Copyright ? 2008 by the Genetics Society of America
Nonsense-Mediated Decay of ash1 Nonsense Transcripts in
Wei Zheng,* Jonathan S. Finkel,* Sharon M. Landers,†Roy M. Long†and
Michael R. Culbertson*,1
*Laboratories of Genetics and Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706 and†Department of Microbiology
and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
Manuscript received August 29, 2008
Accepted for publication September 1, 2008
Nonsense-mediated mRNA decay (NMD) performs two functions in eukaryotes, one in controlling the
expression level of a substantial subset of genes and the other in RNA surveillance. In the vast majority of
genes, nonsense mutations render the corresponding transcripts prone to surveillance and subject to
rapid degradation by NMD. To examine whether some classes of nonsense transcripts escape surveillance,
we asked whether NMD acts on mRNAs that undergo subcellular localization prior to translation. In
Saccharomyces cerevisiae, wild-type ASH1 mRNA is one of several dozen transcripts that are exported from
the mother-cell nucleus during mitotic anaphase, transported to the bud tip on actin cables, anchored at
the bud tip, and translated. Although repressed during transport, translation is a prerequisite for NMD.
We found that ash1 nonsense mutations affect transport and/or anchoring independently of NMD. The
nonsense transcripts respond to NMD in a manner dependent on the position of the mutation. Maximal
sensitivity to NMD occurs when transport and translational repression are simultaneously impaired.
Overall, our results suggest a model in which ash1 mRNAs are insensitive to NMD while translation is
repressed during transport but become sensitive once repression is relieved.
ting aberrant transcripts that contain a nonsense or
frameshift mutation, thereby preventing the accumu-
lation of potentially deleterious dominant-negative pro-
teins. In addition, a subset of functional, error-free
mRNAs accumulate in a manner dependent on the
NMD pathway in the yeast Saccharomyces cerevisiae (Guan
et al. 2006), including transcripts with a small upstream
open reading frame that initiates translation in the 59-
untranslated region (UTR) (Oliveira and McCarthy
reading frame (ORF) is translated due to inefficient
Jacobson 1999), and precursors that undergo ineffi-
cient splicing in which the intron contains an in-frame
stop codon (He et al. 1993).
The UPF1, UPF2, and UPF3 genes are required for
NMD in S. cerevisiae (Leeds et al. 1992). The similarities
of UPF gene orthologs from different classes of organ-
isms coincide with similarities in the pathways for NMD,
including a recruitment step initiated in the nucleus
(Shirley et al. 1998, 2002; Serin et al. 2001), followed by
N eukaryotes, nonsense-mediated mRNA decay
(NMD) plays a role in RNA surveillance by elimina-
translation initiation, premature termination, decapp-
ing, and decay in the cytoplasm. Although NMD can
trigger RNA decay during any round of translation in
yeast (Maderazo et al. 2003), decay is known to occur
during the pioneer round of translation while RNAs
arestillbound tothenuclear cap-bindingcomplex(Gao
et al. 2005).
During pioneer translation, NMD appears to be
temporally and spatially coupled to nuclear export.
However, in S. cerevisiae, .25 transcripts have been
identified where nuclear export and translation are
localize via translocation on actin cables. During trans-
port, translation is repressed. Upon arrival and anchor-
ing at the bud tip, translational repression is relieved
(Long et al. 1997; Takizawa et al. 2000; Shepard et al.
2003; Andoh et al. 2006; Aronov et al. 2007). ASH1
translation appears to utilize specialized ribosomes
containing a specific subset of paralogous ribosomal
proteins (Komili et al. 2007; Warner 2007). These
exceptional transcripts can be exploited to learn more
via actin cables, codes for a transcriptional repressor of
the HO gene, which produces the endonuclease that
initiates homothallicswitching between a- and a-mating
and Feldbru ¨gge 2007). Asymmetric localization of the
1Corresponding author: Laboratory of Molecular Biology, 1525 Linden
Dr., University of Wisconsin, Madison, WI 53706.
Genetics 180: 1391–1405 (November 2008)
ASH1 transcript prior to translation leads to asymmetric
competence to switch mating type (Chartrand et al.
2002). ASH1 is transcribed in the mother-cell nucleus
during anaphase (Long et al. 1997; Takizawa et al.
1997). She2p is hypothesized to bind ASH1 mRNA in
the nucleus (Kruse et al. 2002). Once in the cytoplasm,
the She2p-ASH1 ribonucleoprotein particle associates
with Myo4p (She1p), a type V myosin motor protein,
through the adaptor protein She3p (Gonsalvez et al.
2005). As a result of these associations, ASH1 mRNA is
tethered to a polarized actin cytoskeleton (Long et al.
1997; Takizawaet al. 1997).
During transport, translation of ASH1 mRNA is
slowed by She2p bound at three locations in the ORF
and by two translational repressors, Khd1p and Puf6p,
which bind the mRNA in the 59- and 39-UTR, respec-
tively (Chartrand et al. 2002; Gu et al. 2004; Paquin
et al. 2007; Deng et al. 2008). Another protein, Loc1p,
which affects 60S rRNA processing and ribosome
assembly (Harnpicharnchai et al. 2001; Urbinati
et al. 2006), represses translation and is required for
anchoring at the bud tip (Long et al. 2001). Upon
arrival at the bud tip, ASH1 mRNA is hypothesized to
be anchored and translational repression is relieved
(Gonzalez et al. 1999; Gu et al. 2004; Paquin et al. 2007;
Deng et al. 2008). The ASH1 transcript cofractionates
with membranes, suggesting the possibility that it may
et al. 2000). Locally produced Ash1p is subsequently
imported into the daughter-cell nucleus to repress tran-
scription of HO.
None of the localized mRNAs, including ASH1, are
natural targets of NMD (Diehn et al. 2000), raising the
possibility that localized mRNAs that contain a non-
sense mutation might be also be immune to RNA
surveillance. Support for this idea came from a report
that a nonsense mutation at the 59-end of the ASH1-
coding region had no effect on mRNA abundance
(Gonzalez et al. 1999). To further test whether or not
representative asymmetrically localized transcripts are
prone to RNA surveillance through NMD, we examined
thebehavior ofash1 nonsense mRNAscontainingmuta-
tions that terminate translation prematurely at three
positions in the coding region. The results show that
premature termination of translation affects mRNA
localization independently of NMD. The degree of
sensitivity of ash1 nonsense transcripts to NMD is in-
fluenced by the position of the nonsense mutation, the
transport system, and proteins that mediate transla-
tional repression. Our results are consistent with a
model presented in the discussion that is based on
the postulated existence of two subpopulations of tran-
scripts: a translationally repressed, NMD-insensitive
pool and a translatable, NMD-sensitive pool. The two-
pool model explains many of the phenotypes of ash1
nonsense mutations that are atypical with respect to
MATERIALS AND METHODS
Strains, plasmids, and genetic methods: Strains and plas-
mids used are listed in Tables 1 and 2, respectively. Yeast
transformation was performed by electroporation (Grey and
Brendell 1992) or the LiAc method (Gietz and Woods
2002) Growth media were described previously (Gaber and
Culbertson 1982). Yeast gene deletions were constructed
using the PCR-based gene disruption method (Baudin et al.
