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J. Biol. Chem., Vol. 278, Issue 26, 23553-23560, June 27, 2003
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From the Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Received for publication, January 21, 2003 , and in revised form, April 3, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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).
Unexpectedly, mutation of both cytosolic serine hydroxymethyltransferase
(SHM2) and one-carbon tetrahydrofolate synthase (ADE3) was
required to achieve synthetic lethality with srp40. Shm2p and
Ade3p are cytoplasmic enzymes producing 5,10-methylene tetrahydrofolate in
convergent pathways as the primary source for cellular one-carbon groups.
Nonetheless, point mutants of Shm2p that were catalytically inactive
(i.e. failed to rescue the methionine auxotrophy of a
shm2 ade3 strain) complemented the synthetic lethal
phenotype, thus revealing a novel metabolism-independent function of Shm2p.
The same Shm2p mutants exacerbated a giant cell phenotype observed in the
shm2 ade3 strain suggesting a catalysis-independent
role for Shm2p in cell size control, possibly through regulation of ribosome
biogenesis via SRP40. Additionally, we show that the Sm-like protein
Lsm5p, which as part of Lsm complexes participates in cytosolic and nuclear
RNA processing and degradation pathways, is a multicopy suppressor of the
synthetic lethality and of the specific depletion of box H/ACA small nucleolar
RNAs from the srp40 shm2 ade3 strain. Finally, rat
Nopp140 restored growth and stability of box H/ACA snoRNAs after genetic
depletion of SRP40 in the synthetic lethal strain indicating that it
is indeed the functional homolog of yeast Srp40p. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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200
nucleotides (100 in yeast) by pseudouridylation and
2'-O-methylation. Although apparently nonessential, most of
these modifications occur in functionally important regions of the ribosome
suggesting a role in translation
(1). The nucleotides to be
modified are selected by site-specific base pairing with a similar number of
small nucleolar RNAs
(snoRNAs)1 of the box
H/ACA and box C/D class for rRNA pseudouridylation and
2'-O-methylation, respectively
(2–5).
The same four core proteins associate with each box H/ACA snoRNA to form
separate box H/ACA small nucleolar ribonucleoprotein particles (snoRNPs) and
another set of four core proteins forms box C/D snoRNPs together with each box
C/D snoRNA. One of the core proteins is the pseudouridylase and the methylase,
respectively, catalyzing the modification. In yeast, individual depletion of
most of the snoRNP core proteins leads to instability of the particle and the
respective class of snoRNAs
(6–9).
Although little is known about the maturation of the snoRNPs themselves or
their organization while modifying rRNA, they are concentrated in the
nucleolus and Cajal (coiled) bodies of vertebrate cells. Cajal bodies
generally are enriched in small nuclear RNAs and may be involved in their
maturation, but their function essentially remains elusive
(10).
Vertebrate Nopp140 is the only protein to date that associates with both classes of snoRNPs, although it is not an integral component of either particle (11). Like the snoRNPs, Nopp140 is concentrated in the nucleolus and Cajal bodies (12, 13). In fact, a dominant negative Nopp140 construct specifically chases snoRNPs out of these subnuclear structures indicating an in vivo interaction and a role for Nopp140 in snoRNP localization (14). The interaction of Nopp140 with snoRNPs is reversible and controlled by its unusually high degree of phosphorylation (15). These observations characterized Nopp140 as a chaperone of snoRNPs.
Based on sequence homology and nucleolar localization, Srp40p is the closest Nopp140 relative in yeast (16, 17). Originally identified in genetic screens as a multicopy suppressor of temperature-sensitive mutations in genes involved in rRNA transcription (18, 19), Srp40p concentrates in a nucleolar sub-structure, the nucleolar body, together with box C/D snoRNAs (20). These and other data indicate that Srp40p, like Nopp140, interacts with snoRNPs. Deletion of the nonessential SRP40 leads to the loss of the nucleolar body similar to the dispersal of Cajal bodies by the dominant negative Nopp140 construct (14, 20). These similarities between Srp40p and Nopp140 prompted us to exploit yeast genetics to learn more about the role of these proteins in ribosome and snoRNP biogenesis. Specifically, we report on the unexpected findings in a screen for genes that are synthetically lethal with an SRP40 deletion.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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was used
as the bacterial host for all plasmids and was manipulated using standard
methods (23).
