Degradation of several hypomodified
mature tRNA species in Saccharomyces
cerevisiae is mediated by Met22
and the 5?–3? exonucleases Rat1
Irina Chernyakov, Joseph M. Whipple, Lakmal Kotelawala, Elizabeth J. Grayhack,
and Eric M. Phizicky1
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA
Mature tRNA is normally extensively modified and extremely stable. Recent evidence suggests that
hypomodified mature tRNA in yeast can undergo a quality control check by a rapid tRNA decay (RTD)
pathway, since mature tRNAVal(AAC)lacking 7-methylguanosine and 5-methylcytidine is rapidly degraded and
deacylated at 37°C in a trm8-? trm4-? strain, resulting in temperature-sensitive growth. We show here that
components of this RTD pathway include the 5?–3? exonucleases Rat1 and Xrn1, and Met22, which likely acts
indirectly through Rat1 and Xrn1. Since deletion of MET22 or mutation of RAT1 and XRN1 prevent both
degradation and deacylation of mature tRNAVal(AAC)in a trm8-? trm4-? strain and result in healthy growth at
37°C, hypomodified tRNAVal(AAC)is at least partially functional and structurally intact under these conditions.
The integrity of multiple mature tRNA species is subject to surveillance by the RTD pathway, since mutations in
this pathway also prevent degradation of at least three other mature tRNAs lacking other combinations of
modifications. The RTD pathway is the first to be implicated in the turnover of mature RNA species from the
class of stable RNAs. These results and the results of others demonstrate that tRNA, like mRNA, is subject to
multiple quality control steps.
[Keywords: RNA turnover; mature tRNA; TRM8; TRM4; yeast; tRNAVal(AAC)]
Supplemental material is available at http://www.genesdev.org.
Received January 22, 2008; revised version accepted March 19, 2008.
tRNA molecules are stable RNAs that are exquisitely
adapted for their central role in translation. In all organ-
isms, tRNAs are constructed to be similar enough for
rapid and efficient use in the translation cycle, yet different
enough for accurate discrimination by the translation
machinery, to ultimately deliver the correct amino acid
to the growing peptide chain. tRNA bodies appear to
have evolved together with their corresponding amino
acids to ensure similar binding to components of the
translation apparatus (LaRiviere et al. 2001). tRNA bod-
ies have also evolved for highly specific decoding
through interactions at the anti-codon and at other resi-
dues (Cochella and Green 2005; Olejniczak et al. 2005),
and for highly specific recognition by aminoacyl tRNA
synthetases through multiple determinants to ensure
correct aminoacylation (Giege et al. 1998). In addition,
tRNA bodies have evolved to be extremely stable, with
half-lives measured in hours or days, during which time
each tRNA goes through the translation cycle ∼40 times
per minute (Waldron and Lacroute 1975).
Many of the properties of tRNAs stem from their ubiq-
uitous modifications. About 100 tRNA modifications
have been described, many of which are highly con-
served among different organisms. In the yeast Saccha-
romyces cerevisiae, 25 distinct modifications have been
identified at 34 different positions on cytoplasmic tRNAs,
with an average of 13 modifications per tRNA species
(Sprinzl et al. 1999). Many of the modifications located
in and around the anti-codon in yeast are crucial for
codon–anti-codon interactions or reading frame mainte-
nance (Agris et al. 2007), based on a range of growth and
translation defects of the corresponding yeast mutants
(Huang et al. 2005; Waas et al. 2007; for review, see Hop-
per and Phizicky 2003), and detailed analysis of bacterial
mutants (Urbonavicius et al. 2001) and of ribosome–
tRNA interactions (Weixlbaumer et al. 2007). Modifica-
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GENES & DEVELOPMENT 22:1369–1380 © 2008 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/08; www.genesdev.org1369
tions are also important for correct charging by amino-
acyl tRNA synthetases, as described in yeast for m1G37
of tRNAAsp(Putz et al. 1994) and in Escherichia coli for
s2U34of tRNAGluand lysidine34of tRNAIle(Muramatsu
et al. 1988; Sylvers et al. 1993).
Recent evidence suggests that tRNA, like mRNA
(Doma and Parker 2007) and rRNA (LaRiviere et al.
2006), is subject to quality control steps leading to turnover
in vivo. At least two such pathways are known to exist,
and these appear to act at different stages of tRNA matu-
ration. First, pre-tRNAi
tion of GCD10 or GCD14 is degraded by Rrp6 and the
nuclear exosome, after polyadenylation by Trf4 (Kadaba
et al. 2004, 2006), a component of the TRAMP complex,
which also includes Air1/Air2 and Mtr4 (LaCava et al.
2005; Vanacova et al. 2005; Wyers et al. 2005; Wang et al.