1993; Wach et al. 1994). The accumulation and decay of non-
sense and missense mRNAs were analyzed in congenic strains
expressing the genes from CEN plasmids and/or chromoso-
mally integrated alleles constructed by gene replacement.
Allele construction: Full-length ASH1 was PCR cloned into
the centromeric (CEN) vector pRS314, including 500 nucleo-
tides 59 of the ASH1 ORF and all sequences between the ASH1
stop codon and the start codon of the next downstream gene,
SPE1. Site-directed PCR mutagenesis was performed to gen-
erate nonsense and missense mutations. Base substitutions
were introduced at three sites in the 1750-nucleotide ASH1
ORF: 1308 (site A), 1968 (site B), and 11511 (site C) (Figure
1). Sites were chosen to meet three criteria:
A. Sequences in zip code regions E1, E2A, and E2B were
avoided. These regions are binding sites for She2p and
are required for localization (Chartrand et al. 1999;
Gonzalez et al. 1999).
B. At least one consensus downstream element (TGYYGAT
GYYYYY) thought to be required for NMD (Ruiz-
Echevarria and Peltz 1996) was located within 200
nucleotides 39 of each mutation.
C. The nonsense codons created by base substitution were
followed by an A residue, which results in optimal
translation termination and efficiency of NMD (Bonetti
et al. 1995).
Mutant alleles of ASH1 were chromosomally integrated
using two-step gene replacement (Orr-Weaver and Szostak
1983). The integrity of integrated alleles was confirmed by
DNA sequence analysis. To construct congenic strains, the
following genes were disrupted by one-step gene replacement
(Rothstein 1991): UPF1, UPF2, UPF3, SHE2, SHE3, SHE4,
SHE5, KHD1, PUF6, or LOC1. Strains carrying ASH1 alleles in
a she1D background were identified among progeny from
RNA methods: RNA isolation and Northern blotting were
described previously (Shirley et al. 1998). Rates of RNA decay
were determined by temperature shift of rpb1-1 strains from
28? to 39? or by transcription inhibition using 10 mg/ml
thiolutin (Parker et al. 1991). Cells harvested before temper-
ature shift or drug addition (t0) and at subsequent intervals
were frozen in dry ice/ethanol. Total RNA was extracted and
relative mRNA abundance was determined by quantitative
RT–PCR using 18s rRNA as a loading control or by Northern
blotting using SCR1 mRNA as loading control. Half-lives were
based on average values from three trials. SigmaPlot was used
to evaluate decay data using the mixed exponential decay
formula y ¼ a 3 exp(?b 3 x) 1 c 3 exp(?d 3 x) or the simple
exponential decay formula y ¼ a 3 exp(?b 3 x). Estimations
of b, designated as B, and corresponding standard errors,
designated as SE(B), were used to calculate standard error
(t1/2¼ log(2)/B). t1/26 SE(t1/2) was calculated as [log(2)/
(B 1 SE(B)), log(2)/(B ? SE(B))].
Immunoprecipitation: Immunoprecipitation (IP)ofShe2p-
cmyc or HA-Upf1p was performed as in Irie et al. (2002) with
modifications. Exponentially growing yeast cultures (50 ml)
were harvested at OD600¼ 0.6. Cells were disrupted with
acid-washed glass beads in 500 ml of lysis buffer containing
1392W. Zheng et al.
ash1DTKanMX4 leu2-3,112 his3-11,15 ura3-1 trp1-1
ash1DTKanMX4 upf1DTURA3 leu2-3,112 his3?ura3-1 trp1-1
ash1DTKanMX4 leu2-3,112 his3?ura3-trp1-1 rpb1-1
ash1DTKanMX4 upf1DTURA3 leu2-3,112 his3?ura3?trp1-1 rpb1-1
she2DTURA3 HO-CAN1 ade2-1
ash1DTKanMX4 she2DTURA3 HO-CAN1 ade2-1
leu2-D0 met15-D0 his3-D1 ura3-D0
ash1-A-ns1 leu2-D0 met15-D0 his3-D1 ura3-D0
ash1-A-ns1 khd1DTKanMX4 upf1DTLEU2
ash1-A-ns1 she1DTKanMX4 upf1DTLEU2
ash1-A-ns1 she3DTKanMX4 upf1DTLEU2
ash1-A-ns1 she4DTKanMX4 upf1DTLEU2
ash1-A-ns1 she5DTKanMX4 upf1DTLEU2
ash1-B-ns1 leu2-D0 met15-D0 his3-D1 ura3-D0
aAsh1-C-ns leu2-D0 met15-D0 his3-D1 ura3-D0
ash1-A-ns1 she2DTKanMX4 upf1DTLEU2
ash1-B-ns1 she2DTKanMX4 upf1DTLEU2
ash1-C-ns she2DTKanMX4 upf1DTLEU2
Translation and Turnover of ash1 Nonsense mRNA 1393
25 mm HEPES–KOH (pH 7.5), 150 mm KCl, 2 mm MgCl2
200 units/ml RNasin (Promega), 0.1% NP-40, 1 mm DTT, 0.2
mg/ml heparin, proteinase inhibitor cocktail (Sigma), and
(Sigma) was used to saturate protein-G–agarose beads. IP was
performed by preincubation of monoclonal anti-cmyc or anti-
HA antibodies (Sigma) with protein-G–agarose at 4? over-
night, followed by the addition of cell lysate at 4? for 2 hr. IP
complexes were washed eight times, four with 500 ml of lysis
buffer and four with 500 ml of lysis buffer containing 1 m urea.
RNA recovery from IP and RT–PCR: Protein–RNA com-
plexes were eluted from protein-G–agarose by incubation at
65? for 15 min in 100 ml of elution buffer containing 50 mm
Tris–HCl (pH 8.0), 100 mm NaCl, 10 mm EDTA, and 1% SDS.
RNA was extracted using phenol/chloroform, and the RNA
5.2) overnight at ?20?. The RNA pellet was washed with ice-
cold 70% ethanol and treated with DNase (Ambion, Turbo
DNA-free kit). RNA was quantified by two-step RT–PCR.
Reverse transcription reactions were performed using the
Superscript III cDNA synthesis kit (Invitrogen) or the high-
capacity cDNA reverse transcription kit (Applied Biosystems).
