Exo- and Endoribonucleolytic Activities of Yeast Cytoplasmic and Nuclear RNA Exosomes Are Dependent on the Noncatalytic Core and Central Channel

Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.
Molecular cell (Impact Factor: 14.02). 08/2012; 48(1):133-44. DOI: 10.1016/j.molcel.2012.07.012
Source: PubMed


The RNA exosome is an essential multisubunit ribonuclease (RNase) that contributes to cytoplasmic and nuclear RNA decay and quality control. The 9-subunit exosome core (Exo9) features a prominent central channel formed by stacked asymmetric rings of six RNase PH-like proteins and three S1/KH domain proteins. Exo9 is catalytically inert but associates with Rrp44, an endoribonuclease and processive 3'→5' exoribonuclease, and Rrp6, a distributive 3'→5' exoribonuclease. We show that Exo9 and its central channel modulate all three yeast exosome RNase activities because channel occlusion attenuates RNA binding and RNase activities in vitro and fails to complement exosome functions in vivo. We find that Rrp6 stimulates Rrp44 RNase activities and that Rrp6 is inhibited by a mutation in the Rrp44 exoribonuclease active site in 11-subunit nuclear exosomes. These results suggest the exosome core and central channel is essential because it modulates each of the known RNase activities of the yeast RNA exosome.

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    • "Surprisingly, we found that the uridylyltransferase activity of TUT7 or TUT4 toward pre-miRNAs, regardless of their 3 0 overhang length, was specifically stimulated in vitro by addition of immunoprecipitates from wild-type DIS3, but not RRP6 or catalytically inactive Figure 7. A Positive Feedback Loop Constituted by TUT7, TUT4, and the Exosome in Uridylation and Degradation of Pre-miRNAs (A and B) Decay assay of pre-miR-106b using mixtures of DIS3 with wild-type (WT) or catalytically inactive (Wasmuth and Lima, 2012) TUT7 (A) and TUT4 (B). (C) Uridylation assay using TUT7 and DIS3 with pre-miR-106b harboring 3 0 overhangs of different lengths. "
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    ABSTRACT: MicroRNAs (miRNAs) are essential for regulation of gene expression. Though numerous miRNAs have been identified by high-throughput sequencing, few precursor miRNAs (pre-miRNAs) are experimentally validated. Here we report a strategy for constructing high-throughput sequencing libraries enriched for full-length pre-miRNAs. We find widespread and extensive uridylation of Argonaute (Ago)-bound pre-miRNAs, which is primarily catalyzed by two terminal uridylyltransferases: TUT7 and TUT4. Uridylation by TUT7/4 not only polishes pre-miRNA 3' ends, but also facilitates their degradation by the exosome, preventing clogging of Ago with defective species. We show that the exosome exploits distinct substrate preferences of DIS3 and RRP6, its two catalytic subunits, to distinguish productive from defective pre-miRNAs. Furthermore, we identify a positive feedback loop formed by the exosome and TUT7/4 in triggering uridylation and degradation of Ago-bound pre-miRNAs. Our study reveals a pre-miRNA surveillance system that comprises TUT7, TUT4, and the exosome in quality control of miRNA synthesis.
    Full-text · Article · Aug 2014 · Molecular Cell
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    • "Rrp44, the exosome tenth subunit, interacts with Exo-9 in the nucleus and cytoplasm and is essential for cell viability [5], [9]. It presents endonuclease and processive hydrolytic 3′–5′ exonuclease activities [6], [10], [11], which are modulated by the interaction with the Exo-9 core [12], [13]. In yeast, crystallographic and biochemical studies have shown that the Exo-9 central channel directs the RNA substrate to degradation by Rrp44 which is located on the opposite side of the S1/HK subunits [7], [12]. "
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    ABSTRACT: RRP6 is a 3'-5' exoribonuclease associated to the eukaryotic exosome, a multiprotein complex essential for various RNA processing and degradation pathways. In Trypanosoma brucei, RRP6 associates with the exosome in stoichiometric amounts and was localized in both cytoplasm and nucleus, in contrast to yeast Rrp6 which is exclusively nuclear. Here we report the biochemical and structural characterization of T. brucei RRP6 (TbRRP6) and its interaction with the so-called T. brucei Exosome Associated Protein 3 (TbEAP3), a potential orthologue of the yeast Rrp6 interacting protein, Rrp47. Recombinant TbEAP3 is a thermo stable homodimer in solution, however it forms a heterodimeric complex with TbRRP6 with 1∶1 stoichiometry. The crystallographic structure of the TbRRP6 catalytic core exposes for the first time the native catalytic site of this RNase and also reveals a disulfide bond linking two helices of the HRDC domain. RNA degradation assays show the distributive exoribonuclease activity of TbRRP6 and novel findings regarding the structural range of its RNA substrates. TbRRP6 was able to degrade single and double-stranded RNAs and also RNA substrates containing stem-loops including those with 3' stem-loop lacking single-stranded extensions. Finally, association with TbEAP3 did not significantly interfere with the TbRRP6 catalytic activity in vitro.
    Full-text · Article · Feb 2014 · PLoS ONE
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    • "These observations are consistent with a previous study proposing that Rrp6 has an important noncatalytic role in RNA surveillance [58]. One noncatalytic mechanism by which Rrp6 might promote RNA surveillance is through its interaction with the exosome complex, which promotes channelling of RNA substrates through the core to Rrp44 [19,26]. However, rrp6-1 mpp6∆ double mutants are nonviable [44], suggesting that at least one essential biological process is dependent upon the catalytic activity of either Rrp6 or an Mpp6-dependent activity. "
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    ABSTRACT: Rrp6 is a conserved catalytic subunit of the eukaryotic nuclear exosome ribonuclease complex that functions in the productive 3' end maturation of stable RNAs, the degradation of transiently expressed noncoding transcripts and in discard pathways that eradicate the cell of incorrectly processed or assembled RNAs. The function of Rrp6 in these pathways is at least partially dependent upon its interaction with a small nuclear protein called Rrp47/Lrp1, but the underlying mechanism(s) by which Rrp47 functions in concert with Rrp6 are not established. Previous work on yeast grown in rich medium has suggested that Rrp6 expression is not markedly reduced in strains lacking Rrp47. Here we show that Rrp6 expression in rrp47∆ mutants is substantially reduced during growth in minimal medium through effects on both transcript levels and protein stability. Exogenous expression of Rrp6 enables normal levels to be attained in rrp47∆ mutants. Strikingly, exogenous expression of Rrp6 suppresses many, but not all, of the RNA processing and maturation defects observed in an rrp47∆ mutant and complements the synthetic lethality of rrp47∆ mpp6∆ and rrp47∆ rex1∆ double mutants. Increased Rrp6 expression in the resultant rrp47∆ rex1∆ double mutant suppresses the defect in the 3' maturation of box C/D snoRNAs. In contrast, increased Rrp6 expression in the rrp47∆ mpp6∆ double mutant diminishes the block in the turnover of CUTs and in the degradation of the substrates of RNA discard pathways. These results demonstrate that a principal function of Rrp47 is to facilitate appropriate expression levels of Rrp6 and support the conclusion that the Rrp6/Rrp47 complex and Rex1 provide redundant exonuclease activities for the 3' end maturation of box C/D snoRNAs.
    Full-text · Article · Nov 2013 · PLoS ONE
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