implying that the TRAMP complex polyadenylates them prior to degradation by Rrp6p and the core exosome [ and , (39
)]. However, poly(A)+ forms of 5′-ETS do not accumulate to a significant extent in the ΔC2 mutant, suggesting that Rrp6p removes their poly(A) tails in an core exosome-independent manner (, lane 4). We observed similar results for the 3′ truncated form of 5S rRNA, shown previously to require Rrp6p, TRAMP and the core exosome for its degradation (data not shown) (40
). Thus, in the cases where Rrp6p and the core exosome co-operate to degrade an RNA, it appears that Rrp6p may remove the poly(A) tails in an exosome-independent manner and then participate with the core exosome in the degradation of the body of the transcript.
Our experiments also identified rRNA processing intermediates that appear to be degraded by Rrp6p in a core exosome-independent manner. These poly(A)+ RNAs (27SA2, 23S, 21S, 17S, 5′-S and D-B1L) accumulate in the absence of Rrp6p and in certain Rrp6p deletion mutants, but not upon depletion of core exosome components (, and ). Moreover, while the depletion of Dis3p in an rrp6-Δ strain results in a synergistic enhancement of the accumulation of some poly(A)+ rRNAs, these Rrp6p-specific transcripts are unaffected (). Thus, unlike the 5′-ETS and 3′ truncated 5S rRNAs, these intermediates seem to require only Rrp6p for their degradation. Indeed, they do not accumulate in the ΔC2 mutant suggesting that their degradation does not require the participation of the core exosome. However, the detection of these rare intermediates requires that they bind to oligo(dT) and therefore carry poly(A) tails. Thus, we cannot exclude the possibility that their presence in any of our experiments might reflect the inability of Rrp6p mutants to remove the tails rather than their inability to degrade the body of the RNA. Regardless of the mechanism, the fact that these RNAs do not accumulate in the ΔC2 mutant indicates that disruption of Rrp6p-core exosome interaction does not affect their degradation.
Analysis of unadenylated degradation intermediates of 5.8S rRNA also showed that Rrp6p degrades these RNAs in a core exosome-independent manner. The hydrolysis of these RNAs requires Rrp6p, but not the core exosome or the TRAMP complex (A). The fact that these intermediates do not appear in the ΔC2 mutant indicates that their degradation may not require the interaction of Rrp6p with the core exosome. Notably, these RNAs do not appear in poly(A)+ fractions of RNAs fractionated by oligo(dT) indicating that they do not carry significant poly(A) tails (data not shown). Thus, unlike the RNAs discussed above, they may never have entered, or they may have already exited, the TRAMP-dependent RNA degradation pathway.
The conclusion that the C-terminal portion of Rrp6p plays a critical role in its interaction with the core exosome agrees with experiments on the structure of the human exosome. Two-hybrid system studies of the interaction of human Rrp6p (PMScl-100) with other core exosome components indicated that the C-terminal 276 amino acids of hRrp6p interact with hRrp43 (Oip2) (71
). This fragment includes a region corresponding to the E. coli
HRDC2 domain and shows significant similarity between the yeast and human proteins (33
). Thus, the human interaction data support our conclusion that the C-terminal domain plays an important role in the interaction of Rrp6p with the core exosome.
In summary, the findings reported here provide evidence that Rrp6p may act independently of the core exosome during the degradation of some rRNA processing products. Structure-function analysis of Rrp6p revealed that it requires its C-terminal domain for interaction with the core exosome. When this interaction is disrupted, Rrp6p continues to carry out critical nuclear RNA 3′-end processing and degradation steps, yet fails to degrade RNAs that appear to require the combined action of Rrp6p and the core exosome. These results suggest a functional sub-specialization of core exosome and Rrp6p functions in the nucleus.