The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein

Abstract

The exosome complex is a key component of the cellular RNA surveillance machinery and is required for normal 3′ end processing of many stable RNAs. Exosome activity requires additional factors such as the Ski or TRAMP complexes to activate the complex or facilitate substrate binding. Rrp47p promotes the catalytic activity of the exosome component Rrp6p, but its precise function is unknown. Here we show that recombinant Rrp47p is expressed as an apparently hexameric complex that specifically binds structured nucleic acids. Furthermore, pull-down assays demonstrated that Rrp47p interacts directly with the N-terminal region of Rrp6p that contains the functionally uncharacterized PMC2NT domain. Strains expressing a mutant form of Rrp6p lacking the N-terminal region failed to accumulate Rrp47p at normal levels, exhibited a slow growth phenotype characteristic of rrp47-Δ mutants and showed RNA processing defects consistent with loss of Rrp47p function. These findings suggest Rrp47p promotes Rrp6p activity by facilitating binding via the PMC2NT domain to structural elements within RNA. Notably, characterized Rrp6p substrates such as the 5.8S+30 species are predicted to contain helices at their 3′ termini, while others such as intergenic or antisense cryptic unstable transcripts could potentially form extensive double-stranded molecules with overlapping mRNAs.

Rrp6, Rrp47 and Cofactors of the Nuclear Exosome

“…11,14 Binding occurs via the N-terminal PMC2NT domain of Rrp6 91 and the N-terminal -helical region of Rrp47 (our unpublished observations).…”

“…The C-terminus of Rrp47 and homologous proteins is rich in basic residues and presumably contributes to RNA binding. 11,77 Rrp47 and C1D can both be phosphorylated in vitro 73,78 but the functional relevance of this is not clear. In contrast to other exosome proteins including Rrp6, Rrp47…”

“…11,14,64,65 Research on Rrp47 has focussed on its physical association with the exosome nuclease complex. …”

“…Westerns of epitope-tagged proteins show that Rrp47 and Rrp6 are expressed at comparable levels in yeast, 11 Rrp6 being present at approximately 2,000 molecules per cell. 92 Normal expression levels of Rrp47 in yeast are dependent upon this interaction with Rrp6.…”

“…Like the processing of 7S pre-rRNA to 6S pre-rRNA, the last step, catalyzed by Rrp6, occurs efficiently in an Rrp6 mutant defective for interaction with Exo10, but requires interaction with Rrp47 via the Rrp6 PMC2NT domain. 11,18 Several studies suggest that some portion of mature snoRNAs results from the 3' trimming of extended, polyadenylated forms of snoRNAs precursors, as opposed to their total degradation by an exosome catalyzed surveillance mechanism. [19][20][21] This raises a longstanding question of how Rrp6 and Exo10 recognize which RNA substrates should be destroyed and which should be trimmed to their normal 3' ends.…”