Real-time PCR reactions were performed using the Taqman
universal PCR kit (Applied Biosystems) on an ABI7900HT
cycler. Gene-specific primers and Taqman probes were de-
signed using PrimerExpress software. Background mRNAs
present in mock experiments performed in the absence of
antibodies were 2 3 10?3less abundant relative to the same
mRNAs recovered from IP experiments.
Statistical methods: Two-tailed t-tests assuming equal vari-
ance were performed and P-values were calculated to de-
termine whether the relative levels of mRNA abundance were
the same or different in pairwise sets of strains. The null
abundance equals relative wild-type ASH1 mRNA abundance.
determine whether the relative fold changes in mRNA levels
were the same or different in strains carrying upf1D, upf2D,
or upf3D. The null hypothesis (H0) was defined as the rela-
tive fold change in mutant ash1 or wild-type ASH1 mRNA
abundance is equal in strains carrying upf1D, upf2D, or upf3D.
Pearson’s x2and the corresponding P-value were calculated
to determine whether deletions of genes coding for motor
proteins and/or translational inhibitors affect the magnitude
by which NMD influences the abundance of ash1 nonsense
mRNAs. The null hypothesis (H0) was defined as the effect of
inactivating NMD by deleting UPF1 and the effect of deleting
a gene coding for a motor protein and/or a translational
inhibitor on ash1 nonsense mRNA abundance are indepen-
dent. For all of the statistical tests described above, a P-value of
Strain Relevant genotype
ash1-A-ns1 khd1DTKanMX4 she2DTURA3
khd1DTKanMX4 she2DTURA3 upf1DTLEU2
ash1-A-ns1 khd1DTKanMX4 she2DTURA3 upf1DTLEU2
ash1-A-ns1 puf6D:KanMX4 she2DTURA3
puf6D:KanMX4 she2DTURA3 upf1DTLEU2
ash1-A-ns1 puf6D:KanMX4 she2DTURA3 upf1DTLEU2
leu2-D0 met15-D0 ura3-D04 lys3D puf6DTKanMX4 khd1DTKanMX4
ash1-A-ns1 leu2-D0 met15-D0 ura3-D04 lys3D puf6DTKanMX4 khd1DTKanMX4
upf1DTLEU2 met15-D0 ura3-D04 puf6DTKanMX4 khd1DTKanMX4
ash1-A-ns1 upf1DTLEU2 she2DTURA3 met15-D0 lys3D puf6DTKanMX4 khd1DTKanMX4
upf1DTLEU2 she2DTURA3 met15-D0 lys3D puf6DTKanMX4 khd1DTKanMX4
upf1DTLEU2 she2DTURA3 met15-D0 lys3D puf6DTKanMX4 khd1DTKanMX4
she2DTURA3 leu2-D0 puf6DTKanMX4 khd1DTKanMX4
ash1-A-ns1 upf1DTLEU2 met15-D0 lys3D ura3-D04 puf6DTKanMX4 khd1DTKanMX4
CEN LEU2 ASH1
CEN LEU2 ash1-A-ns1
CEN LEU2 ash1-A-ns2
CEN LEU2 ash1-A-ms1
CEN LEU2 ash1-B-ns1
CEN LEU2 ash1-B-ns2
CEN LEU2 ash1-B-ms1
CEN LEU2 ash1-C-ms1
CEN LEU2 ash1-C-ms2
CEN LEU2 ash1-C-ns1
2m LEU2 ASH1
2m LEU2 ash1-A-ns1
2m LEU2 ash1-A-ns2
2m LEU2 ash1-A-ms1
2m LEU2 ash1-B-ns1
2m LEU2 ash1-B-ns2
2m LEU2 ash1-B-ms1
2m LEU2 ash1-C-ns1
2m LEU2 adh1-C-ms1
CEN HIS3 SHE2
LEU2 ash1-mut A-ns1
LEU2 ash1-mu9 A-ns2
CEN LEU2 UPF1
CEN LEU2 SHE2-myc6
1394W. Zheng et al.
0.05 was used as the standard cutoff. All experiments were
repeated three times (n ¼ 3). The results of the statistical
analyses are described in the supplemental tables.
Cytological methods: Yeast strains W303a and AAY320
were transformed individually with 2m plasmid pC3319 or
derivatives of pC3319, where nonsense or missense mutations
were introduced in the ASH1 ORF. Transformants were grown
to mid-log phase in synthetic liquid medium without leucine.
Cells were fixed and ASH1 mRNA localization was detected
by fluorescent in situ hybridization with probes hybridizing
to different parts of ASH1 mRNA (Long et al. 1997). Fifty
anaphase cells with a visible ASH1 signal were counted and
scored for their localization phenotype. Results were based on
two trials using independent transformants.
Nonsense mutations affect ASH1 mRNA localization:
To achieve localized protein expression, the translation
of ASH1 mRNA is repressed during transport, whereas
the release of translational repression is required for
proper anchoring at the bud tip as a prerequisite for
local translation (Chartrand et al. 2002; Gu et al. 2004;
Paquin et al. 2007; Deng et al. 2008). Since premature
termination of translation caused by a nonsense muta-
tion might interfere with the release of repression and
result in mRNA mislocalization, we performed experi-
ments to assess the effects of nonsense mutations on
localization. We analyzed nonsense mutations at three
sites in ASH1 (Figure 1A). Site A resides upstream of the
E1, E2A, E2B, and E3 binding domains for She2p,
whereas theothertwo sites, B andC,arelocatedbetween
the E1/E2A and E2B/E3 domains, respectively.
A reporter gene was used to monitor the ability of the
alleles to produce functional Ash1p, a transcriptional
repressor of the HO gene. The HO promoter was fused
to CAN1 (HOp-CAN1) (Figure 1B) (Bobola et al. 1996;
Jansen et al. 1996). In ASH1 she2D strains, ASH1 mRNA
mislocalizes, causing repression of HOp-CAN1 in mother
and daughter nuclei and leading to canavanine re-
sistance. Wild-type and mutant alleles of ASH1 were
introduced into she2D strains carrying HOp-CAN1 on a
CEN plasmid. Growth was monitored in the presence
of canavanine (Figure 1C). Strains carrying missense
Ash1p. However, strains carrying nonsense mutations at
the same three sites were sensitive to canavanine, indi-
cating significantly reduced levels of functional Ash1p.
To examine mRNA localization in strains carrying the
ash1 alleles, Cy3-labeled fluorescent probes were used
to detect the mRNAs in situ (materials and methods).
