Exonucleolysis is required for nuclear mRNA quality control in yeast THO mutants

Assenholt, Jannie; Mouaikel, John; Andersen, Kasper R.; Brodersen, D.E.; Libri, Domenico; Jensen, Torben Heick

doi:10.1261/rna.1108008

Cited by 50 publications

(55 citation statements)

References 24 publications

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“…Taken together, these results indicate that when mRNA export is inhibited in the mex67-5 strain at the nonpermissive temperature of 37°C, a large fraction of unspliced transcripts normally degraded by NMD are now retained in the nucleus and targeted for turnover by the nuclear exosome. This observation might be linked to the activation of nuclear RNA surveillance by Rrp6p that was previously shown to occur in RNA export mutants (Jensen et al 2001;Libri et al 2002;Strasser et al 2002;Zenklusen et al 2002;Rougemaille et al 2007;Assenholt et al 2008). These results also demonstrate that the export of several unspliced pre-mRNAs out of the nucleus is dependent on the mRNA export factor, Mex67p, as shown previously for the YRA1 pre-mRNA (Dong et al 2007).…”

Section: Resultssupporting

confidence: 85%

“…In our study, we found that upon genetic inhibition of mRNA export, a large proportion of unspliced precursors are now retained within the nucleus and degraded by the action of the nuclear exosome. This observation might reflect the higher nuclear retention of unspliced transcripts in the nucleus, but could also be due to the activation of nuclear surveillance that was previously shown to occur upon inhibition of mRNA export in a variety of mutant backgrounds (Libri et al 2002;Rougemaille et al 2007;Assenholt et al 2008). Such shifts in the turnover pathways of unspliced transcripts reveal the dynamic nature of RNA turnover that is modulated and/or controlled by the activity of the conserved RNA export factor, Mex67p.…”

Section: Discussionmentioning

confidence: 81%

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Sequential RNA degradation pathways provide a fail-safe mechanism to limit the accumulation of unspliced transcripts in Saccharomyces cerevisiae

Sayani

Chanfreau²

2012

RNA

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The nuclear exosome and the nonsense-mediated mRNA decay (NMD) pathways have been implicated in the degradation of distinct unspliced transcripts in Saccharomyces cerevisiae. In this study we show that these two systems can act sequentially on specific unspliced pre-mRNAs to limit their accumulation. Using steady-state and decay analyses, we show that while specific unspliced transcripts rely mostly on NMD or on the nuclear exosome for their degradation, some unspliced RNAs are stabilized only when both the nuclear exosome and NMD are inactivated. We found that the mechanism of degradation of these unspliced pre-mRNAs is not influenced by promoter identity. However, the specificity in the pre-mRNAs degradation pathways can be manipulated by changing the rate of export or retention of these mRNAs. For instance, reducing the nuclear export of premRNAs mostly degraded by NMD results in a higher fraction of unspliced transcripts degraded by the nuclear exosome. Reciprocally, inactivating the Mlp retention factors results in a higher fraction of unspliced transcripts degraded by NMD for precursors normally targeted by the nuclear exosome. Overall, these results demonstrate that a functional redundancy exists between nuclear and cytoplasmic degradation pathways for unspliced pre-mRNAs, and suggest that the degradation routes of these species are mainly determined by the efficiency of their nuclear export rates. The presence of these two sequential degradation pathways for unspliced pre-mRNAs underscores the importance of limiting their accumulation and might serve as a fail-safe mechanism to prevent the expression of these nonfunctional RNAs.

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Section: Resultssupporting

confidence: 85%

Section: Discussionmentioning

confidence: 81%

Sequential RNA degradation pathways provide a fail-safe mechanism to limit the accumulation of unspliced transcripts in Saccharomyces cerevisiae

Sayani

Chanfreau²

2012

RNA

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“…This led to the proposal that Rrp47 may promote the Rrp6-mediated degradation of structured RNA. However, Rrp6 lacking the PMC2NT domain shows comparable exonucleolytic activity to the full-length protein on the substrates tested in vitro (Assenholt et al 2008;Wasmuth and Lima 2012). Furthermore, the N-terminal Sas10/C1D domain is able to complement the synthetic lethality of an rrp47Δ rex1Δ mutant but is itself insufficient for the RNA-binding activity of Rrp47 (Costello et al 2011).…”

Section: Introductionmentioning

confidence: 96%

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

Garland

Feigenbutz

Turner

et al. 2013

RNA

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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.

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“…Instead, the 39-to-59 exoribonuclease activity of eukaryotic exosomes is achieved by exosome core association with two hydrolytic enzymes: the tenth subunit, Rrp44 or Dis3, and the eleventh subunit, Rrp6 (Briggs et al 1998;Phillips and Butler 2003;Liu et al 2006;Midtgaard et al 2006;Dziembowski et al 2007;Schneider et al 2007;Wang et al 2007;Lorentzen et al 2008b;Bonneau et al 2009). While the activities of budding yeast Rrp44 and Rrp6 have been characterized (Liu et al 2006;Dziembowski et al 2007;Assenholt et al 2008;Callahan and Butler 2008;Bonneau et al 2009;Schaeffer et al 2009;Schneider et al 2009;Callahan and Butler 2010), much less is known about the intrinsic activities of the human paralogs of Rrp44, DIS3 and DIS3L (Staals et al 2010;, or human RRP6.…”

Section: Introductionmentioning

confidence: 99%

Activities of human RRP6 and structure of the human RRP6 catalytic domain

Januszyk¹,

Liu²,

Lima³

2011

RNA

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The eukaryotic RNA exosome is a highly conserved multi-subunit complex that catalyzes degradation and processing of coding and noncoding RNA. A noncatalytic nine-subunit exosome core interacts with Rrp44 and Rrp6, two subunits that possess processive and distributive 39-to-59 exoribonuclease activity, respectively. While both Rrp6 and Rrp44 are responsible for RNA processing in budding yeast, Rrp6 may play a more prominent role in processing, as it has been demonstrated to be inhibited by stable RNA secondary structure in vitro and because the null allele in budding yeast leads to the buildup of specific structured RNA substrates. Human RRP6, otherwise known as PM/SCL-100 or EXOSC10, shares sequence similarity to budding yeast Rrp6 and is proposed to catalyze 39-to-59 exoribonuclease activity on a variety of nuclear transcripts including ribosomal RNA subunits, RNA that has been poly-adenylated by TRAMP, as well as other nuclear RNA transcripts destined for processing and/or destruction. To characterize human RRP6, we expressed the full-length enzyme as well as truncation mutants that retain catalytic activity, compared their activities to analogous constructs for Saccharomyces cerevisiae Rrp6, and determined the X-ray structure of a human construct containing the exoribonuclease and HRDC domains that retains catalytic activity. Structural data show that the human active site is more exposed when compared to the yeast structure, and biochemical data suggest that this feature may play a role in the ability of human RRP6 to productively engage and degrade structured RNA substrates more effectively than the analogous budding yeast enzyme.

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Exonucleolysis is required for nuclear mRNA quality control in yeast THO mutants

Cited by 50 publications

References 24 publications

Sequential RNA degradation pathways provide a fail-safe mechanism to limit the accumulation of unspliced transcripts in Saccharomyces cerevisiae

Sequential RNA degradation pathways provide a fail-safe mechanism to limit the accumulation of unspliced transcripts in Saccharomyces cerevisiae

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

Activities of human RRP6 and structure of the human RRP6 catalytic domain

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