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Molecular and Cellular Biology, October 1998, p. 6121-6130, Vol. 18, No. 10
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Telomere Length Regulation and Telomeric Chromatin
Require the Nonsense-Mediated mRNA Decay Pathway
Jodi E.
Lew,
Shinichiro
Enomoto, and
Judith
Berman*
Department of Plant Biology and Plant
Molecular Genetics Institute, University of Minnesota, St. Paul,
Minnesota 55108
Received 15 April 1998/Returned for modification 15 June
1998/Accepted 10 July 1998
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ABSTRACT |
Rap1p localization factor 4 (RLF4) is a
Saccharomyces cerevisiae gene that was identified in a
screen for mutants that affect telomere function and alter the
localization of the telomere binding protein Rap1p. In rlf4
mutants, telomeric silencing is reduced and telomere DNA tracts are
shorter, indicating that RLF4 is required for both the
establishment and/or maintenance of telomeric chromatin and for the
control of telomere length. In this paper, we demonstrate that
RLF4 is allelic to NMD2/UPF2, a gene required
for the nonsense-mediated mRNA decay (NMD) pathway (Y. Cui, K. W. Hagan, S. Zhang, and S. W. Peltz, Mol. Cell. Biol. 9:423-436,
1995, and F. He and A. Jacobson, Genes Dev. 9:437-454, 1995). The NMD
pathway, which requires Nmd2p/Rlf4p together with two other proteins,
(Upf1p and Upf3p), targets nonsense messages for degradation in the
cytoplasm by the exoribonuclease Xrn1p. Deletion of UPF1
and UPF3 caused telomere-associated defects like those
caused by rlf4 mutations, implying that the NMD pathway, rather than an NMD-independent function of Nmd2p/Rlf4p, is required for
telomere functions. In addition, telomere length regulation required
Xrn1p but not Rat1p, a nuclear exoribonuclease with functional similarity to Xrn1p (A. W. Johnson, Mol. Cell. Biol.
17:6122-6130, 1997). In contrast, telomere-associated defects were not
observed in pan2, pan3, or pan2
pan3 strains, which are defective in the intrinsic
deadenylation-dependent decay of normal (as opposed to nonsense) mRNAs.
Thus, loss of the NMD pathway specifically causes defects at telomeres,
demonstrating a physiological requirement for the NMD pathway in normal
cell functions. We propose a model in which the NMD pathway regulates
the levels of specific mRNAs that are important for telomere functions.
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INTRODUCTION |
Telomeres, the ends of linear
chromosomes, have multiple functions within the cell. Telomeres
stabilize chromosomes by preventing end-to-end fusions (62, 63,
78, 85). Telomerase, an enzyme necessary for the maintenance of
telomeric DNA, is inactive in most somatic cells and is activated in
many malignant cells (reviewed in references 3, 8,
and 25). Furthermore, the lack of telomerase
activity in somatic cells limits the life span of human fibroblasts
(5). In addition, genes adjacent to telomeres are often
packaged in an inaccessible heterochromatin-like structure (23) that results in epigenetic transcriptional
repression termed telomeric silencing or telomere position effect.
Telomeric DNA is composed of simple short repeats in most organisms,
and telomere length is controlled by the actions of multiple gene
products (reviewed in reference 58).
In the yeast Saccharomyces cerevisiae, the most abundant
telomere-binding protein, repressor activator protein (Rap1p) (9, 53, 80), binds double-stranded telomere repeats and is required for both telomeric silencing (43, 83) and telomere length regulation (59, 61, 83). In wild-type cells, Rap1p localizes to a small number of foci located primarily near the nuclear periphery (21, 38), which colocalize with telomeric DNA
(21). A number of mutations, including those in
SIR genes, result in an altered localization of Rap1p
(11) and likely reflect changes in the structure of
telomeric chromatin (19, 21).
To identify additional proteins involved in telomere function, we
performed a genetic screen using circular plasmids that included both
telomeric and centromeric DNA (TEL+CEN plasmids). TEL+CEN plasmids are
highly unstable due to antagonism between the telomere and centromere
sequences (18). We selected for mutant strains in which this
TEL+CEN antagonism was lost (i.e., TEL+CEN plasmids were stabilized).
As a secondary screen, we analyzed the nuclear distribution of Rap1p in
these mutant strains by indirect immunofluorescence microscopy. To
date, we have identified six genes that, when mutated, perturb the
localization of Rap1p. These genes are termed Rap1p localization factor
(RLF) genes (17, 19, 40). The present paper
describes the characterization of RLF4. In addition to the
stabilization of TEL+CEN plasmids and the altered localization of
Rap1p, rlf4 strains are defective in telomeric silencing and
have shortened telomeres. Cloning of RLF4 identified
rlf4-1 as a new allele of NMD2/UPF2, a gene
encoding a component of the pathway required for decay of mRNAs that
prematurely terminate translation (12, 28).
