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-
E-MAIL email@example.com; FAX (585) 271-2683.
Article published online ahead of print. Article and publication date are
online at http://www.genesdev.org/cgi/doi/10.1101/gad.1654308.
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)
Chernyakov et al.
1370GENES & DEVELOPMENT
conclude that deletion of MET22 suppresses the tem-
perature sensitivity of the trm8-? trm4-? strain.
Deletion of MET22 in a trm8-? trm4-? strain prevents
degradation and loss of aminoacylation
To determine if met22-mediated suppression of the
trm8-? trm4-? growth defect is due to an effect on the
amount of functional tRNAVal(AAC), we analyzed the lev-
els and the aminoacylation status of tRNAVal(AAC)in a
trm8-? trm4-? met22-? mutant after growth at 37°C.
Consistent with previous results (Alexandrov et al.
2006), 2 h after temperature shift of the trm8-? trm4-?
parent strain, tRNAVal(AAC)is present at only 20% of
wild-type levels and is only 25% aminoacylated (Fig. 2B
[lane j], C,D). By contrast, tRNAVal(AAC)levels in
the trm8-? trm4-? met22-? strain are maintained at
∼80% of wild-type levels throughout the time course
(Fig. 2B [lanes q–t], C). Strikingly, aminoacylation of
tRNAVal(AAC)is also almost completely stabilized in the
trm8-? trm4-? met22-? strain, remaining at ∼60%–65%
over the time course, compared with ∼75% for wild-type
cells (Fig. 2B [cf. lanes q–t and b–e], D). Since no other
change is observed in the levels or aminoacylation of
each of three control tRNA species in the met22-? or
the trm8-? trm4-? met22-? strain (Fig. 2B), we con-
clude that the met22-? mutation suppresses the tem-
perature sensitivity of the trm8-? trm4-? strain by pre-
venting the degradation and the loss of aminoacylation
of tRNAVal(AAC), rather than by global changes in tRNA
levels or aminoacylation.
Mutation of both RAT1 and XRN1 also suppresses
the temperature-sensitive phenotype of a trm8-?
trm4-? strain and prevents loss of functional
The known biochemical function of Met22 suggests that
it is not directly responsible for degradation and loss of
aminoacylation of tRNAVal(AAC)in the trm8-? trm4-?
strain. Met22 is a phosphatase in the sulfate assimilation
pathway leading to methionine biosynthesis (Fig. 3A), in
which it removes the 3?-phosphate from the byproduct
adenosine 3?,5? bisphosphate (pAp), as well as from the
pathway intermediate 3?-phosphoadenosine 5?-phospho-
sulfate, pApS (Murguia et al. 1995). One possible role of
Met22 in tRNA degradation derives from the observa-
tion that mutation of MET22, or inhibition of Met22 by
Li+treatment, leads to inhibition of 5.8S rRNA process-
ing, snoRNA processing, and rRNA spacer fragment deg-
radation, which is attributed to inhibition of the 5?–3?
exonucleases Rat1 and Xrn1 by pAp (Murguia et al. 1996;
Dichtl et al. 1997). Consistent with this, we find that the
temperature-sensitive phenotype of a trm8-? trm4-?
strain is suppressed on minimal media containing 0.2 M
LiCl, but not on media containing 1 M KCl (Fig. 3B).
To address the possibility that Rat1 and Xrn1 are in-
volved in tRNAVal(AAC)degradation in trm8-? trm4-?
mutants and to identify other possible mechanisms by
which tRNAVal(AAC)degradation is effected, we isolated
33 temperature-resistant suppressors of trm8-? trm4-?
mutants that were not met22 alleles, by starting with a
trm8-? trm4-? strain containing a second copy of the
wild-type MET22 gene. These suppressors belong to at
least three complementation groups, the largest of which
are rat1 mutants, based on three lines of evidence. First,
a CEN RAT1 plasmid complements each of four mutants
tested in this complementation group, restoring the
growth defect of these strains to that of a trm8-? trm4-?
growth defect of trm8-? trm4-? mutants and prevent degrada-
tion and loss of aminoacylation of tRNAVal(AAC). (A) Mutation
of MET22 in the trm8-? trm4-? strain allows growth at 37°C.
Strains were grown overnight in YPD at 28°C, adjusted to
OD600∼ 1, serially 10-fold-diluted, spotted on YPD plates, and
incubated at 18°C, 30°C, and 37°C, as indicated. (B) tRNAVal(AAC)
levels and aminoacylation are stable in the trm8-? trm4-?
met22-? strain. Strains were grown in YPD at 28°C to OD600∼ 2
and shifted to 37°C, and cells were harvested at the indicated
times. Ten micrograms of RNA isolated under acidic conditions
were analyzed by Northern blotting as described in the Materi-
als and Methods. For each strain, one sample was deacylated
prior to gel electrophoresis. Dashed and solid arrows indicate
aminoacylated and deacylated tRNA species, respectively. Note
that tRNAVal(AAC)from strains lacking TRM8 migrates faster
than from other strains. (C) Quantification of the levels of
tRNAVal(AAC). The ordinate shows the ratio of the levels of
tRNAVal(AAC)at each time point relative to its level in the wild-
type strain immediately before temperature shift (each value
itself first normalized to 5S RNA). (D) Quantification of the
percentage of aminoacylation of tRNAVal(AAC).
met22 mutations suppress the temperature-sensitive
Mature tRNA degradation by Rat1 and Xrn1
GENES & DEVELOPMENT1371
mutant at 33°C and above (Fig. 3C; Supplemental Fig.