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Synthetic Lethal Screens—To delete SRP40, the
SRP40 open reading frame including 130 nucleotides 5' and 455
nucleotides 3' in pTM32
(17) was replaced with the
HIS3-containing EcoRI-XhoI fragment of pRS313
generating pYY1. The srp40::HIS3-containing
ApaI-XbaI fragment of pYY1 served to delete the genomic copy
of SRP40 by integrative transformation of YYY2 and YYY3 generating
YYY4 and YYY5, respectively. YYY2 and YYY3 were transformants of YCH125 and
YCH128, respectively (24).
Proper integration of the HIS3 construct at the SRP40 locus
was verified by PCR on genomic DNA with the appropriate primers.
These SRP40-deleted strains were used in the synthetic lethal screens after transformation with pYY5 (SRP40 ADE3 URA3 CEN) to generate YYY12 and YYY14. pYY5 was constructed in two steps. First, ADE3 was cloned from pSW198 (24) into pRS316 (25) as a BamHI-SalI fragment resulting in pRS316A. Second, the SRP40 gene including 1029-nucleotide 5'- and 475-nucleotide 3'-untranslated region, isolated by PCR amplification using yeast genomic DNA as template, was subcloned into the SalI site of pRS316A yielding pYY5. YYY12 and YYY14 produced colonies that exhibited a high degree of sectoring when grown on nonselective medium and were used in a colony-sectoring screening assay. For this purpose, they were mutagenized by random lacZ LEU2 insertions by transformation with a mutagenized genomic yeast library (26) or by exposure to ethylmethane sulfonate (EMS) (24). Colonies were screened for a nonsectoring phenotypes, and candidates were restreaked on YPD plates to confirm the lack of sectoring and on 5-FOA plates to determine that growth indeed depended on the presence of pYY5 (SRP40 ADE3 URA3 CEN). Both screens yielded a single clone that satisfied all criteria.
Identification and Molecular Analysis of the Synthetic Lethal Gene SHM2—The gene mutated by insertional mutagenesis was identified by inverse PCR of the DNA adjacent to the LEU2 transposon (26). Briefly, genomic yeast DNA was digested by RsaI, ligated, and transformed into E. coli. Transformants containing the LEU2 transposon with the E. coli origin of replication and the ampicillin resistance gene were rescued. The genomic DNA adjacent to the LEU2 transposon was amplified by inverse PCR using primers 5'-TAAGTTGGGTAACGCCAGGGTTTTC-3' and 5'-TGTTGCCACTCGCTTTAATG-3' and SHM2 was identified by DNA sequencing followed by data base comparison.
The synthetic lethal gene mutated by EMS was identified by complementation analysis with a yeast genomic library in a centromeric LEU2 vector (gift from Ian Willis, Albert Einstein College of Medicine, Bronx, NY). Transformants were screened for the ability to sector after 7 days of growth at 23 °C on SC medium lacking leucine. Candidates were restreaked on 5-FOA-containing medium to lose pYY5 leading to the isolation of 11 plasmids. Eight contained SRP40 and the others a 3.8-kb genomic fragment with only one intact open reading frame, SHM2.
For complementation and deletion purposes, SHM2 including 494
nucleotides upstream and 289 nucleotides downstream was amplified from genomic
DNA using oligonucleotides 5'-CGGGATCCTCAAAGAGAGCATAGCTCAATTGC-3'
and 5'-GCGGCCGCGAAAACGGTGAAAAAGTGAACAATG-3' and inserted into the
BamHI and NotI sites of pGEM-T (Promega, Madison, WI) to
generate pYY59. pYY45 was constructed by subcloning the
SHM2-containing NotI fragment from pYY59 into pRS315
(25). Nucleotides
115–793 were deleted from the 1407-bp SHM2 coding region in
pYY45 by replacement with the URA3-containing 678-bp
ClaI-BglII fragment from pRS316
(25) to generate pYY60. The
shm2::URA3-containing NotI fragment was used
to disrupt SHM2 in YYX30 to create YYX31.
Precise deletion of the SHM2 and ADE3 open reading frames
was achieved by replacement with the HIS3 and TRP1 markers,
respectively. For this purpose, the markers were PCR-amplified using pRS313
and pRS314 (25) as templates
with oligonucleotides that contained complementarity to the flanking regions
of the target gene (27). The
following oligonucleotides were used for HIS3,
5'-TGTTTTATATATAATGCAGAACTTGCTTCCCTTAGTTTGCGTTCACAGATTGTACTGAGAGTGCA-3'
and
5'-TAATGTTTATTTTTTTGGGATTAAGTGTCGTTGTGGTGATTTCTCATCTGTGCGGTATTTCACA-3',
and for TRP1,
5'-CCAGGTAACGAGACGAACACAACTTTACAAGTCAAATAAGAAATCCAGATTGTACTGAGAGTGCA-3'
and
5'-TGTCTTAGAACAGGCCATCGATCTCACCGTCATCATCGACTTCCCATCTGTGCGGTATTTCACA-3'.