This unique nonsense-mediated decay (NMD) pathway targets nonsense
messages for rapid degradation. Targets of NMD include mRNAs containing
nonsense or frameshift mutations (24, 32, 48, 49, 54, 69),
transcripts with short upstream open reading frames (ORFs) (12,
68, 70, 72), and inefficiently spliced, intron-containing RNAs
that enter the cytoplasm (29, 32). Nonsense mRNAs are
stabilized in cells containing suppressor tRNAs, indicating that NMD
targeting is closely linked to premature translation termination
(4, 24, 54, 70). The NMD pathway requires three elements: a
translational arrest, cis-acting sequences within the 5'
proximal two-thirds to three-quarters of the message, and
trans-acting factors such as the Upf/Nmd proteins. The
Upf/Nmd proteins include Upf1p (13, 49, 71), Nmd2p/Upf2p
(12, 28), and Upf3p (46, 47, 49). Because Upf1p
and Nmd2p, as well as Nmd2p and Upf3p, physically interact
(27) and because single and double mutations in any of the
UPF genes inhibit mRNA decay to the same extent, the NMD
proteins clearly operate together in the NMD pathway (2,
12).
In the present paper, we demonstrate that RLF4 is allelic to
NMD2. Like nmd2/rlf4 strains, both
upf1 and upf3 strains have telomeric defects.
Furthermore, strains lacking Xrn1p, the cytoplasmic exoribonuclease
that degrades mRNAs targeted by the NMD pathway, also have short
telomeres, suggesting that NMD and UPF
genes affect telomere function via Xrn1p-mediated mRNA decay. Our
results indicate that wild-type telomeric chromatin function and
telomere length control require the NMD pathway specifically, rather
than mRNA decay in general.
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MATERIALS AND METHODS |
Yeast and Escherichia coli strains and plasmids.
The genotypes of the yeast strains used in this study are listed in
Table 1. E. coli XL1-blue
(Stratagene, Inc.) was used for all standard plasmid preparations.
Yeast cultures were grown overnight at 30°C in either minimal (SDC)
or rich (YPAD) medium with 2% glucose, unless otherwise stated.
The plasmid pRACUT1 contains an autonomously replicating sequence
(ARS), the
ADE2 gene, centromere sequence from CEN IV,
URA3,
and telomere repeat sequence (
18).
pRS315-NMD2(X-S) contains
the full-length
NMD2 gene on an
XbaI-
SalI fragment (
28), cloned
into
the pRS315 vector backbone (
81).
NMD2 was
disrupted in
the S150-2B background by transforming YJB276 with
SacI/
SalI-digested
pBs-
nmd2::HIS3 (
28). This disruption
replaces 444 bp of the
promoter and 1,286 bp of the coding sequence of
NMD2 with an
XbaI-
ClaI
fragment
containing the
HIS3 gene. Deletion of
NMD2 in
YJB276-
nmd2::HIS3 was confirmed by Southern blot
hybridization, and original disruptants
were back-crossed to YJB1260.
Strains YJB1593 and YJB1594 are
His
+ spores from the
resulting diploid.
URA3 was integrated adjacent to the left telomere of
chromosome VII at the
ADH4 locus (VII-L URA3-TEL)
(
23) and was introduced
into strains containing
nmd2::HIS,
upf1::HIS,
pan2::LEU2,
pan3::HIS,
and
pan2::LEU2 pan3::HIS3, all in the W303
background, by standard
genetic crosses.
Cloning of RLF4.
YJB539 (rlf4-1) cannot
grow on 5-fluoro-orotic acid (5-FOA) due to defective silencing of the
integrated URA3 gene at the left end of chromosome VII
(VII-L URA3-TEL) (23). YJB539 was transformed
(20) with a YCP50-LEU2 library (41), plated onto SDC minus Leu to select for transformants, and then replica plated onto
SDC plus 5-FOA to select for restoration of silencing at telomeres.
5-FOA-resistant colonies were then streaked onto SDC minus Leu, SDC
minus uracil, and SDC plus 5-FOA plates to identify colonies of cells
that contained a library plasmid, the VII-L URA3-TEL marker,
and were consistently 5-FOA resistant. Of ~125,000 colonies screened,
124 colonies met the criteria described above. Plasmids from these
candidates were rescued into E. coli (XL1-blue) cells.
Plasmid p108 contained an ~11-kb insert and conferred 5-FOA resistance on YJB539.
Southern analysis.
Telomere repeat probes were prepared by
end labeling oligonucleotide 238 (CACCACACCCACACACCACCACACCCACACACCACCACAC), which is
identical to 2.5 repeats of the telomere template sequence in
TLC1. DNA was denatured and transferred (60) to a
nylon membrane (Micron Separations, Inc.). Total genomic DNA was
electrophoresed overnight in 1% agarose with Tris-acetate buffer.
Blots were hybridized at 65°C in QuikHyb (Stratagene, Inc.) according
to the manufacturer's instructions and then washed in 2× SSC (1× SSC
is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl
sulfate (SDS) at 65°C for 20 to 30 min and 0.5× SSC-0.1% SDS at
65°C for 10 min.
Northern analysis.
Total RNA was prepared from mid-log-phase
cultures by a glass bead method (22). RNA was subjected to
electrophoresis in 4% formaldehyde-1% agarose gels and transferred
(60) to a nylon membrane (Micron Separations, Inc.).