S2). Second, sequence analysis of the RAT1 gene from two
of the suppressors (rat1-101 and rat1-107) shows that each
has the same A661E mutation in the RAT1 gene. Third,
a CEN rat1-A661E plasmid does not complement the
temperature-resistant phenotype of trm8-? trm4-? rat1
suppressors (Supplemental Fig. S2). Since RAT1 is essen-
tial (Amberg et al. 1992), it was not possible to analyze a
Although the rat1-A661E allele in the rat1-101 and
rat1-107 mutants is the strongest allele isolated and al-
lows the trm8-? trm4-? strain to grow at temperatures
up to 36.5°C (Fig. 3C,D), growth is not observed at higher
temperatures and is not as robust as for a trm8-? trm4-?
met22-? strain (Fig. 3D). Since RAT1 and XRN1 have
been shown previously to have redundant roles in a
number of rRNA and sn(o)RNA processing events, as
well as in mRNA degradation (Henry et al. 1994; Pet-
falski et al. 1998; Geerlings et al. 2000; Danin-Kreisel-
man et al. 2003; Lee et al. 2005), we introduced an xrn1-?
deletion into both the trm8-? trm4-? strain and the
trm8-? trm4-? rat1-107 strain to test the combined ef-
fects of Rat1 and Xrn1 on this tRNA degradation path-
way. We find that the trm8-? trm4-? rat1-107 xrn1-?
strain grows significantly better than the corresponding
trm8-? trm4-? rat1-107 strain at 37.5°C, whereas the
trm8-? trm4-? xrn1-? mutant only grows modestly bet-
ter than its trm8-? trm4-? parent strain at 33°C (Fig. 3D).
Since each of these growth phenotypes is complemented
by introduction of the appropriate XRN1 or RAT1 CEN
plasmid (Supplemental Fig. S3), these results suggest
that mutation of both RAT1 and XRN1 is required to
fully suppress the growth phenotype of a trm8-? trm4-?
Rat1 and Xrn1 both appear to mediate the degradation
trm4-? strain. In a trm8-? trm4-? rat1-107 xrn1-? mutant
strain, there is no observed degradation of tRNAVal(AAC)
(Fig. 4A [lanes h–k], B), and the aminoacylation levels
perature-sensitive phenotype of trm8-? trm4-? mutants. (A)
Schematic of the role of Met22 in the sulfate assimilation path-
way. (B) The temperature-sensitive phenotype of trm8-? trm4-?
mutants is suppressed on media containing LiCl. Wild-type and
trm8-? trm4-? strains were grown in YPD at 28°C, plated as in
Figure 1 on SD media containing 0.2M LiCl or 1M KCl, and
incubated at 30°C or 37°C. (C) RAT1 complements suppressors
of trm8-? trm4-? strain. trm8-? trm4-? suppressor strains and
controls, transformed with a URA3 CEN RAT1 plasmid or a
vector control, were grown at 28°C in SD-Uracil media, plated
on SD-Uracil media, and incubated at temperatures indicated.
(D) Mutation of both RAT1 and XRN1 improves suppression of
the trm8-? trm4-? temperature-sensitive phenotype. Strains
were grown in YPD at 28°C and plated on YPD media at indi-
Mutation of RAT1 and XRN1 suppresses the tem-
tRNAVal(AAC)in the trm8-? trm4-? strain. (A) tRNAVal(AAC)is
stable in trm8-? trm4-? mutants containing mutations in RAT1
and XRN1. Strains were grown in YPD at 28°C to OD600∼ 1.5
and shifted to 37°C, and cells were harvested at the indicated
times. RNA (5 µg) isolated under acidic conditions was analyzed
by Northern blotting. One sample was deacylated prior to gel
electrophoresis. Dashed and solid arrows indicate aminoacyl-
ated and deacylated tRNA species, respectively. (B) Quantifica-
tion of the levels of tRNAVal(AAC). tRNA is quantified as in
Figure 2. (C) Quantification of the percentage of aminoacylation
Both Rat1 and Xrn1 contribute to degradation of
Chernyakov et al.
1372 GENES & DEVELOPMENT
remain constant at 65% after temperature shift, com-
pared with 80% for wild-type cells (Fig. 4A,C). By con-
trast, mutation of RAT1 or XRN1 alone results in partial
prevention of both degradation and loss of aminoacyla-
tion of tRNAVal(AAC). Thus, in a trm8-? trm4-? rat1-107
strain or a trm8-? trm4-? xrn1-? strain, the levels of
tRNAVal(AAC)decrease to ∼40% of wild type after 2 h at
37°C, compared with 20% for the trm8-? trm4-? strain
(Fig. 4A [lanes d–g,l–q], B), and ∼40% of the tRNA re-
mains aminoacylated at this point, compared with 20%
for the trm8-? trm4-? strain (Fig. 4A,C). Since Rat1 and
Xrn1 are known 5?–3? exonucleases, it is likely that
RAT1 and XRN1 are directly responsible for degradation
of tRNAVal(AAC)in the trm8-? trm4-? strain, although
indirect effects cannot be excluded.