In this way SHM2 was deleted in YYX32, a
srp40/srp40 strain generated by mating TMY20
with TMY21 (17), to create
YYX33 and YYX34. ADE3 was deleted in YYX38, an
srp40/srp40
shm2/shm2 strain generated by mating two
haploids obtained from sporulation and dissection of YYX34, to create YYX39.
Correct insertion of the markers was tested by PCR on yeast colonies.
For the construction of a conditional GAL::srp40 strain, pYY38 (pGAL-SRP40) (11) was transformed into the synthetic lethal strain SL1 while shuttling out plasmid pYY5 by growth on 5-FOA-containing medium thus producing YYY206. In the process and for unknown reasons, YYY206 lost its LEU2 marker from the transposon in SHM2.
Plasmids—Site-directed mutagenesis was performed according to the QuickChangeTM protocol (Stratagene, La Jolla, CA) using the following primers for PCR amplification, 5'-GGTGTCGCTATTGGAGCTCCAGCCATGA-3' and 5'-TGGAGCTCCAATAGCGACACCACCTGGA-3' for the R393A mutation and 5'-ACTCACCAGTCTTTGAGAGGTCCCCGTGGTGCTATGA-3' and 5'-ACCACGGGGACCTCTCAAAGACTGGTGAGTGGTGGT-3' for the K248Q mutation of SHM2 generating pYY67 and pYY71, respectively. The underlined nucleotides were introduced to obtain the point mutations, and the nucleotides in boldface reflected silent changes to generate new restriction sites, SacI and DraIII, respectively. The K248Q/R393A double mutation of SHM2 was generated by inserting the BglII-XbaI fragment of pYY67 into pYY71 to create pYY75.
To epitope tag SHM2 and SHM2 (K248Q + K393A), the 5'-terminal SalI fragment in pYY45 and pYY75, respectively, was replaced by a fragment amplified with the oligonucleotides 5'-CCAAGGTCGACGAAGGCT-3' and 5'-ACGCGTCGACTTATCTAGACACAGCCAATGGGTATTCG-3' that generated an in-frame XbaI site in front of the SHM2 stop codon (pYY69 and pYY79, respectively). A triple c-Myc tag from C3003 (gift from Pascal Chartrand, University of Montreal, Montreal, Canada) was subcloned into these XbaI sites generating SHM2-myc (pYY70) and SHM2 (K248Q/K393A)-myc (pYY80), respectively.
GAL4 DNA binding (GBD) and activation domain (GAD) fusion proteins were
constructed by subcloning the respective amplified proteins and/or fragments
thereof into pAS2 and pACT2 (Clontech Laboratories, Inc., Palo Alto, CA).
Thus, the following constructs were generated: GBD-Srp40p (pYY52),
GBD-Shm2p (pYY54), GAD-Srp40p (pYY51), GAD-Shm2p
(pYY53), GAD-NoppN (amino acids 60–704 of Nopp140,
pTM64), GAD-NoppR (60–241, pTM81), and
GAD-NoppR (60–94, pTM83). The other Nopp140
constructs were as described previously
(14). GAD-Lsm5p
(pAEM70) was a gift from Jean Beggs
(28).
RNA Analysis—Srp40p and Cbf5p depletion experiments were performed essentially as described (8, 11). Briefly, strains were grown in galactose-containing medium to mid-log phase before switching to glucose-containing medium in which they were maintained in log phase by dilution. At 0 and 24 h in glucose, total RNA was prepared (29). For Northern blotting, gel loading of RNA was adjusted to yield approximately equal quantities of U4 snRNA in each lane of 8% polyacrylamide gels. SnoRNAs were detected by hybridization with the following 32P-labeled oligonucleotides: snR10, 5'-CATGGGTCAAGAACGCCCCGGAGGGG-3'; snR30, 5'-GCCGTTGTCCGAAGCGCC-3'; snR42, 5'-TCAAACAATAGGCTCCCTAAAGCATCACAA-3'; snR190, 5'-CGAGGAAAGAAGAGACACCATTATC-3'; U24, 5'-ATTGGTATGTCTCATTCGGATCTCAAAGTTCCATCTGA-3'; U4, 5'-CGAATCCTCACTGATATG-3'; and those published previously (11). Blots were quantitated using a PhosphorImager (Amersham Biosciences).