CYH2 probe was prepared by random priming (Oligolabelling
kit; Pharmacia, Inc.) of an ~0.5-kb EcoRI-HindIII fragment from pGEM-4Z-CYH2
(28). The blots were incubated with the CYH2
probe at 42°C in hybridization solution containing 50% formamide,
5× Denhardt's solution, 5× SSPE (1× SSPE is 0.18 M NaCl, 10 mM
NaH2PO4, and 1 mM EDTA [pH 7.7]), 0.1-mg/ml single-stranded herring sperm DNA, 0.2% SDS, and 5.5% dextran sulfate. The blots were washed at 42°C in 2× SSC-0.1% SDS for 20 to 30 min and analyzed on a Molecular Dynamics Storm 840 PhosphorImager with Image Quant version 1.11 software. Ratios were determined by
dividing the corrected volumes of pre-mRNA signal by the corrected volumes of mature mRNA signal for each individual strain.
Antisera and indirect immunofluorescence microscopy.
Indirect immunofluorescence was performed with anti-Rap1p antiserum
exactly as described previously (19).
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RESULTS |
Telomeric chromatin is altered in rlf4-1 mutants.
In S. cerevisiae, telomere repeats, like centromere
sequences, stabilize circular plasmids (52). In contrast,
circular plasmids containing both telomere and centromere sequences
exhibit TEL+CEN antagonism, i.e., they are highly unstable
(18). Genes that affect telomeric chromatin (e.g.,
RAP1, SIR2, SIR3, and SIR4) also affect the stability of TEL and TEL+CEN plasmids (18,
52), suggesting that telomeric DNA on plasmids has some of the
properties of chromosomal telomeres. The rlf4-1 mutation was
identified in a genetic screen (18, 19) for mutants in which
TEL+CEN antagonism was lost, i.e., TEL+CEN plasmids were stabilized.
When pRACUT1, a TEL+CEN plasmid carrying ADE2, was
transformed into an ade2
strain, the instability of the
TEL+CEN plasmid could be visualized by the predominance of red sectors
in the colonies (Fig. 1A). In contrast,
ade2 strains carrying the rlf4-1 allele gave
rise to colonies with predominantly white sectors, indicating that the
TEL+CEN plasmid was quite stable (i.e., antagonism was lost) (Fig. 1A).
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FIG. 1.
rlf4-1 mutant strain exhibits reduced TEL+CEN
antagonism and altered localization of Rap1p. (A) TEL+CEN antagonism
assay. WT (YJB276) and rlf4-1 (YJB539) strains were
transformed with pRACUT1. The left panel is a transformant of pRACUT1
into YJB276, and the right panel is a transformant of pRACUT1 into
YJB539. Transformants were streaked on complete medium to assay loss of
pRACUT1, which is indicated by the red. (B) Anti-Rap1p indirect
immunofluorescence assay. Images are confocal laser micrographs of the
equatorial Z section of each nucleus. WT (YJB492), rlf4-1
(YJB1260), nmd2::HIS3 (YJB1274), and
rlf4-1 + pNMD2 (YJB1275) strains are shown. Arrows
point to nuclei with characteristic WT Rap1p distribution.
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The localization of Rap1p within yeast nuclei, which is thought to
reflect the state of telomeric chromatin (
19,
21),
was
altered in
rlf4-1 strains. In wild-type (WT) strains, Rap1p
stains as a small number of discrete foci that localize primarily
near
the nuclear periphery (
19,
38) (Fig.
1B). In contrast,
in
most
rlf4-1 cells, the Rap1p foci appeared less discrete,
such
that it was difficult to count the exact number of foci within
a
single nucleus. Diffuse Rap1p staining within the central portion
of
the nuclei was often evident in
rlf4-1 cells (Fig.
1B).
Alterations
in Rap1p localization in other strains with defects in
telomeric
chromatin function have been observed (e.g.,
sir3,
sir4, and
rlf2 strains) (
19,
21).
Genes inserted adjacent to telomeres are silenced in
S. cerevisiae (
23). Telomeric silencing was measured by
determining
the proportion of cells in the population that repressed a
telomeric
copy of
URA3 by plating the cells on 5-FOA, which
kills cells
expressing
URA3 (
23). In WT cells,
VII-L URA3-TEL is subject
to epigenetic silencing; more than
half of the cells in the population
expressed
URA3 and thus
could grow on medium lacking uracil; yet
a significant proportion (1 to
50%) of the cells also grew on
SDC containing 5-FOA, indicating
that the
URA3 gene was silenced
(Fig.
2). In the
rlf4-1 strain, a
very small proportion (10
4 to 10
5) of the
cells silenced
VII-L URA3-TEL (Fig.
2). Thus, almost
all of
the cells were unable to grow on 5-FOA, indicating that
RLF4
is required for telomeric silencing.
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FIG. 2.
Telomeric silencing assay. rlf4-1 mutant
strain exhibits reduced telomeric silencing. (Top panels) WT (YJB492),
rlf4-1 (YJB1338), rlf4-1 + p108 (YJB1222),
and rlf4-1 + pNMD2 (YJB2541); (bottom panels) WT
(YJB492), rlf4-1 (YJB1260), and
nmd2::HIS3 (YJB2763). 1:10 serial dilutions of
fresh overnight cultures were spotted onto the indicated media and
grown for 48 h at 30°C. Reduced growth on SDC containing 5-FOA
indicates reduced silencing of VII-L URA3-TEL. Growth on SDC
plates (not shown) was similar to growth on SDC plates lacking
uracil.
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Telomere length control is altered in rlf4-1
mutants.
In wild-type S. cerevisiae cells, the length
of telomere repeat DNA is maintained within a narrow size range
(79). Although the average telomere lengths are slightly
different in different strain backgrounds, in most strains telomere
repeats (TG1-3/C1-3A) are ~250 to 350 bp.
