The RNA Exosome Channeling and Direct Access Conformations Have Distinct In Vivo Functions

Han, Jaeil; Hoof, Ambro van

doi:10.1016/j.celrep.2016.08.059

Cited by 34 publications

(39 citation statements)

References 67 publications

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“…Because deletion of Rrp43L1 enhances activity in a manner dependent on Rrp43L2, we posit that Rrp44 conformational changes are important for degrading both long and short substrates independent of whether RNA enters the exosome through the central channel (long RNA) or via direct binding to Rrp44 (short RNA). It remains unclear if this could be a regulated feature of the RNA exosome, or if it serves to inhibit Rrp44 activities when RNA is not presented through the Exo9 central channel, the presumed route for most cellular RNAs (Drazkowska et al, 2013; Han and van Hoof, 2016; Wasmuth and Lima, 2012b). …”

Section: Resultsmentioning

confidence: 99%

“…A recent study in S. cerevisiae showed that expressing a sole copy of Rrp44 containing mutations predicted to disrupt its channel-independent conformation was viable, though it exhibited a growth defect relative to the WT enzyme (Han and van Hoof, 2016). This strain accumulated certain RNA species such as hypomodified tRNA i Met and the rRNA 5’ externally transcribed spacer (5’ ETS).…”

Section: Discussionmentioning

confidence: 99%

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Nuclear RNA Exosome at 3.1 Å Reveals Substrate Specificities, RNA Paths, and Allosteric Inhibition of Rrp44/Dis3

2016

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SUMMARY The eukaryotic RNA exosome is an essential and conserved 3’ to 5’ exoribonuclease complex that degrades or processes nearly every class of cellular RNA. The nuclear RNA exosome includes a nine-subunit non-catalytic core that binds Rrp44 (Dis3) and Rrp6 subunits to modulate their processive and distributive 3’ to 5’ exoribonuclease activities, respectively. Here, we utilize an engineered RNA with two 3’ ends to obtain a crystal structure of an eleven-subunit nuclear exosome bound to RNA at 3.1 Å. The structure reveals an extended RNA path to Rrp6 that penetrates into the non-catalytic core, contacts between the non-catalytic core and Rrp44 that inhibit exoribonuclease activity, and features of the Rrp44 exoribonuclease site that support its ability to degrade 3’ phosphate RNA substrates. Using reconstituted exosome complexes, we show that 3’ phosphate RNA is not a substrate for Rrp6, but is readily degraded by Rrp44 in the nuclear exosome.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Nuclear RNA Exosome at 3.1 Å Reveals Substrate Specificities, RNA Paths, and Allosteric Inhibition of Rrp44/Dis3

2016

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“…The potential importance for these two Dis3 conformations was underscored by two recent studies (Han and van Hoof 2016;Zinder et al 2016). In one case, investigators analyzed the in vivo effects in a strain expressing a dis3 allele that contained point mutations predicted to destabilize the direct access Dis3 conformation (Han and van Hoof 2016).…”

Section: Yeast Dis3 Conformations and Rna Pathsmentioning

confidence: 99%

“…In one case, investigators analyzed the in vivo effects in a strain expressing a dis3 allele that contained point mutations predicted to destabilize the direct access Dis3 conformation (Han and van Hoof 2016). Among many observations, they showed that cells expressing this dis3 allele were viable, albeit with a slow growth phenotype and defects in the degradation of structured substrates.…”

Section: Yeast Dis3 Conformations and Rna Pathsmentioning

confidence: 99%

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

2017

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The eukaryotic RNA exosome is an essential and conserved protein complex that can degrade or process RNA substrates in the 3 ′ -to-5 ′ direction. Since its discovery nearly two decades ago, studies have focused on determining how the exosome, along with associated cofactors, achieves the demanding task of targeting particular RNAs for degradation and/or processing in both the nucleus and cytoplasm. In this review, we highlight recent advances that have illuminated roles for the RNA exosome and its cofactors in specific biological pathways, alongside studies that attempted to dissect these activities through structural and biochemical characterization of nuclear and cytoplasmic RNA exosome complexes.