Immunochemical Methods and Microscopy—Indirect immunofluorescence microscopy was performed essentially as described (17, 30) using mouse monoclonal antibodies 9E10 (31) against the Myc epitope (culture supernatant at a 1:3 dilution). Total protein extracts were prepared according to standard methods (32), separated by 9% PAGE, and transferred to nitrocellulose. The blots were probed with 12CA5 anti-HA ascites fluid (33) at a 1:5000 dilution.
For cell size analysis, strains were grown in SC media to log phase. The pelleted cells were resuspended in 0.1 M potassium phosphate buffer by sonication in a water bath sonicator for 5 min. The cells were fixed with 3.7% formaldehyde and observed on an inverted Nikon microscope equipped with a JVC KY55B color CCD camera. Cell size was determined manually on digitized images by measuring the maximal diagonal length of at least 100 cells using NIH Image software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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deletion strain to identify genes that were functionally related to
SRP40. For this purpose, we used a colony-sectoring approach looking
for colonies whose growth depended on the presence of an SRP40 ADE3
URA3 plasmid, which turned the colonies uniformly red and rendered them
inviable on medium containing 5-FOA
(34). In two independent
screens, the srp40 strain was mutagenized either by random
lacZ LEU2 insertions through transformation with a mutagenized
genomic yeast library (26) or
by exposure to EMS. Although 100,000 and 177,000 mutagenized colonies were
screened, respectively, only one each contained a mutation in a single gene
that conferred lethality in conjunction with the srp40
deletion. After plasmid rescue, sequencing from the inserted lacZ
gene identified the disrupted gene as cytosolic serine
hydroxymethyltransferase, SHM2, formerly referred to as LES2
(lethal with srp40; see Ref.
11). The insertion occurred at
amino acid 119 out of 469 for the full-length protein and generated a stop
codon 5 amino acids downstream. The EMS mutagenized gene was identified by
complementation with a yeast genomic library as the only predicted full-length
open reading frame in a 3.8-kb insert corresponding to SHM2. Indeed,
SHM2 and SRP40 restored growth to the synthetic lethal
strain (srp40 shm2 pSRP40 URA3) on 5-FOA-containing
medium when provided under their own promoters on LEU2 plasmids
(Fig. 1A). Thus both
screens identified one and the same gene, SHM2.
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Cytosolic serine hydroxymethyltransferase converts THF into 5,10-methylene
THF by transfer of a hydroxymethyl group from serine while liberating glycine
and water (Fig. 1B).
5,10-Methylene THF is the major one-carbon source for methionine, thymidylate,
and purine synthesis. Although identified in two independent screens, no
connection between this cytoplasmic housekeeping gene and the nucleolar
SRP40 was apparent. To confirm further the synthetic lethal
relationship between these two genes, therefore, we deleted one of the genomic
copies of SHM2 by replacement with the URA3 gene in an
SRP40-deleted diploid strain
(srp40/srp40). Sporulation and tetrad
dissection of the resulting shm2/SHM2
srp40/srp40 strain yielded only two viable spores
each, all uracil auxotroph, confirming the synthetic lethality
(Fig. 1C).
5,10-Methylene THF, the product of Shm2p, can also be generated from formate and THF by the cytoplasmic trifunctional C1-THF synthase, Ade3p (Fig. 1B). Coincidentally, both our synthetic lethal strains were ade3 mutants as required for screening with the colony sectoring assay (34). Therefore, we tested if ADE3 also was involved in the synthetic lethal phenotype. Indeed, like SRP40 and SHM2, ADE3 restored growth to the synthetic lethal strain on 5-FOA-containing medium when provided on a LEU2 plasmid (Fig. 1A). To corroborate this finding, we deleted both genomic copies of SRP40 and SHM2 in an ADE3 wild type diploid strain. This strain exhibited no major growth defects. However, after deletion of one of the genomic copies of ADE3 from this strain, sporulation and tetrad dissection yielded only two viable spores in each case, confirming the participation of ADE3 in the synthetic lethal phenotype (Fig. 1D). Thus, our synthetic lethal strains exhibit triple synthetic lethality between SRP40, SHM2, and ADE3.
A Noncatalytic Function for SHM2—The surprising identification of two major cytosolic enzymes involved in one-carbon metabolism in a synthetic lethal relationship with a nucleolar chaperone suggested that the metabolites could be part of this genetic interaction. We addressed this possibility in two ways, by the complementation of the lethal phenotype by addition of either metabolites or catalytically inactive SHM2. Addition of increased concentrations of serine, glycine, or methionine to the 5-FOA-containing medium failed to rescue growth of the synthetic lethal strain (Fig. 2A). Additionally all media were supplemented with adenine. These data suggested that the growth defect was not caused by a simple lack of metabolites.
