Different nuclease requirements for exosome-
mediated degradation of normal and nonstop mRNAs
Daneen Schaeffer and Ambro van Hoof1
Department of Microbiology and Molecular Genetics, University of Texas Health Science Center, Houston, TX 77030
Edited by Joan A. Steitz, Howard Hughes Medical Institute, New Haven, CT, and approved January 3, 2011 (received for review September 4, 2010)
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′ exoribonu-
clease and endoribonuclease activity, and the available evidence
suggests that the exonuclease activity is required for the degra-
dation 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 exonucle-
ase activities of the exosome are both capable of rapidly degrad-
ing nonstop mRNAs and ribozyme-cleaved mRNAs. Additionally,
the endonuclease activity of the exosome is not required for endo-
nucleolytic cleavage in no-go decay. In vitro, the endonuclease
domain of the exosome is active only under nonphysiological con-
ditions, 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 mo-
lecular function of the core exosome that is not disrupted by inac-
tivating its exonuclease activity.
essential for viability, but catalytically inactive (1–4). The
catalytic activity is provided by a 10th essential subunit, Rrp44p
(2, 5, 6). This protein is similar to RNase II in that it contains
three putative RNA binding domains that flank an RNB domain
(3, 7–10). The RNB domain is responsible for the 3′ exonuclease
activity of the exosome (3). In addition, the N terminus of
Rrp44p contains an endonucleolytic PIN domain. Either active
site is sufficient for viability; however, simultaneous inactivation
of both nuclease activities results in a lack of cell growth (11–13).
Although the biological substrates of the Rrp44p endonuclease
have not been fully elucidated, the synthetic lethality observed
upon inactivation of the Rrp44p nucleases implies that they have
overlapping functions. The exosome is involved in RNA pro-
cessing and RNA degradation, and several of these reactions have
been shown to be defective if the exonuclease activity of the
exosome is disrupted by a point mutation [including 5.8S rRNA
and snoRNA processing and 5′ external transcribed spacer (ETS)
and cryptic unstable transcripts (CUT) degradation]. In contrast,
mutating the endonuclease active site of Rrp44p has minor or no
effect on the exosome functions that have been tested (11–13).
The exosome is present in both the nucleus and the cytoplasm,
and the cytoplasmic exosome plays a dual role in gene expres-
sion. First, the exosome is involved in one of two redundant
decay pathways for normal mRNAs. The initiating step for decay
of normal mRNAs is shortening of the poly(A) tail (14, 15).
Removal of the poly(A) tail predominantly triggers cytoplasmic
5′-to-3′ decay in yeast (16–19). Deadenylation can also trigger
the degradation of an mRNA from its 3′ end, in a process cat-
alyzed by the cytoplasmic exosome (20).
he core eukaryotic exosome contains nine subunits that are
The second role of the cytoplasmic exosome is to maintain the
fidelity of gene expression by degrading aberrant mRNAs. Ab-
errant transcripts arise through mistakes in gene expression, in-
cluding genetic mutations, defects in transcription or splicing, or
premature polyadenylation at incorrect or cryptic sites. In one of
the exosome-mediated mRNA surveillance pathways, mRNAs
that lack in-frame termination codons are targeted to the non-
stop decay pathway (21, 22). In the current model of nonstop
decay, a translating ribosome stalls at the 3′ end, which triggers
exosome-mediated decay (22).
endonuclease cleavage [e.g., no-go decay, RNAi, and nonsense-
mediated mRNA decay (23–31)]. For example, in no-go decay,
cleaved by an unknown endonuclease (25, 29). Similar cleavage
products can be generated by inserting a ribozyme into an mRNA
(32). Though these pathways are initiated by a variety of endo-
by common exonucleases. Specifically, the 5′ fragments are de-
graded by the cytoplasmic exosome, and the 3′ fragments are
degraded by the 5′-to-3′ exonuclease Xrn1p (25–28, 32–34).
Understanding the molecular mechanisms that are responsible
for the degradation of aberrant transcripts is needed to un-
derstand how these mRNAs are preferentially targeted for rapid
degradation and how the fidelity of gene expression is main-
tained. Additionally, the recent identification of a second nu-
clease active site in the exosome means that the role of the
Rrp44p endonuclease must be examined in the known functions
of the exosome. To address these issues, we tested the role of the
Rrp44p nuclease activities in general mRNA degradation and in
mRNA surveillance. Here we report that nonstop mRNAs and
ribozyme-cleaved mRNAs can be degraded by either of the Rrp44p
nuclease activities. Additionally, we show that the Rrp44p endo-
nuclease is not responsible for endonucleolytic cleavage in no-go
decay, which suggests that endonuclease-mediated nonstop decay is
distinct from no-go decay. Our results indicate that the exonuclease
activity of Rrp44p is needed for the cytoplasmic exosome-mediated
turnover of normal cellular transcripts, but not for the degradation
of nonstop mRNAs.
Individual Mutations That Disrupt the Endo- or Exonuclease Activity
of Rrp44p Do Not Affect Expression of Nonstop Reporters. Mutations
that inactivate the cytoplasmic exosome stabilize transcripts that
lack stop codons, suggesting that the exosome degrades such
mRNAs (21, 22). However, the cytoplasmic exosome contains
two RNase domains, and it is unknown which domain degrades
Author contributions: D.S. and A.v.H. designed research; D.S. performed research; D.S.
and A.v.H. analyzed data; and D.S. and A.v.H. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| February 8, 2011
| vol. 108
| no. 6www.pnas.org/cgi/doi/10.1073/pnas.1013180108
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Schaeffer and van Hoof PNAS
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| no. 6