Anaphase cells were classified into three distinct local-
ization phenotypes: crescent (localization at the bud
tip), full bud (diffuse localization in the bud), and
delocalized (diffuse localization in mother and bud)
(Figure 2A). In asynchronous cultures of Nmd1strains
Figure 1.—Expression of nonsense and mis-
sense alleles of ASH1. (A) The map of ASH1
shows the locations of missense and nonsense
mutations. Purple boxes indicate the locations
of regions (zip codes) required for She2p/
RNA binding. Red lines and red letters indicate
the locations and mutational changes at sites A,
B, and C. Numbers refer to nucleotides in the
ASH1 open reading frame. (B) Structure of a re-
porter used to assay for the function of ASH1
alleles. (C) The reporter was integrated in the
genome as a replacement of the wild-type
CAN1 gene. Wild-type or mutant ASH1 alleles
were introduced into the reporter strain on a
CEN plasmid. Transcriptional repression of the
HO promoter by Ash1p confers canavanine resis-
tance. Growth was assayed on plates containing
synthetic defined medium plus 100 mg/ml cana-
vanine using 1:5 serial dilutions of mid-log cul-
Translation and Turnover of ash1 Nonsense mRNA 1395
crescent at the bud tip in ?60% of cells (Figure 2B).
Nonsense mutationsat allthree sites caused areduction
to ,5% of anaphase cells showing a crescent localiza-
tion pattern, whereas the percentage of cells showing
a delocalized signal rose significantly. Missense muta-
tions at sites A, B, and C had no discernible effects on
localization. The results indicate that shifts from the
crescent to the delocalized pattern in cells carrying the
nonsense mutations are most likely due to premature
termination of translation.
In addition to the increased percentage of cells
showing the delocalized signal, nonsense mutations at
sites A and B caused a modest but statistically significant
increase in the percentage of anaphase cells showing
suggest that the corresponding nonsense mRNAs were
transported to the bud tip, but failed to anchor. The
changesinlocalizationcausedbythe nonsense mutation
at site C also indicate a failure to anchor, but the effects
were more dramatic. Almost 90% of cells showed a de-
localized pattern. In She2p/RNA-binding experiments
described in a later section, we show that nonsense
while remaining bound to She2p. The mRNAs are there-
fore still tethered to actin cables. The nonsense mutation
at site C causes release of the mRNA from She2p. The
mRNA probably exhibits more pronounced mislocaliza-
tion because it is no longer tethered to actin cables.
The changes in localization caused by the nonsense
mutations could affect the ratio of translationally re-
pressed mRNAs engaged in transport and the translat-
able mRNAs that are anchored at the bud tip. Since
NMD requires translation, a shift toward more transla-
tionally repressed mRNA at the expense of translatable
mRNA could reduce the overall sensitivity of the non-
sense mRNAs to NMD. Furthermore, NMD itself might
affect localization. When the nonsense mutations were
examined in Nmd?strains, the patterns of mislocaliza-
tion were similar to those observed for Nmd1strains
(Figure 2C). Given limits on information that can be
gained from cytological analysis, additional approaches
described below were pursued to assess the potential
impact of NMD on translation termination, decay, and
localization of ash1 nonsense mRNAs.
Changes in mRNA levels associated with premature
translation termination and NMD: Since the acceler-
ated decay of nonsense mRNAs caused by NMD is a
direct consequence of premature termination of trans-
Figure 2.—Effects of nonsense and missense mutations on
ASH1 mRNA localization. (A) Nonsense codons affect the in-
tracellular distribution of ASH1 mRNA. Yeast strain W303a
was individually transformed with 2m plasmids carrying differ-
ent alleles of ASH1 and analyzed by fluorescent in situ hybrid-
ization (materials and methods; Long et al. 1997). In cells
classified as ‘‘delocalized,’’ ASH1 mRNA was symmetrically dis-
tributed between the mother cell and the bud. In cells classi-
fied as ‘‘full bud,’’ ASH1 mRNA was distributed throughout
the daughter cell. In cells classified as ‘‘crescent,’’ ASH1
mRNA formed a tight crescent at the bud tip. (B) Quantifica-
tion of the effects of premature termination codons on ASH1
mRNA localization. The phenotype for 50 late-anaphase cells
in A expressing mutant alleles of ASH1 mRNA were counted
and classified as localized, full bud, or delocalized. Two inde-
pendent trials were performed.
1396W. Zheng et al.
mRNAs would be unaffected by the loss of NMD.
Consistent with expectation, missense mutations at sites
A, B, or C (ash1-A-ms1, ash1-B-ms1, ash1-C-ms1, and ash1-
C-ms2) had no effect on mRNA abundance either in
Nmd1strains or in Nmd?strains carrying upf1D, a de-
letion that inactivates NMD (Table 3).
SinceNMD acceleratesthe decay ofnonsensemRNAs,
the accumulation of nonsense mRNAs are typically re-
duced compared to the corresponding wild-type mRNA,
whereas the wild-type level is restored when NMD is
inactivated.However, the levels ofash1nonsense mRNAs
could deviate from expectation in the event of a shift
NMD-insensitive mRNAs. The cytological evidenceshow-
ing that the nonsense mRNAs cause mislocalization was
suggestive of this possibility.
Whentranscriptsproduced from thenonsensealleles
were analyzed in Nmd1strains, the reduced levels
typical of most nonsense mRNAs were not observed
(Table 3). Instead, we found that the relative levels of
ash1-A-ns1 and ash1-A-ns2 mRNAs were indistinguish-
able from the wild-type ASH1 mRNA. In Nmd?strains
ash1-B-ns1 and ash1-C-ns1 mRNAs behaved differently.
In Nmd1strains, they were 1.5- to 2-fold more abundant
than ASH1 mRNA. In Nmd?strains, the same excess
accumulation was observed as in Nmd1strains but
without any further changes that could be attributed
to the inactivation of NMD.
To summarize the data, we found that nonsense
mutations at all three sites cause increased mRNA
Fold changes in mRNA abundance in UPF1 and upf1D strains
Effect on abundance
ASH1 alleleFold change ash1/ASH1Pa
ash1 allele NMD
1.00 6 0.08
0.92 6 0.25
0.92 6 0.25
0.98 6 0.08
4.07 6 0.16
4.15 6 0.16
0.97 6 0.42
2.84 6 0.55
2.93 6 0.57
0.72 6 0.09
0.82 6 0.20
1.14 6 0.28
1.54 6 0.23
1.52 6 0.10
0.99 6 0.06
1.21 6 0.46
0.89 6 0.12
0.74 6 0.26
1.87 6 0.26
1.73 6 0.28
0.93 6 0.15
1.44 6 0.67
0.85 6 0.28
0.59 6 0.19
1.18 6 0.44
0.93 6 0.52
0.79 6 0.44
aStatistical analyses were performed using two-tailed t-tests (materials and methods). H0: mutant ash1
mRNA abundance equals wild-type ASH1 mRNA abundance. H1: mutant ash1 mRNA abundance does not equal
wild-type ASH1 mRNA abundance. P-values ,0.05 (italic) indicate a change in abundance.
bExperiments were performed by expressing ash1 alleles that were integrated at the ASH1 locus by gene
cExperiments were performed by expressing ash1 alleles from a centromeric plasmid.