At least 15 genes that affect average telomere length have been
identified (reviewed in references 56, 58, and
86). We measured average telomere length by
digesting chromosomal DNA with PstI, which digests ~520 bp
from the telomere proximal end of Y' repeats, which are present at
~50% of the chromosome ends (57). Digestion of the WT
strain released an ~800-bp terminal fragment (Fig.
3, arrow), indicating that telomere
repeats are an average of ~280 bp in the S150-2B strain background
(Fig. 3, lane 1). In rlf4-1 strains, the terminal
PstI fragment was ~700 bp (Fig. 3, lane 2), indicating
that the telomere repeat tract was ~100 bp shorter than the telomere
repeat tract in the parental strain. An ~100-bp reduction in telomere
length was consistently observed in all rlf4-1 progeny and
always cosegregated with the TEL+CEN antagonism and telomeric silencing
phenotypes of rlf4-1 strains. Taken together, the phenotypes
of rlf4-1 strains indicate that both telomeric chromatin
function and telomere length control are altered in rlf4-1
strains.
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FIG. 3.
rlf4/nmd2 mutants exhibit shortened telomere
tracts. Total genomic DNA was digested with PstI and
separated in 1% agarose. The arrow points to the terminal
PstI fragment of ~800 bp, which includes ~520 bp of the
Y' TAS element and ~180 to 280 bp of telomere repeat sequence
TG1-3/C1-3A. Other bands are terminal X
element fragments and/or internal X and Y' fragments. Lanes 1 to 5, WT
(YJB492), rlf4-1 (YJB1338), nmd2::HIS3
(YJB2763), rlf4-1 + p108 (YJB1222), and
rlf4-1 + pNMD2 (YJB2541), respectively.
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Cloning of RLF4.
RLF4 was cloned by
complementation of the telomeric silencing defect in rlf4-1
strains (see Materials and Methods). One genomic clone, p108, restored
TEL+CEN antagonism (data not shown), telomeric silencing (Fig. 2), and
WT telomere length (Fig. 3, lane 4) to the rlf4-1 strain
YJB539.
Restriction and DNA sequence analysis of the insert in p108 indicated
that the plasmid contained an ~11-kb fragment of chromosome
VIII
including at least portions of four ORFs:
ORF76W,
NMD2,
ORF78W,
and
IRE1. A plasmid copy
of
NMD2 [(pRS315-NMD2(X-S) (
28)], which
is
hereafter called pNMD2, was sufficient to restore TEL+CEN antagonism,
telomeric silencing, Rap1p localization, and telomere length control
to
YJB539 (Fig.
1,
2, and
3; data not shown). Furthermore,
nmd2::HIS3 strains had defects in telomeric
chromatin function and telomere
length control like those observed in
the
rlf4-1 strain; TEL+CEN
plasmids were stabilized (data
not shown), Rap1p appeared more
diffuse (Fig.
1B), telomeric silencing
was reduced (Fig.
2), and
telomeric DNA was shorter than that in the
isogenic WT strain
(Fig.
3, lane 3), suggesting that
NMD2
was the gene on p108 responsible
for the suppression of
rlf4-1 phenotypes.
Mutations in genes encoding components of the NMD pathway cause
steady-state accumulation of a number of RNAs that normally
have very
short half-lives (reviewed in reference
32). One of
these messages is PPR1, a positive regulator of
URA3
(
55). Thus,
it was possible that derepression of the
VII-L URA3-TEL in YJB539
occurred due to upregulation by
Ppr1p, rather than by reduced
telomeric silencing. To distinguish
between these possibilities,
we monitored the expression of an
ADE2 gene integrated adjacent
to the right end of chromosome
V (
V-R ADE2-TEL) (
23) in a strain
lacking the
normal chromosomal copy of
ADE2. The
ADE2 gene is
not upregulated by Prp1p (
82) or by mutation of
NMD2 (
49a).
Red and white colony sectoring was
used to monitor the status
of the telomeric
ADE2 gene. In
these strains, expression of
ADE2 leads to the cells being
colored white and lack of
ADE2 expression
results in cells
being colored red. WT and
nmd2::HIS3 tetrad
progenies
that carried
V-R-ADE2-TEL were isolated from the
heterozygous
diploid YJB2239. WT (
NMD2) progeny gave rise to
red-white sectored
colonies (Fig.
4),
which are characteristic of the normal epigenetic
silencing of
telomere-adjacent genes (
23). In contrast,
nmd2::HIS3 progeny gave rise to solid white
colonies (Fig.
4), which are
indicative of extensive derepression of
the telomeric
ADE2 gene.
Thus, despite the possible
upregulation of the
URA3 gene in
nmd2/rlf4 strains, telomeric silencing is perturbed in these strains due
to
alteration of the telomeric chromatin.
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FIG. 4.
Silencing of a telomere-proximal V-R ADE2-TEL
gene is lost in an nmd2::HIS3 mutant strain. Fresh
overnight cultures of WT (YJB2263) and nmd2::HIS3
(YJB2262) strains were diluted 1:105 in sterile water and
then plated on complete medium to assay expression of V-R
ADE2-TEL. Red sectors indicate silencing of V-R
ADE2-TEL, and white sectors indicate expression of V-R
ADE2-TEL.