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To inactivate Shm2p, we targeted two evolutionary conserved amino acid
residues required for internal aldimine formation with its cofactor pyridoxal
phosphate and for binding of the substrate carboxyl group. Specifically, the
lysine at position 248 of Shm2p was mutated to a glutamine (K248Q) and
arginine 393 to alanine (R393A), individually and combined. Mutation of each
of the corresponding residues in the E. coli enzyme alone abolished
its activity with no apparent impact on its tertiary structure
(35,
36). To confirm the catalytic
inactivation of the yeast enzyme by these point mutations, their ability was
tested to complement a strain rendered methionine-auxotrophic by disruption of
the genomic copies of SHM2 and ADE3
(37). Although wild type
SHM2 supported growth of the shm2 ade3
strain on medium lacking methionine, the single (K248Q and R393A) and double
(K248Q/R393A) point mutants failed to confer growth strongly suggesting that
they were catalytically inactive (Fig.
2C). Surprisingly, when these apparently inactive
constructs were transformed into the synthetic lethal strain, they, like wild
type SHM2, complemented growth on 5-FOA-containing medium
(Fig. 2D). However, a
construct truncated at amino acid 203, SHM2C, failed
to restore growth in both cases demonstrating the specificity of the effect of
the point mutations (Fig. 2, C and
D). We conclude that a block in folate-mediated
one-carbon metabolism did not cause the synthetic lethality and, consequently,
that Shm2p serves two cellular functions, a catalytic and a noncatalytic one.
These findings further imply that Ade3p too may harbor an additional,
noncatalytic function linking it in some manner to Srp40p and Shm2p.
Effects of Shm2p Expression on Localization and Cell Size—
Shm2p has been characterized as a cytoplasmic protein, mainly based on the
subcellular fractionation of its orthologs in other species. The unexpected
synthetic lethal relationship of Shm2p with the nucleolar Srp40p led us to
reevaluate its localization. In particular, it was interesting to examine if
the catalytically inactive Shm2p, freed of its predicted cytoplasmic duty,
localized to the nucleolus. For this purpose, Myc-tagged copies of
SHM2 or SHM2 (K248Q/R393A) were transformed into an
shm2 strain and observed by indirect immunofluorescence
(Fig. 2, E and
F). Both constructs were situated in the cytoplasm and
appeared excluded from nuclei (left panels), which were identified by
DNA staining with 4,6-diamidino-2-phenylindole (right panels).
Although, these data did not exclude the possibility of a minor fraction of
wild type or mutant Shm2p making its way into nuclei, it demonstrated the same
localization for both constructs, which was clearly distinct from that of the
nucleolar Srp40p (11).
Although the localization of the wild type and the catalytically inactive
Shm2p appeared identical, we noted a significant increase in the size of the
cells expressing mutant Shm2p (Fig. 2,
compare E and F). This striking phenotype was
investigated by quantitative analysis of cell size in images of
logarithmically growing strains (Fig.
3). First, the effect of the genetic background of the strains
with respect to the three synthetic lethal genes was tested. As recently
reported (38), single
disruption of SHM2, SRP40, and ADE3 had no effect on cell
size (not shown). However, double disruption of SHM2 and
ADE3 caused a near doubling in cell size
(Fig. 3, compare A to
B) whereas disruption of SHM2 in conjunction
with SRP40 had no effect (Table
II). Because cell diameter is reported here
(Table II), cell volume
actually increased 7–8-fold. The size increase was unlikely caused by
changes in osmotic behavior because it even occurred in the presence of 1
M sorbitol (not shown). Exogenous expression of wild type Shm2p
rescued the giant cell phenotype of the shm2 ade3
strain, whereas that of the catalytically inactive Shm2p (K248Q/R393A)
increased the cell size from 200 to 250% of wild type
(Fig. 3, C and
D; Table
II). In fact, when compared with vector control, expression of
mutant Shm2p (K248Q/R393A) consistently and significantly enlarged cell size
in a variety of strains (Table
II). Thus, the exaggeration of the giant cell phenotype was
apparently caused by a catalysis-independent function of this basic metabolic
enzyme. Additionally, the impact on cell size of the mutant Shm2p
(K248Q/R393A) was not based on a dominant negative effect because it occurred
even in the absence of wild type Shm2p, i.e. in shm2
strains (Fig. 3D;
Table II). To test if
SRP40 played a role in the giant cell phenotype, cell size of the
synthetic lethal strain was analyzed when SRP40 on plasmids was
expressed from its own promoter and in addition from the GAL10
promoter. Although the former situation was meant to mimic the SRP40
shm2 ade3 strain, its cell size was not increased
(Fig. 3B and
Table II). However,
overexpression of Srp40p in that background significantly enlarged the cells
suggesting its participation in the phenotype
(Table II). These data,
together with the fact that the catalytically inactive Shm2p (K248Q/R393A)
increased cell size and rescued the synthetic lethality, were consistent with
similar signaling pathways controlling both cell size and synthetic lethal
phenotypes.