Translation and Turnover of ash1 Nonsense mRNA 1397
accumulation, but the only mRNAs that responded to
the inactivation of UPF1 were those carrying nonsense
mutations at site A. Similar experiments were per-
formed using congenic sets of strains carrying deletions
of UPF1, UPF2, or UPF3. The same results were obtained
regardless of which UPF gene was deleted (Figure 3,
Table 4). The NMD-dependent increases in mRNA
abundance observed for mutations at site A are there-
fore most likely due to the inactivation of NMD rather
than to the loss of function of a specific UPF gene.
Transcript selection and decay of ash1-A-ns1 mRNA:
The phenotypes described above for nonsense muta-
tions at site A deviate from what has been observed for
the typical nonsense mutation in the typical gene where
nonsense mutations at site A produced transcripts that
exhibit NMD-sensitive increases in accumulation, we
performed additional experiments to confirm a role for
NMD and to explain the underlying reasons for the
deviations from expectation.
We asked whether Upf1p preferentially binds to the
ash1-A-ns1 nonsense transcript, which is a diagnostic
indicator of NMD targeting (Johansson et al. 2007).
Using IP/quantitative RT–PCR (materials and meth-
ods), we found that the amount of ash1-A-ns1 mRNA
associated with Upf1p was more than six-fold higher
compared to ASH1 mRNA (Figure 4B). The IP was
performed with lysates from strains that carried chro-
mosomal upf1D and an allele of UPF1 on a CEN vector
Upf1p). The ratios of ASH1 and ash1-A-ns1mRNAs were
calculated relative to RDR1 mRNA.
transcript should become more stable when NMD is
inactivated and less stable compared to the wild-type
mRNA in Nmd1strains. The decay rates were examined
using the temperature-shift method for transcriptional
shutoff followed by Northern blotting at intervals
following shutoff (materials and methods). Biphasic
decay was observed for both the wild-type and the non-
sense mRNA irrespective of NMD (Figure 4C). The
initial phase was characterized by rapid mRNA disap-
pearance (phase I) followed by a phase of apparent
stability (phase II). The phase I decay rates for ASH1
mRNA in Nmd1and Nmd?strains were indistinguish-
able (2.8 6 0.8 and 2.7 6 0.7 min, respectively), indi-
cating that NMD had no effect on the decay of the
wild-type mRNA. The nonsense mRNA was stabilized in
Nmd?strains. Phase I decay rates for ash1-A-ns1 mRNA
in Nmd1and Nmd?strains were 3.6 6 0.1 and 7.2 6 2.5
An unexpected result was observed when thedecay of
the wild-type and nonsense mRNAs were compared.
The phase I decay rates were statistically similar (2.8 6
0.8 and 3.6 6 0.1, respectively) in Nmd1strains, but the
nonsense mRNA should have a faster decay rate if it is
targeted by NMD. By comparison, the accumulation of
the nonsense mRNA was higher than wildtype in Nmd1
strains, but NMD targeting should cause it to be lower.
One possible explanation for the deviation from
expectation is suggested by the changes in localization
described above that are associated with nonsense
mutations. If the mutations cause a shift toward trans-
lationally repressed mRNA at the expense of translat-
Figure 3.—Accumulation and decay of ash1
nonsense mRNAs. The relative fold changes in
mRNA levels of the nonsense mRNAs compared
to ASH1 mRNA are shown. The accumulation of
ASH1 mRNA was compared with the accumula-
tion of representative ash1 nonsense mRNAs in
strains carrying null alleles of UPF1, UPF2, and
UPF3. The mRNAs were detected by Northern
blotting using a probe that anneals to ASH1
mRNA. The RNA levels were normalized to
RPS3 mRNA, which is not affected by NMD
(Guan et al. 2006).
Relative mRNA abundance in Nmd?strains carrying different upf gene deletions
1.00 6 0.10
0.86 6 0.09
1.33 6 0.14
1.92 6 0.16
1.00 6 0.31
2.96 6 0.06
1.64 6 0.44
1.64 6 0.36
1.00 6 0.23
3.32 6 0.18
2.41 6 0.05
1.77 6 0.52
1.00 6 0.23
2.44 6 0.19
1.88 6 0.17
1.68 6 0.15
aData were evaluated using F-tests (materials and methods).
1398 W. Zheng et al.
able mRNA, a greater proportion of the nonsense
mRNA pool would be insensitive to NMD as compared
to the pool of wild-type mRNA. Since the estimated
phase I decay rates are composite averages of the decay
would cause the composite decay rate of the nonsense
mRNAtoappearartificially higherthan the decay rateof
the wild-type mRNA. An increase in the proportion of
more stable, NMD-insensitive mRNA might also affect
phase II decay rates, but phase II is more difficult to
assess because of the potential contribution of low level,
residual transcription caused by incomplete inhibition
of transcription. Residual transcription contributes in a
minor way to phase I decay, but is more significant in
phase II because low-level, ongoing transcription in
phase II represents a higher proportion of the mRNAs
remaining after transcriptional shutoff. Residual tran-
scription is typically not .10% of the total mRNA
(Lelivelt and Culbertson 1999; Guan et al. 2006).
Relative pool size of She2p-bound mRNA: To un-
derstand the underlying causes of deviations from
expectation as described above, we examined the
transcript pool that binds to She2p. Since these tran-
scripts are mostly if not entirely engaged in transport via
actin cables, the She2-bound mRNA pool most likely
corresponds to the translationally repressed, NMD-
insensitive pool. If the relative proportion of nonsense
this might explain the unexpectedly high levels of
accumulation observed for nonsense mRNAs and the
The relative amounts of She2p bound to wild-type
ASH1 and three ash1 nonsense transcripts were de-
termined by IP/quantitative RT–PCR. Lysates were
prepared for IP from Nmd1and Nmd?strains carrying
an allele of SHE2 that produces a functional epitope-
ods). The amount of RNA recovered by RT–PCR was
normalized to a control mRNA, IST2, which localizes on
actin cables but is not affected by NMD (Lelivelt and
Culbertson 1999; Guan et al. 2006).
In Nmd1strains, the relative amount of ash1-A-ns1
mRNA that copurified with She2p-cmyc was increased
Figure 4.—Co-immunoprecipitation of ASH1
and ash1-A-ns1 mRNA with Upf1p. (A) Anti-HA
antibodies immunoprecipiate HA-UPF1 as de-
tected by Western blotting of a post-IP lysate.