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A rlf4-1 strain is defective in nonsense-mediated mRNA
decay.
Because the nmd2::HIS3 strain had
defects in telomeric functions similar to those of rlf4-1
strains, we asked whether an rlf4-1 strain had defects in
nonsense-mediated mRNA decay similar to those of an
nmd2::HIS3 strain. To ensure that genetic
background did not contribute to any differences observed, we disrupted
NMD2 in the S150-2B background (see Materials and Methods)
and compared mRNA stabilities between isogenic WT, rlf4-1,
and nmd2::HIS3 strains.
The
CYH2 transcript is a poorly spliced RNA, and the
CYH2 pre-mRNA accumulates in strains defective in
nonsense-mediated mRNA
decay (
29). We prepared total RNA and
asked whether
CYH2 pre-mRNA
accumulated in the
rlf4-1 strain as it did in the
nmd2::HIS3 strain
by analyzing Northern blots with
a
CYH2 probe (Fig.
5). The
ratio
of
CYH2 pre-mRNA to mature mRNA was approximately
twofold higher
in the
rlf4-1 strain than the ratio of
pre-mRNA to mature mRNA
(0.31) in the WT strain (Fig.
5, compare lanes 1 and 2). Introduction
of pNMD2 into the
rlf4-1 strain (Fig.
5, lane 4) reduced the
ratio
of pre-mRNA to mRNA to 0.27, suggesting that the aberrant
accumulation
of pre-mRNA in the
rlf4-1 strain was due to
loss of Nmd2p function.
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FIG. 5.
A rlf4-1 strain exhibits defective
nonsense-mediated mRNA decay. Total RNA was isolated from mid-log-phase
cells and separated in 1% agarose-4% formaldehyde. The Northern blot
was probed with an ~500 bp radiolabeled fragment of CHY2
from pGEM-4Z-CYH2.
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rlf4-1 is an allele of NMD2.
Since the
mutant phenotypes of rlf4-1 and
nmd2::HIS3 were similar in all cases, and since
pNMD2 complemented all phenotypes of rlf4-1 strains, we
performed genetic linkage analysis to ask whether rlf4-1 and
nmd2::HIS3 were allelic. Haploid rlf4-1
strain YJB1260 was crossed to haploid nmd2::HIS3
strain YJB1593, the resulting diploid was sporulated, and TEL+CEN
antagonism was scored in the tetrad progeny with pRACUT1. In 10 complete tetrads and more than 60 other individual spores, no
restoration of TEL+CEN antagonism was observed, which is consistent
with the idea that rlf4-1 and
nmd2::HIS3 are allelic.
We then determined the nature of the
nmd2 allele from the
rlf4-1 mutant strain by PCR amplification and subsequent DNA
sequencing
of overlapping segments covering the complete
NMD2 coding sequence.
Comparison of the
rlf4-1
allele to the
NMD2 sequence in the GenBank
database revealed
that the AAG lysine codon at amino acid 304
of
NMD2 had been
changed to a UAG stop codon in
rlf4-1. Thus,
the predicted
product of
rlf4-1 includes only the N-terminal one-third
of
the Nmd2p peptide sequence. These data, together with the phenotypic
similarities of the
rlf4-1 and
nmd2::HIS3 alleles, indicate that
RLF4
and
NMD2 are allelic.
Telomere functions require the NMD pathway.
Nmd2p is a member
of the NMD pathway that targets nonsense messages for
exoribonucleolytic decay (27, 28). We asked whether the
telomeric defects of rlf4 strains were caused by loss of NMD function or rather by loss of a putative NMD-independent function of
Nmd2p. If the telomeric defects of nmd2 strains are due to inactivation of the NMD pathway, then we expected that mutation of
genes encoding other members of this pathway, UPF1 and
UPF3, would result in similar telomeric defects.
We generated an
upf1 strain containing
VII-L
URA3-TEL in the W303 background and assayed telomeric silencing in
this strain.
The silencing defect caused by an
nmd2::HIS3 mutation is less
severe in this genetic
background (~10- to 100-fold reduction
in 5-FOA resistance [Fig.
6A]) than in S150-2B (~100- to
1,000-fold
reduction in 5-FOA resistance [Fig.
2]). Similar to an
nmd2::HIS3 strain, the
upf1 VII-L
URA3-TEL strain exhibited an ~100-fold
reduction in 5-FOA
resistance relative to the WT strain, suggesting
a modest silencing
defect (Fig.
6A). Thus, telomeric chromatin
is disrupted to the same
degree in
upf1 and
nmd2 strains.
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FIG. 6.
upf1 and upf3 strains exhibit
telomeric defects. (A) upf1 telomeric silencing assay.
Serial dilutions (1:10) of fresh overnight cultures were spotted onto
the indicated media and grown for 48 h at 30°C. Reduced growth
on SDC containing 5-FOA indicates reduced silencing of VII-L
URA3-TEL. WT (YJB1680), nmd2::HIS3 (YJB2299),
and upf1::HIS3 (YJB2297) are shown. Growth on SDC
plates (not shown) was similar to growth on SDC plates lacking uracil.