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LSM5 Is a Multicopy Suppressor of the Triple Synthetic
Lethality—A genome-wide two-hybrid analysis revealed an interaction
between Shm2p and Lsm5p (39).
As part of Sm-like complexes, Lsm5p has been implicated in several nuclear
activities including normal processing of pre-tRNAs
(40). Pre-tRNAs, unlike mature
tRNAs, are often localized to nucleoli through a poorly understood process
involving Cbf5p, one of the four core proteins of box H/ACA snoRNPs
(8,
41). Therefore, Shm2p, via
Lsm5p, may display some distant relationship to box H/ACA snoRNAs, which are
specifically depleted in the synthetic lethal strain
(11). For these reasons, we
tested if overexpression of Lsm5p rescued the srp40 shm2
ade3 synthetic lethality on 5-FOA-containing medium. Indeed, providing
additional copies of LSM5 under the ADH1 promoter on a
multicopy plasmid partially restored growth on solid medium
(Fig. 4A). This was
consistent with the reported Shm2p-Lsm5p interaction between full-length Lsm5p
and the carboxyl terminus of Shm2p
(39), although the two
full-length proteins failed to interact in a two-hybrid assay (data not
shown).
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Nevertheless, we further investigated the LSM5-SHM2 connection in
our srp40 shm2 ade3 strain rendered conditional for
growth on galactose by carrying a plasmid copy of
GAL::srp40. Indeed, extra copies of LSM5 partially
restored growth to this strain on glucose
(Fig. 4B). As reported
previously (11), growth arrest
of this conditional SRP40 strain was at least partially caused by the
specific depletion of box H/ACA snoRNAs, because after 24 h snR3, snR10,
snR11, snR42, and the essential snR30 were depleted (Fig.
4C and
5C, lanes 4).
In contrast, the stability of the box C/D snoRNAs U3, U14, U24, and the
spliceosomal snRNA U4 was unaffected (Fig.
4C, lane 4). Overexpression of LSM5 in
this GAL::srp40 strain in glucose-containing liquid medium
stabilized the tested box H/ACA snoRNAs snR3 and snR10
(Fig. 4C, lane
6). Restoration of growth under these conditions contrasted that of the
synthetic lethal strain with the genomic copy of SRP40 deleted,
growth of which in liquid medium was not rescued by LSM5
overexpression (data not shown). This apparent discrepancy is most likely
explained by residual expression of minute amounts of Srp40p in the
GAL::srp40 strain, even in the presence of glucose, as we
observed previously (11) for a
GAL::cbf5 strain. Nevertheless, these results supported a
genetic link between Lsm5p and box H/ACA snoRNPs.
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Rat Nopp140 Is a Functional Homolog of Yeast Srp40p— Based
on sequence comparison and nucleolar localization, we previously established
that yeast Srp40p was the closest homolog to rat Nopp140
(17). The generation of a
yeast strain conditional for SRP40 allowed us to test if Nopp140 also
was a functional homolog. For this purpose, we tested if Nopp140 rescued
growth on glucose of our srp40 shm2 ade3 strain
carrying a plasmid copy of GAL::srp40. Indeed, Nopp140
restored growth unlike a vector control
(Fig. 5A). Nopp140
consists of three major domains, the unique amino and carboxyl termini
separated by the signature central repeat domain containing 11 half-acidic and
half-basic repeats (Fig.
5B). The carboxyl terminus is most closely related to
yeast Srp40p with 59% sequence identity between the last 50 amino acids,
whereas the repeat domain is structurally related to the rest of Srp40p
(17). Surprisingly, deletion
of the conserved carboxyl terminus had no effect on the ability of Nopp140 to
restore growth on glucose (Fig.