(B) The relative amounts of ASH1 and ash1-A-
ns1 mRNAs recovered in the post-IP lysates were
assayed quantitatively using real-time RT–PCR.
Relative RNA amounts were normalized to RDR1
RNA. (C) The rates of decay of ASH1 and ash1-
A-ns1 mRNAs were determined by measuring
RNA levels after inhibition of transcription using
the temperature-shift method in strains carrying
rpb1-1 (materials and methods). Real-time
RT–PCR was used to quantify the relative amounts
of RNA. 18s rRNA was used as loading control.
The graphs represent the relative amounts of
mRNA remaining following the inhibition of tran-
scription at t0. The error bars show standard devi-
ations based on three trials.
Translation and Turnover of ash1 Nonsense mRNA 1399
5A) even though both transcripts were similar in total
abundancewhen assayedbyNorthernblotting (Table 3,
Figure 3). In Nmd?strains, the relative amount of
She2p-bound ash1-A-ns1 mRNA was increased 3.7- 6
0.5-fold compared to the wild-type mRNA with a
corresponding 3- to 4-fold increase in total ash1-A-ns1
mRNA abundance (Table 3, Figure 3). Since both
premature termination of translation at the A site and
loss of NMD contribute to an increased proportion of
She2p-bound mRNA, the results support a model
developed further in the discussion in which a higher
proportion of the nonsense transcript is protected from
NMD by translational repression, which causes distor-
tions in the expected effects of NMD.
In Nmd1strains, the relative amount of She2p-bound
ash1-B-ns1 nonsense mRNA was twice that of ASH1
mRNA (Figure 5B) compared with a 1.5-fold increase
in total mRNA (Figure 3, Table 3). However, in Nmd?
strains, the relative amount of She2-bound ash1-B-ns1
was the same as wild-type mRNA, possibly because the
loss of NMD has no effect on the total abundance of
ash1-B-ns1 mRNA (Table 3, Figure 3). Thus, premature
translation termination caused by the ash1-B-ns1 and
ash1-A-ns1 mutations have similar effects on She2-
bound pool size, but the two nonsense transcripts differ
in their sensitivity to NMD, possibly due to the binding
(Figure 1A). She2p bound at E1 might affect the ability
of ribosomes to reach the termination codon at site B.
The ash1-C-ns1 mutation differed dramatically from
the nonsense mutations at sites A and B. Although total
ash1-C-ns1 mRNA increased twofold compared to the
wild-type mRNA (Figure 3, Table3), the relative amount
of mRNA bound to She2p-cmyc was reduced by 90%
(Figure 5B). Themislocalization ofash1-C-ns1 nonsense
mRNA (Figure 2) presumably differs from the misloc-
alization of mRNAs containing nonsense mutations at
sites A and B because the ash1-C-ns1 nonsense mRNA
is no longer bound to the actin cytoskeleton, whereas
nonsense mRNAs containing mutations at sites A and B
show increased binding to the actin cytoskeleton.
Behavior of ash1 nonsense transcripts in transport-
defective mutants: To see if disruption of the transport
machinery affects the abundance and decay of ash1
mRNAs were examined in Nmd1and Nmd?strains
carrying she2D (supplemental Table S1). In Nmd1
strains, the relative levels of wild-type, ash1-A-ns1, and
ash1-B-ns1 nonsense mRNAs were significantly reduced
compared to SHE2 strains. The ash1-C-ns1 mRNA was
modestly elevated, but with marginal statistical signifi-
cance (P ¼ 0.049). In Nmd?strains, the ash1-A-ns1
mRNA was equally sensitive to NMD in both SHE2 and
she2D strains. The ash1-C-ns1 mRNA was unaffected by
NMD in both SHE2 and she2D strains.
Although the ash1-B-ns1 mRNA level was not affected
by NMD in SHE2 strains (Table 3), a twofold increase in
abundance was observed in she2D Nmd?strains (sup-
plemental Table S1). Decay rates were compared in
SHE2 UPF1, she2D UPF1, SHE2 upf1D, and she2D upf1D
strains (Figure 6). In all four strains, biphasic decay was
observed, indicating the existence of at least two pools
of transcripts that decay at different rates. In SHE2
strains, the ash1-B-ns1 mRNA was insensitive to NMD
(Figure 6A). However, in she2D strains, it was NMD
sensitive. The phase I half-lives were 1.7 6 0.1 and 2.6 6
0.6 min in the Nmd1and Nmd?strains, respectively,
which corresponds to a 1.5-fold, statistically significant
difference. Since two pools of differentially decaying
transcripts were detected in SHE2 strains and since the
She2-bound pool was eliminated in the she2D strains,
the NMD-insensitive pool in she2D strains consists of
transcripts that are no longer tethered to the actin
cytoskeleton but that remain translationally repressed.
As an alternative approach to disrupt transport, we
made use of a previously reported allele called ash1-
MUT, which contains multiple mutations in the zip
changing the amino acid sequence of the protein
product. The ash1-MUT mRNA is delocalized because
(Chartrand et al. 2002). The nonsense mutations in
ash1-A-ns1 and ash1-A-ns2 were combined with ash1-
MUT to produce ash1-MUT-A-ns1 and ash1-MUT-A-ns2
and then integrated at the ASH1 locus by gene re-
placement in congenic Nmd1and Nmd?strains. When
mRNA levels were assayed by Northern blotting, we
with She2p. (A) Anti-cmyc antibod-
ies immunoprecipitate She2p-cmyc
as detected by Western blotting of
a post-IP lysate. (B) The relative
amounts of ASH1 and ash1-A-ns1
mRNAs recovered in the post-IP ly-
sates from Nmd1and Nmd?strains
were assayed quantitatively using
quantitative RT–PCR. Relative RNA
amounts were normalized to IST2
tivation of UPF genes.
1400W. Zheng et al.
found that the inactivation of NMD had the same effect
on mRNA accumulation regardless of whether tether-
ing to the transport system was disrupted by deletion
of SHE2, as described above, or by disruption of She2p
binding at the zip codes (supplemental Table S2).
We wanted to know whether a more general disrup-
tion of transport affects the behavior of ash1 nonsense
mRNA. The relative levels of wild-type ASH1 and ash1-A-
ns1 nonsense mRNAs were determined by Northern
blotting in strains carrying deletions of genes required
for transport, including she1D (type V myosin motor
protein), she3D (actin–myosin adaptor protein), she4D
(regulator of She1p), and she5D (actin filament assem-
bly). For each set of sheD strains, the inactivation of
NMD caused a three- to fivefold increase in ash1-A-ns1
mRNA abundance compared to ASH1 mRNA abun-
dance (supplemental Table S3). Chi-square tests in-
dicated that the magnitudes of change caused by the
inactivation of NMD were similar regardless of which
SHE gene was deleted (supplemental Table S4). The
effects of deleting these genes were similar to effects
of she2D or the mutations in ash1-MUT that prevent
tethering of the mRNA to the transport system. Overall,
the results indicate that disruption of transport did
not cause increased sensitivity of ash1-A-ns1 nonsense
mRNA to NMD. These results might be explained if
mRNAs that are not tethered to the actin cytoskeleton
remain translationally repressed.