(B) upf1 and upf3 telomere length assay. Total
genomic DNA was digested with PstI and separated in 1%
agarose. The arrow points to the terminal PstI fragment of
~800 bp, which includes ~520 bp of the Y' TAS element and ~180 to
280 bp of telomere repeat sequence
TG1-3/C1-3A. (Left panel) Lanes 1 to 3, WT
(YJB447), upf1::HIS3 (YJB1324), and
nmd2::HIS3 (YJB1274), respectively; (right panel)
lanes 1 to 4 WT (YJB2889), upf3::TRP1 (YJB2890),
upf3::TRP1 + pUPF3 (YJB2891), and
upf3::TRP1 + vector (YJB2892),
respectively.
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We also assayed telomere length in both an
upf1 strain and
an
upf3 strain. Like telomeres in
nmd2 strains,
the bulk of the
telomeres in the
upf1 and
upf3
strains was ~100 bp shorter than
that in the isogenic WT strains
(Fig.
6B).
upf1 and
upf3 strains
had terminal
PstI restriction fragments that were similar in size
to
those of
nmd2 mutants and were significantly shorter than
those
of the isogenic WT strains. Addition of plasmid-born
UPF3 to the
upf3 strain restored normal telomere
length regulation (Fig.
6B,
right panel, lanes 2 and 3). Taken
together, the silencing defect
in the
upf1 strain and the
telomere length reduction in the
upf1 and
upf3
strains suggest that it is the NMD pathway, rather than
Nmd2p alone,
that contributes to telomere functions in WT cells.
A strain deficient in cytoplasmic exoribonucleolytic activity also
has short telomeres.
The NMD pathway could contribute to
telomere functions by mediating the decay of specific mRNAs.
Alternatively, this pathway could perform a novel function at
telomeres that is unrelated to its role in mRNA decay. To distinguish
between these possibilities, we analyzed average telomere lengths in
strains lacking the major exoribonuclease (Xrn1p) required for
cytoplasmic RNA decay (44, 65, 67). If the decay of mRNAs
targeted by the NMD pathway is responsible for the telomere-associated
phenotypes of upf1, nmd2, and upf3
mutants, then Xrn1p should also contribute to telomere functions.
Consistent with the hypothesis that telomeric defects arise in an
rlf4/nmd2 strain due to defective nonsense-mediated mRNA
decay, an xrn1 strain (34) had telomeres
~100 bp shorter than those in the isogenic WT strain (Fig.
7, left panel, lane 2).
View larger version (52K):
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|
FIG. 7.
An xrn1 strain, but not a
rat1-1 strain, exhibits defective telomere length control.
Total genomic DNA was digested with PstI and separated in
1% agarose. The arrow points to the terminal PstI fragment
of ~800 bp, which includes ~520 bp of the Y' TAS element and ~180
to 280 bp of telomere repeat sequence
TG1-3/C1-3A. (Left panel) Lanes 1 and 2, WT
(YJB2793) and xrn1 (YJB2794), respectively; (right
panel), lanes 1 and 2, WT (YJB2796) and rat1-1 (YJB2797),
respectively. All strains were grown at 30°C.
|
|
Xrn1p, which is exclusively cytoplasmic (
30), is
functionally similar to Rat1p, a nuclear exoribonuclease that performs
an essential function in
S. cerevisiae. Localization of
Rat1p
to the cytoplasm allows it to complement an
xrn1
mutation (
33,
73). Telomeres are, by definition, nuclear in
S. cerevisiae,
because the nuclear membrane remains intact
throughout the cell
cycle. Because telomeres are nuclear, we asked
whether Rat1p might
play a role in telomere function by analyzing
telomere length
in a
rat1-1 strain. In contrast to the
xrn1 strain, the
rat1-1 strain did not exhibit
any significant alteration in the length
of the terminal
PstI fragment when cells were grown at permissive
(23°C)
and semipermissive (30 and 33°C) temperatures (Fig.
7 and
data not
shown). Since Xrn1p and Rat1p can be functionally redundant
yet differ
in cellular localization (
33), the finding that the
xrn1 strain had a defect in telomere length control and
that
the
rat1-1 strain did not suggests that telomere
defects arise
due to perturbation of mRNA degradation by a cytoplasmic
exoribonuclease
rather than due to mRNA degradation by a nuclear
exoribonuclease.
These results also support the hypothesis that the
telomere defects
of an
rlf4/nmd2 strain occur due to
inactivation of the NMD pathway,
which targets mRNAs for degradation by
Xrn1p.
Telomere functions are not perturbed in strains deficient in the
decay of normal messages.
Like the NMD pathway, the intrinsic mRNA
decay pathway relies on Xrn1p activity for final exoribonucleolytic
activity (44, 65, 67). However, the intrinsic mRNA decay
pathway requires progressive deadenylation followed by 5' decapping,
whereas the NMD pathway bypasses the deadenylation requirement for mRNA
decay (14, 65, 66, 76). Poly(A) nuclease (PAN) deadenylates mRNA in a poly(A)-binding-protein-dependent manner (6, 77). To ask if it is intrinsic mRNA decay or NMD pathway-specific decay that
is required for telomere functions, we analyzed pan2 and pan3 mutants. PAN2 and PAN3 encode two
subunits of the PAN nuclease, and pan2 and
pan3 mutant strains accumulate mRNAs with average poly(A) tails longer than those of WT strains (6, 7). We asked whether strains that might be hampered in the processes of normal
mRNA turnover would exhibit defects in telomeric functions similar to
those of strains deficient in nonsense-mediated mRNA decay. We analyzed
telomeric silencing and telomere length control in strains carrying
deletions of PAN2 and PAN3. Neither the
pan2 strain, the pan3 strain, nor a double mutant
pan2 pan3 strain exhibited a reduction in telomeric
silencing relative to an isogenic WT strain (Fig.