5A, NoppC). Similarly, a
deletion of the amino terminus did not impair the complementation ability of
Nopp140 (NoppN), and the amino terminus alone appeared insufficient for
complementation, although it is uncertain to what extent the latter construct
was expressed (Fig. 5, A and
C, NoppN). Furthermore, the conserved carboxyl
terminus of Nopp140 alone failed to complement, whereas the repeat domain
alone fully restored growth on glucose
(Fig. 5A,
NoppC and NoppR). Even the first four Nopp140 repeats alone
were sufficient to fully restore growth, whereas a single repeat was less
efficient (NoppR and NoppR, respectively). To ensure that
these results were not a mere reflection of expression levels of the
heterologous proteins in yeast, their expression was verified by Western
blotting. With the exception of NoppN, all constructs expressed detectable
amounts of proteins that migrated at their expected positions
(Fig. 5C). Although
the expression levels varied, they were unrelated to the ability of the
constructs to complement growth on glucose
(Fig. 5, compare A and
C).
Depletion of Srp40p by growth in glucose-containing medium led to a specific loss of box H/ACA but not C/D snoRNAs (11). To test if, like the growth defect, this phenotype was complemented by Nopp140 or its repeat domain, Northern blots of total RNA from the corresponding strains were probed for small nuclear RNAs (Fig. 5D). The box H/ACA snoRNAs snR3, snR10, snR11, and snR42 were depleted after 24 h of growth in glucose-containing medium in the presence of vector control, whereas the box C/D snRNAs U3, U14, and snR190 and the spliceosomal snRNA U4 remained unaffected (Fig. 5D, lane 4). Complementation with full-length Nopp140 restored the levels of all RNAs tested (Fig. 5D, lane 6). However, despite complementation of growth on solid medium (Fig. 5A), in liquid medium the repeat domain alone barely restored growth (not shown) and only marginally stabilized box H/ACA snoRNAs (Fig. 5D, lane 8). Nevertheless, these data suggest that Nopp140 can functionally replace Srp40p and that this occurs mostly via its repeat domain but not its conserved carboxyl terminus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Shm2p has been highly conserved throughout evolution and has a well
established role in one-carbon metabolism, in addition, our studies now
suggest that it acquired a noncatalytic function linking it to ribosome
biogenesis. The point mutations selected for inactivation of Shm2p were based
on mutations previously shown to abolish serine hydroxymethyltransferase
activity in the bacterial ortholog
(35,
36). They affect its substrate
carboxyl-binding site and the internal aldimine formation with its prosthetic
group pyridoxal phosphate. These residues have been conserved from bacteria to
man at the amino acid level as well as in their position within the crystal
structure (43,
44). The lack of
complementation of the methionine auxotrophy of an shm2
ade3 strain by Shm2p with the equivalent mutations (K248Q/R393A)
suggested that these positions in the yeast enzyme serve the same functions
and that their mutation indeed inactivated the enzyme. Therefore, the
complementation of the synthetic lethality by the mutant enzyme was likely
caused by an additional catalysis-independent function of the protein.
A function independent of amino acid and folate substrate binding has also been reported for the human Shm2p ortholog (45). Thus, human cytoplasmic serine hydroxymethyltransferase inhibits translation of its own mRNA by binding to the 5'-untranslated region. Such an activity has not been documented for yeast Shm2p but illustrates that this metabolic enzyme can perform other functions. Although we did not test if enzymatically impaired Ade3p would rescue the synthetic lethality, the results with mutant Shm2p and the lack of complementation by metabolites suggest that it too might exhibit a noncatalytic function. In fact, Ade3p plays a noncatalytic role in de novo purine biosynthesis (46). Thus, Shm2p and Ade3p may display a nonmetabolic interaction, which may be indirect and which may link them both to Srp40p. What exactly constitutes the noncatalytic function of Shm2p remains to be determined, but insight might be gained from the analysis of interacting proteins such as Lsm5p.
We identified Lsm5p as a candidate communicator between Shm2p and Srp40p. Apparently, the latter two proteins did not interact directly because we failed to detect any physical interaction in two-hybrid and coimmunoprecipitation assays (data not shown). This was supported by the localization of the two proteins in separate cellular compartments. Lsm5p, however, was present in both cytoplasmic and nuclear complexes and interacted with Shm2p in a genome-wide two-hybrid screen (28, 39, 47, 48). Indeed overexpression of LSM5 stabilized box H/ACA snoRNAs, which were specifically depleted from the synthetic lethal strain, and presumably thereby rescued the synthetic lethality. Although depletion of Lsm proteins affects the stability of pre-rRNA and rRNAs, it does not impact snoRNAs (49). Therefore, the effect of LSM5 overexpression on the stability of box H/ACA snoRNAs was likely indirect and could have been mediated via Srp40p, thus closing the chain between SHM2 and SRP40.
