Mol. Cells, Vol. 6, No. 5, pp. 615-621
Translational Control of the SPP2 Gene Expression in
Saccharomyces cerevisiae
Hyunsic Choi, Kwanghark Park, Donghwa Kim, Chang-Jin Lim and Kyunghoon Kim*
Division of Life Sciences, College of Natural Sciences, Kangwon National University,
Chunchon 200-701, Korea
(Received on August 28, 1996)
There are four AUG codons in two short open reading frames within 54 bases upstream
of the initiation codon of the SPP2 gene, which is involved in pre-mRNA splicing of the
budding yeast Saccharomyces cerevisiae. Fifteen different combinations of the four AUG
codons were mutated to other codons. Then, SPP2-1acZ gene fusions retaining a various
number of the AUGs were constructed, and the activities of ~-galactosidase expressed
from the fused genes were measured in yeast. The ~-galactosidase activities from the mutated fusions were higher than that from the wild type fusion: the more mutated AUG codons, the higher the enzyme activities. This result strongly suggests that the AUG codons
present on the 51 leader region of the SPP2 transcript are cis-acting regulatory elements
for SPP2 gene expression on a translational level.
Primary transcripts of most eukaryotic genes contain nontranslated introns as well as translated exons
(Green, 1991; Newman, 1994). Thus pre-mRNA splicing, in which introns are removed from nuclear
mRNA precursors (pre-mRNAs) and remaining exons
are spliced accurately, is essential for eukaryotic gene
expression. The pre-mRNA splicing involves two consecutive trans-esterification reactions and takes place
in a multimolecular complex called the spliceosome.
At least four small nuclear ribonucleoproteins (U1, U
2, U4/U6, and U5 snRNPs) and the pre-mRNA are assembled into a spliceosome through an ordered multistep pathway requiring ATP and additional extrinsic
factors (Cheng and Abelson, 1987).
In the budding yeast Saccharomyces cerevisiae, almost 40 PRP (pre-mRNA processing) gene products
have been identified to be involved in pre-mRNA
splicing (Maddock et al. , 1996). Many of the PRP
gene products are protein components of snRNPs in
the spliceosome or are involved in the assembly and
disassembly of the spliceosome (Abovich et ai, 1990;
Banroques and Abelson, 1989; Chang et aI., 1988;
Company et aI., 1991; Kao and Siliciano, 1996; Lossky et ai., 1987; Tam et al., 1993). Five of the PRP
products, however, have been identified as a family of
putative ATP-dependent RNA helicase, termed DEAD!
H box proteins (Burgess et aI., 1990; Chen and Lin,
1990; Company et ai., 1991; Dalbadie-McFarland and
Abelson, 1990; Schwer and Guthrie, 1991). Prp5p and
Prp28p contain the DEAD motif, while Prp2p, Prp16p,
and Prp22p contain the DEAR one (Schmid and Linder, 1992). Prp2p, Prp16p and Prp22p share additional
* To whom correspondence should be addressed.
sequence similarities in their carboxy termini (Chen
and Lin, 1990; Company et al., 1991).
The SPP2 (suppressor of pre-mRNA processing)
gene had been isolated as a multicopy suppressor of
temperature-sensitive prp2 mutations (Last et al.,
1987). It is essential for cell viability and its gene product is involved in pre-mRNA splicing in vivo and in
vitro (Roy et al., 1995). The Spp2p interacts with the
Prp2p and promotes the first trans-esterification reaction (Roy et aI. , 1995). There are four AUG codons
in two short open reading frames within 54 bases
upstream of the initiation codon of the SPP2 gene. In
eukaryotes, a ribosome scans for the first AUG from
the 51 cap of mRNA instead of using a ribosome binding site for initiation (Kozak, 1989). Thus the four
AUG codons may act as titrators of scanning ribosomes to inhibit the translation of the SPP2 message.
In this study, we show that these AUG codons are
indeed cis-acting regulatory elements for SPP2 gene
expression on a translational level: the more AUG codons are changed to other codons, the higher translations of the SPP2 gene are achieved.