8A). Similarly, telomere length control
in a pan2 strain, a pan3 strain, and a pan2
pan3 double mutant strain was indistinguishable from that of the
isogenic WT strain (i.e., terminal telomere tracts were not shortened
[Fig. 8B]). Thus, mutants with defects
in the deadenylation process required for efficient poly(A) shortening-dependent mRNA turnover do not exhibit altered telomeric chromatin or telomere length control. These results are consistent with
the idea that the NMD pathway specifically, rather than the intrinsic
pathway that targets mRNA for degradation, is required for normal
telomere function.
View larger version (43K):
[in this window]
[in a new window]
|
FIG. 8.
pan mutants do not exhibit telomere defects.
(A) Telomeric silencing assay. Serial dilutions (1:10) of fresh
overnight cultures were spotted onto the indicated media and grown for
48 h at 30°C. WT (YJB2827), pan2::LEU2
(YJB2823), pan3::HIS3 (YJB2824),
pan2::LEU2 pan3::HIS3 (YJB2826), and
nmd2::HIS3 (YJB1593) are shown. Growth on SDC
plates (not shown) was similar to growth on SDC plates lacking uracil.
(B) Telomere length assay. Total genomic DNA was digested with
PstI and separated in 1% agarose. The arrow points to the
terminal PstI fragment of ~800 bp, which includes ~520
bp of the Y' TAS element and ~180 to 280 bp of telomere repeat
sequence TG1-3/C1-3A. Lanes 1 to 4, WT
(YJB2739), pan2::LEU2 (YJB2735),
pan3::HIS3 (YJB2736), and
pan2::LEU2 pan3::HIS3 (YJB2740),
respectively.
|
|
| |
DISCUSSION |
rlf4-1 is an allele of the nonsense-mediated decay gene
NMD2.
RLF4, which was isolated by virtue of its
defects in telomere-associated phenotypes, is allelic with
NMD2/UPF2 by several criteria. First, NMD2
complemented all of the mutant phenotypes of an rlf4-1
strain. Second, nmd2 and rlf4 strains exhibited
similar telomere-associated phenotypes as well as a similar defect in nonsense-mediated decay. Third, genetic linkage analysis indicated that
rlf4-1 and nmd2::HIS3 cosegregate.
Finally, the DNA sequence of rlf4-1 indicated that it is a
nonsense allele that would encode only the first one-third of Nmd2p.
The phenotypic similarities of rlf4-1 and the
nmd2::HIS3 deletion allele suggest that the rlf4-1 allele is effectively a null allele of
NMD2.
How does NMD2/RLF4 affect telomere functions?
NMD2/RLF4 encodes a protein necessary for nonsense-mediated
mRNA decay (12, 28). The NMD pathway targets mRNAs for rapid turnover when the translational machinery encounters a premature termination codon during translation (reviewed in references
32, 69, and 70).
NMD2/RLF4 is one of three genes, the other two being
UPF1 and UPF3, that are required for
nonsense-mediated decay. Like nmd2/rlf4 strains,
upf1 and upf3 strains had telomere-associated defects (Fig. 6), suggesting that the NMD pathway, rather than an
NMD-independent function of Nmd2/Rlf4p, is required for normal telomere
function.
We envision two general models to explain why the NMD pathway proteins
might be required for telomere functions: a direct
model and an
indirect model. In the direct model, NMD proteins
have a second
function at telomeres in the nucleus. In the indirect
model, the NMD
pathway affects telomere function by altering the
stability of one or
more specific mRNAs that are important for
telomere chromatin function
and/or telomere length control. A
number of genes, such as
RAP1 and
RIF1, affect both telomere length
control and telomeric silencing (
24a,
59). It is also
possible
that the NMD pathway affects telomere functions even more
indirectly,
by altering the level of an mRNA that encodes a protein
that then,
in turn, regulates telomere-related genes. Nmd2p has a
putative
bipartite nuclear localization sequence that is required for
function
(
26,
28). Upf3p also has predicted nuclear
localization sequences
and nuclear export sequences (
46,
79a). Thus, at least two
of the NMD proteins may have an
opportunity to interact directly
with telomeres, at least transiently.
To begin to distinguish between the direct and indirect models of NMD
protein involvement in telomere function, we asked whether
Xrn1p is
required for telomere function.
XRN1 has been isolated
from
a wide range of biochemical and genetic screens and is also
known as
KEM1,
SEP1,
DST2,
RAR5, and
SKI1 (
15,
36,
37,
39,
84). We found that, like
upf1,
nmd2, and
upf3 mutants, the
xrn1 mutant had short telomeres (Fig.
7, left panel). Our
work confirms
a previous report that telomeres are shorter in
xrn1/kem1 strains
(
51). Unlike the previous
report, we found that
xrn1 mutants
did not display a
senescence phenotype (data not shown); rather,
in keeping with the
observations of others (
16,
33,
36,
45),
xrn1
cells exhibited a slow-growth phenotype. The fact
that Xrn1p is
exclusively cytoplasmic (
30,
33) supports the
indirect model
that Xrn1p (and Upf1p, Nmd2p, and Upf3p) affects
telomeres via message
degradation.