In addition to the cytosolic serine hydroxymethyltransferase, eukaryotes contain a mitochondrial enzyme that is about 60% identical, encoded by SHM1 in yeast (50–52). Shm1p is part of a parallel system for interconversion of one-carbon units in mitochondria. Due to its sequestration in mitochondria, it is unlikely that Shm1p participates in the synthetic lethality. Indeed all strains used in this study contained a wild type copy of SHM1. Because Shm1p can contribute to the cellular need of one-carbon units in the absence of Shm2p, these data further support our observation that the synthetic lethality is not based on impaired metabolism but on a defunct noncatalytic role of Shm2p (37, 53, 54).
The synthetic lethality in our strains strictly required the simultaneous disruption of all three genes, SRP40, SHM2, and ADE3. Disruption of SHM2 together with ADE3 impaired growth on medium lacking methionine. These findings are in conflict with recent results showing synthetic lethality between shm2 and ade3 alone (55). This discrepancy could be explained by different genetic backgrounds of the strains used in these studies. Regardless, our studies are consistent with at least two previous reports (37, 54) showing that shm2 ade3 strains are methionine auxotrophs.
The most striking phenotype of the shm2 ade3
strain was its increase in cell size. This phenotype was reversed by
expression of wild type Shm2p but exaggerated by catalytically inactive Shm2p
suggesting a role for Shm2p in cell growth. Based on these observations, the
effect of Shm2p was apparently catalysis-independent and not due to a dominant
negative mechanism. It is interesting to note that mammalian serine
hydroxymethyltransferases were recently identified as Myc targets in the
regulation of cell growth bolstering the role of these enzymes in this
signaling pathway (56). One
potential mechanism for the regulation of cell size during growth is the
control of ribosome biogenesis
(57,
58). Srp40p, through snoRNPs,
could provide a link between Shm2p, ribosome biogenesis, and consequently cell
size. This is consistent with the fact that the same Shm2p mutant increased
cell size and rescued the triple synthetic lethality. Additionally,
overexpression of SRP40 increased cell size in the triple synthetic
lethal background suggesting a role for Srp40p in the regulation of cell size.
Although these connections are highly speculative, they are tantalizing.
Further investigation is required to sort out which, if any, of these
interactions are involved in the control of cell size.
The synthetic lethal strain provided us for the first time with the opportunity to engineer an SRP40 conditional strain by placing its expression under the conditional GAL promoter. We had previously taken advantage of this fact to demonstrate that Srp40p depletion in the synthetic lethal background leads to the specific depletion of box H/ACA but not box C/D snoRNAs. Because box H/ACA snoRNA snR30 is essential for rRNA processing and consequently viability of yeast (59), its depletion is the likely cause for growth arrest after Srp40p depletion. Indeed, depletion of Srp40p caused a defect in early pre-rRNA cleavages at sites A0, A1, and A22 that is characteristic for the depletion of box H/ACA snoRNAs (8, 59, 60).
Growth and stability of box H/ACA snoRNAs of the conditional SRP40 strain could be restored by expression of rat Nopp140 suggesting that it is indeed the functional homolog of Srp40p. It was the central repeat domain of Nopp140 and not its evolutionary most highly conserved carboxyl terminus, which complemented the Srp40p depletion. Although surprising at first, this may reflect the capacity of the repeat domain to interact with box H/ACA snoRNPs in a phosphorylation-dependent manner (14, 15). Complementation of growth by the repeat domain was strong on solid but only partial in liquid medium. This is reminiscent of the fact that yeast only when grown on solid medium exhibits a nucleolar body, which contains Srp40p, snoRNAs, and the cap methylase Tgs1p (20, 61). This nucleolar body, which may be related to the mammalian Cajal body, is lost in an srp40 null background. Therefore, it is interesting to speculate that the nucleolar body may be involved in the NoppR complementation mechanism. Whether Nopp140 in mammalian cells also exhibits a connection to cytosolic serine hydroxymethyltransferase awaits further experimentation.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Anatomy and Structural
Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY
10461. Tel.: 718-430-3294; Fax: 718-430-8996; E-mail:
meier{at}aecom.yu.edu.
1 The abbreviations used are: snoRNA, small nucleolar RNA; 5-FOA,
5-fluoroorotic acid; EMS, ethylmethane sulfonate; GAD, GAL4 activation domain;
GBD, GAL4 DNA binding domain; SC, synthetic complete; snoRNP, small nucleolar
ribonucleoprotein particle; THF, tetrahydrofolate; snRNA, small nuclear RNA;
HA, hemagglutinin.
2 D. Lafontaine, personal communication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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