Rrp47 functions in RNA surveillance and stable RNA processing when divorced from the exoribonuclease and exosome-binding domains of Rrp6

Abstract

The eukaryotic exosome exoribonuclease Rrp6 forms a complex with Rrp47 that functions in nuclear RNA quality control mechanisms, the degradation of cryptic unstable transcripts (CUTs), and in the 3 ′ end maturation of stable RNAs. Stable expression of Rrp47 is dependent upon its interaction with the N-terminal domain of Rrp6 (Rrp6 NT ). To address the function of Rrp47 independently of Rrp6, we developed a DECOID (decreased expression of complexes by overexpression of interacting domains) strategy to resolve the Rrp6/Rrp47 complex in vivo and employed mpp6Δ and rex1Δ mutants that are synthetic lethal with loss-of-function rrp47 mutants. Strikingly, Rrp47 was able to function in mpp6Δ and rex1Δ mutants when separated from the catalytic and exosome-binding domains of Rrp6, whereas a truncated Rrp47 protein lacking its C-terminal region caused a block in cell growth. Northern analyses of the conditional mutants revealed a specific block in the 3 ′ maturation of box C/D snoRNAs in the rex1 rrp47 mutant and widespread inhibition of Rrp6-mediated RNA surveillance processes in the mpp6 rrp47 mutant. In contrast, growth analyses and RNA northern blot hybridization analyses showed no effect on the rrp47Δ mutant upon overexpression of the Rrp6 NT domain. These findings demonstrate that Rrp47 and Rrp6 have resolvable functions in Rrp6-mediated RNA surveillance and processing pathways. In addition, this study reveals a redundant requirement for Rrp6 or Rex1 in snoRNA maturation and demonstrates the effective use of the DECOID strategy for the resolution and functional analysis of protein complexes.

The Exosome Cofactor Rrp47 Is Critical for the Stability and Normal Expression of Its Associated Exoribonuclease Rrp6 in Saccharomyces cerevisiae

“…Overexpression of Rrp6 suppressed RNA processing and degradation defects observed in the rrp47∆ mutant and complemented the synthetic lethality of rrp47∆ mpp6∆ and rrp47∆ rex1∆ double mutants. Furthermore, analyses of RNA from the rrp47∆ mpp6∆ and rrp47∆ rex1∆ double mutants are consistent with studies proposing that either the Rrp6/Rrp47 complex or an Mpp6-dependent activity is required for RNA surveillance pathways and the degradation of CUTs, while the Rrp6/Rrp47 complex and Rex1 provide redundant activities for the 3’ maturation of box C/D snoRNAs [29,44]. …”

“…This construct expresses the Rrp6 fusion protein from the RRP4 promoter. An analogous MPP6 construct has been recently reported [44]. Mutant variants of the RRP6 construct that express the catalytically inactive rrp6-1 (D238N) derivative (p389) [24] or just the N-terminal domain truncation (L197X) (p287) were generated by site directed mutagenesis with appropriate primers [43].…”

“…The rex1∆::KANMX4 rrp47∆::KANMX4 double mutant strain has been described previously [28] and was made by crossing rex1∆ and rrp47∆ single mutants, transforming the diploid strain with a plasmid encoding a wild-type copy of the RRP47 gene, and isolating meiotic progeny bearing both null alleles. Strains expressing the plasmid-borne zz-Rrp6 fusion protein as the sole form of the protein and lacking either the MPP6 or REX1 gene have been recently reported [44]. …”

Limited Portability of G-Patch Domains in Regulators of the Prp43 RNA Helicase Required for Pre-mRNA Splicing and Ribosomal RNA Maturation in Saccharomyces cerevisiae

“…Removal of this element does, however, promote the accumulation of slightly extended snR18 RNAs, a characteristic seen with several other pxr1 mutant alleles and proposed to result from aberrant 39-end processing . In fact, 39-extended snoRNAs, including polyadenylated species, also accumulate when snoRNA transcriptional termination is blocked, snoRNP assembly is disrupted by Nop58 or Nop1 depletion, or exosome activity is impaired (see Allmang et al 1999;Grzechnik and Kufel 2008;Costello et al 2011;Garland et al 2013). While commonly thought to target stable RNAs for degradation (Lacava et al 2005;Vanacova et al 2005;Wyers et al 2005), some 39-extended RNAs escape this fate (Grzechnik and Kufel 2008), and the snR18+3 transcripts found in the pxr1 mutants appear stable and accumulate to levels equivalent to those of the properly processed RNAs in the WT background.…”

Targeting RNA for processing or destruction by the eukaryotic RNA exosome and its cofactors

“…2B; Schuch et al 2014), while the C-terminal region of C1D interacts with protein components of box C/D snoRNPs (Costello et al 2011). With that said, the NTD of C1D fully rescues growth in synthetic-lethal Δrex1Δrrp47 and Δmpp6Δrrp47 S. cerevisiae strains, suggesting that the most critical functions for C1D may pertain to Mtr4/TRAMP recruitment and stabilization of Rrp6 (Costello et al 2011;Garland et al 2013).…”