A second exoribonuclease in
S. cerevisiae is encoded by the
essential gene
RAT1 (
1,
33,
35). Rat1p localizes
predominantly
to the nucleus; yet this nuclear localization appears
saturable,
such that multicopy expression of
RAT1 results in
both nuclear
and cytoplasmic localization of Rat1p and partially
restores the
slow growth and RNA turnover phenotypes of an
xrn1 strain (
33,
73). In contrast to the
xrn1 mutant, the
rat1-1 mutant had normal
telomere length control (Fig.
7), suggesting that, despite the
similarities between Xrn1p and Rat1p, the NMD proteins do not
affect
telomere functions via nuclear Rat1p activity. This result
is again
consistent with the indirect model that NMD proteins
are required for
telomere function because they target specific
mRNAs for decay by
Xrn1p.
Telomere functions could be indirectly affected by all mRNA decay
pathways or by the NMD pathway specifically. To distinguish
between
these possibilities, we analyzed the telomere-associated
phenotypes of
pan strains which are deficient in the
poly(A)-binding-protein-dependent
PAN. PAN is the only characterized
deadenylase in yeast, and poly(A)
tail deadenylation is a prerequisite
for targeting of a message
for the intrinsic pathway of mRNA turnover
(
6,
7,
14,
77). Pan2p and Pan3p interact physically, and
pan2 and
pan3 mutant strains have mRNA
populations with average poly(A) tail
lengths longer than those of WT
strains (
7). The fact that
neither a
pan2, a
pan3, nor a
pan2 pan3 strain exhibited reduced
telomeric silencing or shortened telomere tracts (Fig.
8) is consistent
with the hypothesis that it is the NMD pathway specifically, rather
than intrinsic mRNA decay, that is required for telomere function.
Because some (inefficient) deadenylation occurs in strains lacking
Pab1p, it is possible that other Pab1p-independent PANs exist
in yeast
(
7,
10,
76), and, thus, we cannot exclude the
possibility
that a Pab1p-independent PAN may contribute to telomere
function.
Despite this caveat, we propose the model that the levels
of mRNAs
important for telomere functions are inappropriately
increased in
upf and
nmd mutants, leading to defects in
telomeric
silencing and length control. This model implies that in WT
cells,
the level of these mRNAs is regulated by the NMD pathway. While
our data support this indirect model, we cannot rule out the
possibility
that the NMD proteins have a second, more direct, role in
the
nucleus that is independent of their cytoplasmic function and
of
the function of Rat1p and Xrn1p.
Is there a role for the nonsense-mediated decay pathway in
regulating normal rather than nonsense mRNAs?
Sequence features
that the NMD pathway recognizes include upstream ORFs (uORFs) and
frameshifts. Interestingly, a number of messages encoding
telomere-associated proteins include uORFs or frameshifts. For example,
TEL2, EST1, and EST2 (genes required for telomerase activity and/or telomere length control) contain clusters of uORFs within 100 bp of the initiation codons (50, 74), and EST3 requires a frameshift for translation of
a full-length functional protein (64). Neither uORFs nor
frameshifts alone are sufficient to ensure that mRNAs containing
them are targeted by the NMD pathway (71). For example, the
GCN4 mRNA, which is regulated by a mechanism involving four
short uORFs, is not targeted for decay by the NMD pathway (reviewed in
reference 31). Analysis of specific mRNA levels and
mRNA half-lives will be required to determine whether the NMD pathway
specifically and directly regulates the expression of genes that encode
proteins known to be required for telomeric silencing and/or telomere
length control.
The NMD pathway has been presumed to target defective rather than
normal mRNAs for degradation (
70). The telomere-associated
phenotypes of
upf1,
nmd2,
upf3, and
xrn1 mutants implicate the
NMD pathway in the regulation of
normal mRNAs involved in telomere
function. The half-lives of
mRNAs are thought to be governed by
a number of competing
stabilizing and destabilizing forces (reviewed
in references
32,
70 and
75). Upf-mediated
decay may be
one of the destabilizing forces that contribute to the
regulation
of mRNA half-life for one or more normal mRNAs important for
telomere
functions.
| |
ACKNOWLEDGMENTS |
We thank Maryam Gerami-Nejad for technical assistance and David
Gartner and Mark Sanders of the University of Minnesota Imaging Center
for assistance with digital imaging. We thank Feng He, Allen Jacobson,
Alan Sachs, and Michael Culbertson for providing strains and plasmids.
We thank Steve Johnston, Cathy Asleson, Michael Lelivelt, and Jeff
Dahlseid for critical reading of the manuscript and many helpful
suggestions.
This work was supported by a grant from the National Institutes of
Health (GM38636) to J.B.
| |
ADDENDUM IN PROOF |
Recently, CTF13, a gene important for centromere
function, was found to be regulated, indirectly, by the NMD pathway (J. N. Dahlseid, J. Puziss, R. L. Shirley, A. L. Atkin, P. Hieter, and M. R. Culbertson, Genetics, in press).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Plant Biology and Plant Molecular Genetics Institute, 220 Biological Sciences Center, 1445 Gortner Ave., University of Minnesota, Twin Cities Campus, St. Paul, MN 55108. Phone: (612) 625-1971. Fax: (612)
625-1738. E-mail: judith{at}biosci.cbs.umn.edu.
| |
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Abstract
Introduction