Behavior of ash1-A-ns1 nonsense mRNA in the
absence of translational repressors: The protein prod-
ucts of PUF6, KHD1, and LOC1 have been implicated
in mediating translational repression of ASH1 mRNA
during transport (Long et al. 2001; Irie et al. 2002;
Gu et al. 2004). The effects of deleting genes cod-
ing for translational repressors were investigated in
Nmd1and Nmd?strains expressing ash1-A-ns1 mRNA
(supplemental Tables S4–S6) to see if relief of trans-
of transcripts that are sensitive to NMD. We found that
puf6D caused an overall reduction in the abundance of
ash1-A-ns1 mRNA, but the effect was unrelated to NMD.
The reduction could be due to either direct or indirect
effects ofPuf proteinsonmRNAstability(Wickenset al.
2002). Single deletions of KHD1 or LOC1 had no
in sensitivity to NMD was observed as the result of
deleting these translational repressors one at a time.
These experiments were extended by analyzing khd1D
and puf6D double deletions in combination with a
she2D deletion to see if ash1-A-ns1 mRNAs that are not
tethered to the actin cytoskeleton are more sensitive to
NMD in the absence of translational repressors. Once
again, no increase in sensitivity was observed (supple-
mental Table S6).
One possible explanation for the results described
above is that the translational repressors have redun-
dant effects on translation. Maximal sensitivity to NMD
might occur only in transport-defective mutants where
thegenes for translational inhibitors are simultaneously
deleted. To test this, ash1-A-ns1 mRNA was analyzed in
she2D strains carrying deletions of UPF1, KHD1, and
PUF6 in multiple combinations. When mRNA levels
were compared, we found, as expected from previous
results, that disruption of NMD caused increased accu-
mulation of ash1-A-ns1 mRNA (Figure 7A). In Nmd1
strains, a trend toward higher accumulation of ash1-A-
strain simultaneously deleted for UPF1, KHD1, PUF6,
To assess whether changes in mRNA abundance in
the quadruple mutant reflect underlying changes in
mRNA half-life, we measured the kinetics of decay of
Figure 6.—Effects of she2D on
the rate of ash1-B-ns1 mRNA de-
cay in Nmd1and Nmd?strains.
The decay rate of ash1-B-ns1
mRNAs was determined by mea-
mRNA detected by Northern blot-
ting following the inhibition of
transcription with 15 mg/ml thio-
lutin (materials and methods).
(A) Decay rates in Nmd1and
Nmd?strains that carry SHE2.
(B) Decay rates in Nmd1and
Nmd?strains that carry she2D.
Standard error was based on
Translation and Turnover of ash1 Nonsense mRNA 1401
ash1-A-ns1 mRNA in Nmd1and Nmd?strains carry-
ing simultaneous deletions of KHD1, PUF6, and SHE2
(Figure 7B). The nonsense mRNA decayed with bi-
phasic kinetics in the Nmd1strain. The phase I decay
rate was extremely rapid with an estimated half-life of
0.13 6 0.02 min. The kinetics of decay also show the
presence of a more slowly decaying pool of mRNAs with
ahalf-life of 27 6 2.4 min. In the Nmd?strain, decay was
monophasic. A dramatic stabilization was observed in
which the overall half-life was 15.4 6 2.0 min. A NMD-
insensitive mRNA subpopulation corresponding to
phase II could still be present in the Nmd?strain, but
might be obscured by the predominant NMD-sensitive
subpopulation. Compared with previous observations,
these results suggest that the ash1-A-ns1 nonsense tran-
script is hypersensitive to NMD when it cannot tether to
the transport system and when it can be more efficiently
translated in the absence of the Khd1p and Puf6p
To our knowledge, there have been no studies that
address whether particular classes of transcripts are
immune to or sequestered from the effects of NMD. We
studied ASH1 mRNA to assess how mRNAs that are
transported by the actin cytoskeleton prior to trans-
lation are affected by blocks in translation and whether
to previously proposed models (Chartrand et al.
2002), tight regulation exists between two temporally
incompatible events: translation and the transport of
mRNAs tethered to the transport machinery through
the binding of She2p to the mRNAs. Our data support a
model inwhichfull-lengthtranslation ofASH1 mRNAis
an integral part of the maturation pathway. Blocks in
translation cause mislocalization. Furthermore, ash1
nonsense mRNAs are prone to NMD, but sensitivity to
NMD depends on the position of the mutation.
The ashA-ns1 nonsense mRNA accumulated and
responded to loss of NMD in a manner atypical of
transcripts encoded by nonsense alleles of other genes.
Typically, nonsense mRNA abundance is reduced com-
pared to the corresponding wild-type mRNA. The
reduction is caused by an acceleration of the mRNA
half-life due to targeting of the mRNA by NMD. When
NMD is inactivated, the abundance rises back to the
same level as the wild-type mRNA (Leeds et al. 1992). By
contrast, the wild-type ASH1 and nonsense ash1-A-ns1
mRNAs were equally abundant in Nmd1strains. When
NMD was inactivated, the ash1-A-ns1 nonsense mRNA
An explanation for the atypical behavior comes from
the finding that the relative proportion of ash1-A-ns1
mRNA bound to She2p, and therefore tethered to the
actin cytoskeleton, rose twofold in Nmd1strains and
fourfold in Nmd?strains. These results reflect an
anchoring defect caused by the nonsense mRNA that
is accentuated when NMD is inactivated. The results
are summarized in Figure 8. The underlying cause of
appears to be related to the division of ASH1 mRNA
into two pools. One pool, which is bound to She2p, is
engaged in transport. These mRNAs are translationally
repressed. Since NMD requires translation, the She2p-
bound pool is insensitive to NMD. The other pool is
anchored at the bud tip. These mRNAs are translation-
ally derepressed and sensitive to NMD. This could be
Figure 7.—Accumulation and decay of ash1-A-ns1 mRNA
in she2D strains lacking inhibitors of ASH1 translation. (A)
Levels of accumulation were compared by Northern blotting.
(B) The decay of ash1-A-ns1 mRNA was determined in Nmd1
and Nmd?strains carrying upf1D, khd1D, puf6D, and she2D as
described in Figure 6 and materials and methods.