Materials and Methods
Strains and plasmids
The Escherichia coli strains, Saccharomyces cerevisiae strains, and plasrnids used in this study are listed
in Table 1. The E. coli strains HB101 and JM109
The abbreviations used are: BPB, bromphenol bule; EDTA,
ethylenediaminetetraacetic acid; ONPG, o-nitrophenyl galactopyranoside; PIPES, piperazine-N,NI -bis(2-ethanesulfonic
acid.
.
@ 1996 The Korean Society for Molecular Biology
Mol. Cells
Translational Control of the Yeast SPP2 Gene
616
were routinely grown in Luria-Bertarti broth (LB broth:
1 % tryptone, 0.5 % yeast extract, 0.5% NaCl). E. coli
BMH71-18 was used for site-directed mutagenesis
(Promega). M9 minimal medium was used for the
q
maintenance of F'(traD36 proA +B + lacI lacZL1M15)
within the JM109 (Miller, 1972). Ampicillin (50 mgll)
was added to the LB media, if necessary. S. cerevisiae
strains were cultivated in YEPD (1 % yeast extract, 2%
peptone, 2% glucose), or synthetic complete broth
without a particular amino acid or base (Fink and
Hicks, 1982). For solid and soft agar media, 1.5% and
0.7% agar were included, respectively.
et al. (1972) and Ito et al. (1983), respectively.
Isolation of yeast total RNA
Yeast total RNA was prepared by the method of
Hereford and Rosbash (1977).
S1 mapping
Manipulation of plasmid DNA
Plasmid DNA was prepared from overnight culture
of E. coli by the boiling method of Holmes and Quigley (1981), or by the large scale alkaline extraction
method of Marko et al. (1982). Single-stranded
phagemid DNAs were prepared according to Schreier
and Cortese (1979), and used for DNA sequencing or
site-directed mutagenesis. Various restriction endonucleases, T4 DNA ligase, and Sequenase T7
DNA polymerase were purchased from Boehringer
Mannheirn or United States Biochemical. Enzyme
reactions were performed according to the supplier' s
instructions. Agarose or polyacrylamide gel electrophoresis of DNA was performed by the procedure
of Sambrook et al. (1989). For DNA size markers,
HindIII-digested lambda phage DNA (Allet and Bukhari, 1975) · and Hinfl-digested pBR322 DNA
(Sutcliffe, 1978) were used. Restriction fragments of
plasmid DNA were eluted from agarose gels (Dretzen
et al., 1981).
Sl mapping of the 5' terminal nucleotide of the
SPP2 transcript was performed by the procedure of
Sambrook et al. (1989). An SspI restriction fragment
spanning - 185 bases and +48 bases from the SPP2
initiation codon (Fig. 1) was treated with y_p 32 _ATP
and polynucleotide kinase to label the 5' end of the
5
DNA. This fragment (5 ng, 5 X 10 CPM) and yeast
cellular RNA (0.2 mg) were dissolved in 10 /-ll hybridization solution (0.4 M NaCl, 40 mM PIPES, 1
mM EDTA, pH 6.4). In order to make DNA-RNA
hybrids, the mixture was incubated for 3 min at 100
°c, for 60 min at 65°C, and for 60 min at 37 °c . 200
/-ll of Sl buffer (0.25 M NaCl, 30 mM sodium acetate, 1 mM ZnS0 4 , 0.002% salmon sperm DNA, 5%
glycerol, pH 4 .6) and 280 units of Sl endonuclease
were then added to the mixture. After 60 min at 37
°c, 10 /-ll of Sl stop solution (0.25 M EDTA, pH 8.0)
was added to the mixture. Sl-protected DNA was precipitated with ethanol, resuspended in 15 /-ll of Sl
loading buffer (80% formamide, 0.01 N NaOH,
1 mM EDTA, 0.025 % BPB, 0.025 % xylene cyanole),
and incubated for 3 min at 100 °C. The DNA was
subjected to a 7% urea polyacrylamide gel electrophoresis, and visualized by autoradiography. Appropriately prepared sequencing ladders were used as
size markers.