The rat1-107 allele acts primarily by preventing
degradation of mature tRNAVal(AAC)and not
by altering transcription or affecting pre-tRNA
Because a RAT1 allele was previously implicated as an
activator of tRNA transcription (Di Segni et al. 1993), it
was possible that stabilization of tRNAVal(AAC)in the
trm8-? trm4-? rat1-107 xrn1-? strain was due to a tran-
scription effect. To test this, we examined tRNAVal(AAC)
levels after temperature shift in the presence of thiolu-
tin, which inhibits RNA polymerases including pol III
(Jimenez et al. 1973). As expected, thiolutin treatment
results in rapid disappearance of intron-containing pre-
tRNAPhe(GAA)and pre-tRNATyr(GUA)species (Fig. 5A).
However, since levels of tRNAVal(AAC)and control tRNAs
are similar in the presence or absence of thiolutin in wild-
type and trm8-? trm4-? rat1-107 xrn1-? cells (Fig. 5A [cf.
lanes a–h and q–x], B), we conclude that the rat1-107
allele does not act as an activator of tRNAVal(AAC)tran-
scription but instead stabilizes tRNAVal(AAC)levels in
the trm8-? trm4-? rat1-107 xrn1-? strain by preventing
This experiment also underscores two points about
the tRNAVal(AAC)degradation in trm8-? trm4-? mutants
at 37°C. First, since tRNAVal(AAC)degradation in trm8-?
trm4-? mutants is at least as fast in thiolutin-treated
cells as in untreated cells (Fig. 5A [cf. lanes i–l and m–p],
B), this demonstrates that the vast majority of loss of
tRNAVal(AAC)in trm8-? trm4-? mutants is due to deg-
radation of the mature tRNA, rather than pre-tRNA.
This finding is consistent with our previous argument
that the observed tRNAVal(AAC)degradation is too fast to
be accounted for by degradation of pre-tRNA (Alexan-
drov et al. 2006), as occurs for pre-tRNAi
Rrp6 pathway (Kadaba et al. 2004, 2006). Second, since
tRNAVal(AAC)degradation occurs to a much larger extent
in thiolutin-treated trm8-? trm4-? mutants than in un-
treated cells, resulting in just 6% of wild-type tRNAVal(AAC)
25% in untreated cells (Fig. 5A [cf. lanes l and p], B), we
conclude that the full extent of tRNAVal(AAC)degrada-
tion is masked by transcription of new tRNAVal(AAC). We
had previously speculated that the slow phase of
tRNAVal(AAC)degradation that begins ∼30 min after tem-
Metin the Trf4/
perature shift of trm8-? trm4-? mutants might be due to
the presence of a resistant subpool of tRNAVal(AAC)or to
compensatory synthesis of new tRNA (Alexandrov et al.
2006). The disappearance of almost all of the tRNAVal(AAC)
in thiolutin-treated trm8-? trm4-? cells implies that
there is no pool of resistant tRNA in the pre-existing
Mutation of the RTD pathway suppresses the growth
defect of strains lacking different combinations
The results described above demonstrate that the RTD
pathway mediates degradation of mature tRNAVal(AAC)
lacking m7G46and m5C49in trm8-? trm4-? mutants
through Met22, Rat1, and Xrn1, a pathway that is dis-
tinct from the Trf4/Rrp6-dependent pathway responsible
for degradation of pre-tRNAi
m1A58(Kadaba et al. 2004, 2006; Alexandrov et al. 2006).
To further explore the scope of these tRNA degradation
pathways, we investigated the effect of met22-?, trf4-?,
and rrp6-? mutations on the temperature-sensitive phe-
notypes of several other strains bearing different combi-
nations of mutations affecting tRNA modifications. As
shown below, we find that the growth defect of each of
Metin mutants lacking
levels in a trm8-? trm4-? rat1-107 xrn1-? strain. (A) Northern
blot analysis of tRNA after treatment with thiolutin. Strains were
grown at 28°C to OD600∼ 1.5, treated with 5 µg/mL thiolutin for
10 min, and then shifted to 37°C for indicated times before har-
vest, RNA preparation, and analysis of 5 µg of RNA by Northern
blotting. (B) Quantification of the levels of tRNAVal(AAC). Thiolu-
tin-treated samples are indicated by dashed lines and open sym-
bols, and untreated samples are indicated by solid lines and
closed symbols. Wild type is indicated by squares, trm8-?
trm4-? is indicated by circles, and trm8-? trm4-? rat1-107
xrn1-? is indicated by triangles.
Transcription inhibition does not affect tRNAVal(AAC)
Mature tRNA degradation by Rat1 and Xrn1
GENES & DEVELOPMENT1373