2008). Second, mature tRNAVal(AAC)lacking m7G46and
degraded and deacylated at 37°C by a rapid tRNA decay
(RTD) pathway that is independent of TRF4/RRP6, lead-
ing to a temperature-sensitive growth defect of the
trm8-? trm4-? strain (Alexandrov et al. 2006). In addi-
tion, there are several other cases where reduced levels of
tRNA species are observed, but in each case the mecha-
nism of tRNA loss is largely unknown: Certain muta-
tions of tRNAArg(CCG)result in Trf4-independent reduc-
tion in tRNA levels (Copela et al. 2006); reduced levels of
tRNASer(CGA)are observed in strains with a tRNASermu-
tation that also lack m5U54or ?55due to deletion of
TRM2 or PUS4 (Johansson and Bystrom 2002); and re-
duced levels of tRNASer(CGA)and tRNASer(UGA)are ob-
served at high temperature in strains lacking Um44and
ac4C12due to deletion of TRM44 and TAN1 (Fig. 1; Ko-
telawala et al. 2008). Thus, although tRNA is among the
most stable RNA species in vivo, it appears to undergo
turnover both during and after biosynthesis when the
sequence or modifications of the tRNA are altered.
In this study, we investigate the components of the
RTD pathway by which tRNAVal(AAC)lacking m7G46
and m5C49is rapidly degraded and deacylated in a trm8-?
trm4-? strain at high temperature. We show that loss of
Metlacking m1A58due to muta-
functional mature tRNAVal(AAC)in this strain is medi-
ated by Met22 and the 5?–3? exonucleases Rat1 and Xrn1,
since deletion of MET22, or mutation of RAT1 in com-
bination with deletion of XRN1 prevents degradation of
mature tRNAVal(AAC). The involvement of Met22 in tRNA
degradation is likely indirect, since loss of Met22 func-
tion has previously been proposed to inhibit Rat1 and
Xrn1 through accumulation of its metabolite substrate
adenosine 5?,3? bisphosphate (Dichtl et al. 1997). The
involvement of Rat1 and Xrn1 in degradation of mature
tRNAVal(AAC)is the first case in which these proteins
have been implicated in degradation of a mature RNA
species from the class of stable noncoding RNA. Surpris-
ingly, mutation of components of the RTD pathway also
prevents the loss of aminoacylation of tRNAVal(AAC)that
is observed in a trm8-? trm4-? strain at high tempera-
ture, suggesting that the tRNA is at least partially func-
tional and structurally intact under these conditions and
that degradation of the tRNA is more complicated than
simply the nonspecific removal of waste RNA. Finally,
we provide evidence that the RTD pathway is a general
tRNA quality control pathway that acts on multiple hy-
pomodified mature tRNA species.
Mutation of MET22 suppresses the temperature
sensitivity of a trm8-? trm4-? strain
We have shown previously that degradation of hypo-
modified tRNAVal(AAC)in trm8-? trm4-? mutants does
not occur via the nuclear pre-tRNA surveillance path-
way, since deletion of RRP6 or TRF4 does not prevent
degradation of tRNAVal(AAC)or rescue growth of the
trm8-? trm4-? strain at 37°C (Alexandrov et al. 2006).
To identify components of the RTD pathway by which
tRNAVal(AAC)is degraded and deacylated, we isolated
and analyzed 26 spontaneous suppressors of the tempera-
ture-sensitive phenotype of the trm8-? trm4-? strain. All
of these suppressors belong to a single complementation
group, are cold-sensitive, and are methionine auxotrophs
(data not shown).
We cloned the wild-type allele of the suppressor gene
by complementation of the methionine auxotrophy. We
transformed a suppressor strain (revertant 13) with the
genomic movable ORF (MORF) collection of yeast ORF-
containing plasmids, each of which expresses an indi-
vidual ORF under PGALcontrol (Gelperin et al. 2005).
Each of three plasmids that conferred methionine proto-
trophy in media containing galactose encoded the
MET22 gene, and expression of this gene also suppressed
both the temperature resistance and cold sensitivity of
the suppressor strain (Supplemental Fig. S1A). Further-
more, a trm8-? trm4-? met22-? strain grows as well on
plates at 37°C as the original revertant 13 strain (now
named trm8-? trm4-? met22-13), is cold-sensitive (Fig.
2A), and is a methionine auxotroph, and introduction of
a single-copy (CEN) plasmid expressing MET22 from its
own promoter complements all three phenotypes of the
strain (Supplemental Fig. S1B; data not shown). Thus, we
and tRNASer(CGA). tRNA sequences are as described (Sprinzl et al.
1999). Modifications described in the text are highlighted, and
the proteins that catalyze their formation are indicated.
Schematic of the secondary structure of tRNAVal(AAC)
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