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“…RNA substrates can be threaded through the central channel of the core ring from the EXOSC3/cap side Bonneau et al 2009;Malet et al 2010;Makino et al 2013). Additional RNA entry points have also been identified (Liu et al 2014;Wasmuth et al 2014;Han and van Hoof 2016) that could provide insight into how the RNA exosome balances precise processing and complete degradation of RNA. The RNA exosome is present in both the nucleus and the cytoplasm.…”

mentioning

confidence: 99%

Insight into the RNA Exosome Complex Through Modeling Pontocerebellar Hypoplasia Type 1b Disease Mutations in Yeast

et al. 2017

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Pontocerebellar hypoplasia type 1b (PCH1b) is an autosomal recessive disorder that causes cerebellar hypoplasia and spinal motor neuron degeneration, leading to mortality in early childhood. PCH1b is caused by mutations in the RNA exosome subunit gene, EXOSC3. The RNA exosome is an evolutionarily conserved complex, consisting of nine different core subunits, and one or two 39-59 exoribonuclease subunits, that mediates several RNA degradation and processing steps. The goal of this study is to assess the functional consequences of the amino acid substitutions that have been identified in EXOSC3 in PCH1b patients. To analyze these EXOSC3 substitutions, we generated the corresponding amino acid substitutions in the Saccharomyces cerevisiae ortholog of EXOSC3, Rrp40. We find that the rrp40 variants corresponding to EXOSC3-G31A and -D132A do not affect yeast function when expressed as the sole copy of the essential Rrp40 protein. In contrast, the rrp40-W195R variant, corresponding to EXOSC3-W238R in PCH1b patients, impacts cell growth and RNA exosome function when expressed as the sole copy of Rrp40. The rrp40-W195R protein is unstable, and does not associate efficiently with the RNA exosome in cells that also express wild-type Rrp40. Consistent with these findings in yeast, the levels of mouse EXOSC3 variants are reduced compared to wild-type EXOSC3 in a neuronal cell line. These data suggest that cells possess a mechanism for optimal assembly of functional RNA exosome complex that can discriminate between wildtype and variant exosome subunits. Budding yeast can therefore serve as a useful tool to understand the molecular defects in the RNA exosome caused by PCH1b-associated amino acid substitutions in EXOSC3, and potentially extending to disease-associated substitutions in other exosome subunits.KEYWORDS pontocerebellar hypoplasia type 1b; RNA exosome; EXOSC3; Rrp40; EXOSC2; RNA processing/degradation T HE RNA exosome is an evolutionarily conserved ribonuclease complex that is responsible for several essential RNA processing and degradation steps (Mitchell et al. 1997;Allmang et al. 1999;Schneider and Tollervey 2013). Major exosome substrates include mRNA, rRNA, and small RNAs. In addition to degrading aberrant and unneeded transcripts, the RNA exosome also trims precursor RNAs, including 5.8S rRNA (Mitchell et al. 1996). The RNA exosome thus plays critical roles in both RNA degradation and maturation.The subunits of the RNA exosome complex are evolutionarily conserved and many were first identified in Saccharomyces cerevisiae in a screen for ribosomal RNA processing (rrp) mutants (Mitchell et al. 1996(Mitchell et al. , 1997Allmang et al. 1999). S. cerevisiae Rrp subunits correspond to human EXOSC subunits. The functions of the RNA exosome have been extensively characterized in budding yeast (Sloan et al. 2012), and many are conserved in humans (Schilders et al. 2005;Staals et al. 2010;Lubas et al. 2011Lubas et al. , 2015 the RNA exosome, six subunits comprise a core ring, and three putative RNA-binding sub...

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The RNA Exosome Channeling and Direct Access Conformations Have Distinct In Vivo Functions

Cited by 34 publications

References 67 publications

Nuclear RNA Exosome at 3.1 Å Reveals Substrate Specificities, RNA Paths, and Allosteric Inhibition of Rrp44/Dis3

Nuclear RNA Exosome at 3.1 Å Reveals Substrate Specificities, RNA Paths, and Allosteric Inhibition of Rrp44/Dis3

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

Insight into the RNA Exosome Complex Through Modeling Pontocerebellar Hypoplasia Type 1b Disease Mutations in Yeast

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