1402 W. Zheng et al.
explained by changes in the relative sizes of the two
pools caused by premature termination of translation
According to the model (Figure 8), impaired anchor-
a change in the relative sizes of the two pools in Nmd1
strains, favoring an increase in the size of the transport
pool at the expense of the anchored pool. This reduces
the proportion of mRNAs that are sensitive to NMD.
Because of the shift toward NMD-insensitive mRNAs,
the measured half-life of ash1-A-ns1 (Figure 4) appears
to be similar to wild-type ASH1 mRNA, but in reality the
shift masks morerapiddecay ofthe NMD-sensitive pool.
The data on accumulation, decay, and the relative
binding ofash1-A-ns1mRNAwithShe2p are predictable
outcomes of the model. Most notably, twice as much
ash1-A-ns1 mRNA is bound to She2p compared to ASH1
mRNA in Nmd1strains, whereas four times as much is
bound in Nmd?strains (Figure 5). Furthermore, the
total abundance of the nonsense and wild-type mRNAs
are the same in Nmd1strains, but in Nmd?strains the
nonsense mRNA is more abundant (Figure 3).
In Nmd?strains, mRNAs in the anchored pool are
more stable, which increases the size of the pool. If
anchoring of the nonsense mRNA is a rate-limiting
bottleneck for entry into this pool, slower decay of the
anchored pool might slow entry into the pool and cause
a rise in the size of the She2p-bound pool. Alternatively,
in the absence of Upf1p, less efficient termination
might also slow entry into the anchored pool since
Upf1p is known to promote efficient termination of
translation in conjunction with the release factors eRF1
and eRF3 as a prerequisite for decapping and decay
(Wang et al. 2001). In either case, our data support the
idea that anchoring depends not only on translation but
To reveal the full potential of NMD to degrade ash1-
A-ns1 mRNA, we disrupted the transport system by
deleting five different SHE genes and by analyzing
nonsense alleles in which the ash1-A-ns1 and ash1-A-
ns2 mutations were combined with multiple mutations
in the zip codes to prevent the binding of She2p.
Although none of the mutations had any added effect
on sensitivity to NMD by themselves or in combination
with single deletions of genes coding for translational
repressors of ASH1, we found that ash1-A-ns1 mRNA
was hypersensitive to NMD in she2D strains carrying
simultaneous deletions of PUF6 and KHD1. The rapid
decay in the triple-deletion strain demonstrates the full
efficacy of NMD-mediated degradation of ash1-A-ns1
is in an actin-detethered, translationally derepressed
pool that does not normally exist.
The nonsense mutations at sites B and C differ from
site A since the latter is located upstream of all She2p-
binding domains whereas the former are located
downstream of one or more of the binding domains.
Like the mutations at site A, mutations at sites B and C
cause increased mRNA abundance relative to wild type
in Nmd1strains, but unlike the mutations at site A, the
corresponding mRNAs are insensitive to the inactiva-
tion of NMD in otherwise wild-type strains. The non-
sense mutation at site B causes a mislocalization
phenotype similar to nonsense mutations at site A, but
the mutation at site B confers more severe mislocaliza-
tion. Like ash1-A-ns1, the ash1-B-ns1 mutation causes a
shift toward increased amounts of mRNA bound to
increase was observed when NMD was inactivated,
consistent with its insensitivity to NMD. Reduced bind-
ing to She2p was observed for a nonsense mutation at
site C in both Nmd1and Nmd?strains.
Figure 8.—Dual-pool model. Nonsense mutations at the A site (Figure 1) cause a change in the relative proportion of two pools
of mRNA: (1) She2p-bound mRNAs, which are tethered to the actin cytoskeleton, are engaged in transport, and are translationally
repressed and NMD insensitive and (2) mRNAs anchored at the bud-tip cortex, which are translatable and sensitive to NMD. The
numbers in the circles in A and B represent the relative pool sizes on the basis of experimental evidence shown in Figure 5. The
same relative ratios are shown in tabular form in C. Changes in the relative pool sizes due to premature termination at site A and to
the inactivation of NMD are consistent with data presented in the text.
Translation and Turnover of ash1 Nonsense mRNA1403
Most importantly, the ash1-B-ns1 nonsense mRNA,
which was insensitive to NMD in SHE2 strains, became
sensitive in she2D strains as evidenced by changes in
mRNA abundance and decay rate in Nmd?strains. The
difference in phenotypes between mutations at sites A
and B might be explained by their locations relative to
positions of the binding sites for She2p. The sequence
if repression of translation initiation is relieved before
the mRNA is released from binding to She2p, trans-
lation could initiate and proceed unimpeded to site A
where translation terminates. However, ribosomes
would have to transit across the E1-binding domain
for She2p to reach a termination codon at site B. If
(Chartrand et al. 2002), this could result in inefficient
terminationat downstream siteB.It hasbeenshownthat
reduced rates of translational elongation cause ineffi-
cient termination leading to readthrough (Sandbaken
and Culbertson 1988). Since efficient termination is
required for NMD (Bonetti et al. 1995), sensitivity to
NMD might be negatively affected.
The nonsense mutation at site C was insensitive to
NMD in all strain backgrounds tested. The insensitivity
356 nucleotides upstream of the normal stop codon
(Figure 1). Mutations near the normal stop codon can
be insensitive to NMD. For example, the HIS4 frame-
upstream of the normal stop codon and which gener-
ates a premature stop codon immediately after the site
of frameshifting, is NMD insensitive (Mathison and
Culbertson 1985; Leeds et al. 1991). The proposed
explanation for insensitivity is that A/U-rich down-
stream sequence elements required for NMD are not
present in between the 39-proximal premature stop
codon and the normal stop codon (Ruiz-Echevarria
and Peltz 1996). We identified a potential downstream
element, but it might not be functional. In that case,
cells might recognize the ash1-C-ns1 premature stop
codon as a normal stop codon.
When nonsense transcripts produced by alleles car-
rying mutations at sites A or B fail to anchor, they
mislocalize as She2-bound transcripts. However, when
ash1-C-ns1 nonsense mRNA fails to anchor, it dissociates
from She2p and mislocalizes without being tethered to
the actin cytoskeleton. By the time a ribosome reaches
site C, it has passed through and presumably displaced
She2p binding at three of the four binding domains,
leaving the mRNA tethered only through binding in
the 39-UTR. If termination occurs at the normal stop
of the bud tip. However, if termination occurs at site C,
the mRNA dissociates from She2p without anchoring.
The results suggest the existence of a mechanism to
ensure that anchored transcripts can be translated full
This is Laboratory of Genetics paper no. 3635. This work was
supported by the University of Wisconsin College of Agricultural and
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Targeting, disruption, replacement, and allele
Utilizing the GCN4
Mutations in elon-
The role of
An internal open reading
mRNA trafficking in fungi.
Communicating editor: M. Hampsey
Translation and Turnover of ash1 Nonsense mRNA 1405