Different nuclease requirements for exosome-mediated degradation of normal and nonstop mRNAs

Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, TX 77030, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2011; 108(6):2366-71. DOI: 10.1073/pnas.1013180108
Source: PubMed


Two general pathways of mRNA decay have been characterized in yeast. In one pathway, the mRNA is degraded by the cytoplasmic form of the exosome. The exosome has both 3' to 5' exoribonuclease and endoribonuclease activity, and the available evidence suggests that the exonuclease activity is required for the degradation of mRNAs. We confirm here that this is true for normal mRNAs, but that aberrant mRNAs that lack a stop codon can be efficiently degraded in the absence of the exonuclease activity of the exosome. Specifically, we show that the endo- and exonuclease activities of the exosome are both capable of rapidly degrading nonstop mRNAs and ribozyme-cleaved mRNAs. Additionally, the endonuclease activity of the exosome is not required for endonucleolytic cleavage in no-go decay. In vitro, the endonuclease domain of the exosome is active only under nonphysiological conditions, but our findings show that the in vivo activity is sufficient for the rapid degradation of nonstop mRNAs. Thus, whereas normal mRNAs are degraded by two exonucleases (Xrn1p and Rrp44p), several endonucleases contribute to the decay of many aberrant mRNAs, including transcripts subject to nonstop and no-go decay. Our findings suggest that the nuclease requirements for general and nonstop mRNA decay are different, and describe a molecular function of the core exosome that is not disrupted by inactivating its exonuclease activity.

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    • "To further investigate whether the mutations of Rrp44 disrupt exosome-mediated mRNA decay we also tested for synthetic lethality with xrn1Δ, essentially as previously described [37]. Briefly, plasmids encoding the rrp44 point mutations were transformed into the rrp44Δ xrn1Δ strain containing a URA3 plasmid encoding wild-type Rrp44. "
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    ABSTRACT: In eukaryotes, the exosome plays a central role in RNA maturation, turnover, and quality control. In Saccharomyces cerevisiae, the core exosome is composed of nine catalytically inactive subunits constituting a ring structure and the active nuclease Rrp44, also known as Dis3. Rrp44 is a member of the ribonuclease II superfamily of exoribonucleases which include RNase R, Dis3L1 and Dis3L2. In this work we have functionally characterized three residues located in the highly conserved RNB catalytic domain of Rrp44: Y595, Q892 and G895. To address their precise role in Rrp44 activity, we have constructed Rrp44 mutants and compared their activity to the wild-type Rrp44. When we mutated residue Q892 and tested its activity in vitro, the enzyme became slightly more active. We also showed that when we mutated Y595, the final degradation product of Rrp44 changed from 4 to 5 nucleotides. This result confirms that this residue is responsible for the stacking of the RNA substrate in the catalytic cavity, as was predicted from the structure of Rrp44. Furthermore, we also show that a strain with a mutation in this residue has a growth defect and affects RNA processing and degradation. These results lead us to hypothesize that this residue has an important biological role. Molecular dynamics modeling of these Rrp44 mutants and the wild-type enzyme showed changes that extended beyond the mutated residues and helped to explain these results.
    PLoS ONE 11/2013; 8(11):e76504. DOI:10.1371/journal.pone.0076504 · 3.23 Impact Factor
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    • "We show that in the cytoplasm, when 5′–3′ decay pathways were inactivated, the Rrp414M mutation drastically increased the half lives of all reporter mRNAs. Interestingly, the substrates of NSD, which requires either endo- or exonucleolytic Dis3 activities (27), were also strongly stabilized. This is the first in vivo indication that the channel recruits the substrates to the Dis3 in the cytoplasm and that not only exonucleolytic but also endonucleolytic activity of the protein is controlled by the central channel. "
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    ABSTRACT: The RNA exosome is an essential ribonuclease complex involved in RNA processing and decay. It consists of a 9-subunit catalytically inert ring composed of six RNase PH-like proteins forming a central channel and three cap subunits with KH/S1 domains located at the top. The yeast exosome catalytic activity is supplied by the Dis3 (also known as Rrp44) protein, which has both endo- and exoribonucleolytic activities and the nucleus-specific exonuclease Rrp6. In vitro studies suggest that substrates reach the Dis3 exonucleolytic active site following passage through the ring channel, but in vivo support is lacking. Here, we constructed an Rrp41 ring subunit mutant with a partially blocked channel that led to thermosensitivity and synthetic lethality with Rrp6 deletion. Rrp41 mutation caused accumulation of nuclear and cytoplasmic exosome substrates including the non-stop decay reporter, for which degradation is dependent on either endonucleolytic or exonucleolytic Dis3 activities. This suggests that the central channel also controls endonucleolytic activity. In vitro experiments performed using Chaetomium thermophilum exosomes reconstituted from recombinant subunits confirmed this notion. Finally, we analysed the impact of a lethal mutation of conserved basic residues in Rrp4 cap subunit and found that it inhibits digestion of single-stranded and structured RNA substrates.
    Nucleic Acids Research 02/2013; 41(6). DOI:10.1093/nar/gkt060 · 9.11 Impact Factor
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    • "We have also shown previously that the rrp44–CR3 mutant has a defect in the rapid degradation of mRNAs that lack a stop codon (22). Because the rrp44–CR3 mutant stabilizes the his3-nonstop mRNA, a rrp44–CR3 his3-nonstop strain produces sufficient histidine to support growth in the absence of added histidine, whereas an isogenic RRP44 his3-nonstop strain fails to grow under these conditions (22). "
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    ABSTRACT: The 10-subunit RNA exosome is involved in a large number of diverse RNA processing and degradation events in eukaryotes. These reactions are carried out by the single catalytic subunit, Rrp44p/Dis3p, which is composed of three parts that are conserved throughout eukaryotes. The exosome is named for the 3' to 5' exoribonuclease activity provided by a large C-terminal region of the Rrp44p subunit that resembles other exoribonucleases. Rrp44p also contains an endoribonuclease domain. Finally, the very N-terminus of Rrp44p contains three Cys residues (CR3 motif) that are conserved in many eukaryotes but have no known function. These three conserved Cys residues cluster with a previously unrecognized conserved His residue in what resembles a metal-ion-binding site. Genetic and biochemical data show that this CR3 motif affects both endo- and exonuclease activity in vivo and both the nuclear and cytoplasmic exosome, as well as the ability of Rrp44p to associate with the other exosome subunits. These data provide the first direct evidence that the exosome-Rrp44p interaction is functionally important and also provides a molecular explanation for the functional defects when the conserved Cys residues are mutated.
    Nucleic Acids Research 07/2012; 40(18):9298-307. DOI:10.1093/nar/gks693 · 9.11 Impact Factor
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