Transformation
E. coli and S. cereVlszae were transformed with
plasmid DNA according to the procedures of Cohen .
Mutagenic oligonucleotides
Mutagenic oligonucleotides were designed to have
only one mismatch in the middle of 25 bases com-
Table 1. Strains and plasmids used in this study
Strains and plasmids
Escherichia coli
HE101
JM109
BMH71-18 mutS
Saccharomyces cerevisiae
KY106
RL92
Plasmids
pALTER-1
YEp357R
pJDB207-SPP2
pALTER -1-SPP2
pALTER-1-SPP2'
Relevant properties
hsdS hsdM supE44 ara14 galK2 lacYl
proA2 rpsL20 xyl-5 mtl-l recA13
endAl recAl gyrA96 thi hsdR17(rk- m/ )
reLAl supE44 A- ~(lac proAB) [F' traD36
proA +B lacrZ~M15]
thi supE~(lac proAB) [mutS::Tn10]
[F' proA +B + lacrZ~M15]
Source or reference
Sarnbrook et al. (1989)
Sambrook et al. (1989)
Prom ega
a leu2 ura3 his3 trpl lys2
a prp2-l leu2 ura3
This study
Last et al. (1987)
ColE1 replicon, f1 ori, Amps, Tel'
ColE1 rep licon, 211 replicon, URA3
ColE1 replicon, 211 replicon, LEU2
ColE1 replicon, f1 ori, Amps, Tet',
No mutaion in the SPP2 DNA
ColE1 replicon, f1 ori, Amp', Tel',
A mutation in the SPP2 DNA
Promega
Myers et al. (1986)
Roy et al. (1995)
This study
This study
Vol. 6 (1996)
Hyunsic Choi et al.
617
plementary to a region around the AUGs present at
- 54, - 40, - 28, and - 7 bases, respectively,
upstream of the SPP2 initiation codon. The base sequence of the mutagenic oligonucleotide I complementary to a region at the - 54 base is 5'GCfCGACGTGCAATATTACCITAAT-3'. This sequence is for a mutation of ATG to TTG. That will
also create a new SspI restriction site for easier screening of the mutation. The base sequence of the mutagenic oligonucleotide II complementary to a region
at the - 40 base is 5'-CATTTTACCfACGATCGCTCGACGT-3'. This would not only cause a mutation
of ATG to ATC, but also create a new PvuI restriction site for easier screening of the mutation. Sequence of the mutagenic oligonucleotide ill complementary to a region at the - 28 base is 5'-ATGCCTCTAGACGTITTACCTACCA-3'. This would cause
a mutation of ATG to ATe. Sequence of the mutagenic oligonucleotide IV complementary to a region
at the - 7 base is 5'-TTGCTCATTCTAGATCCTAGATATG-3'. This would cause a mutation of ATG to
ATC and create a Sau3AI restriction site for easier
screening of the mutation.
phorylated with ATP and T4 polynucleotide kinase.
One /-11 (4 pmol) of the phosphorylated oligonucleotide, 1 /-11 (0.05 pmol) of single-stranded
phagemid template DNA, 1 /-11 (0.25 pmol) of ampicillin repair oligonucleotide, 2 /-11 of 10 X annealing
buffer (0.5 M NaCl, 0.1 M MgCI2, 0.2 M Tris-HCI,
pH 7.5), and 15 /-11 of distilled water were mixed and
incubated for 5 min at 70 °C. The mixture was incubated further for 15 min at room temperature and
then put on ice. To this annealing mixture, 3 /-11 of extension buffer (5 mM dNTPS' 10 mM ATP' 20 mM
DTT, 0.1 M Tris-HC1, pH 7.5), 5 /-11 of distilled water, 1 /-11 of T4 DNA polymerase, and 1 /-11 of T4 DNA
ligase were added, and the mixture was incubated for
2 h at 37°C . The reaction mixture was used to
transform a mismatch correction deficient E. coli
BMH71-18 to ampicillin resistance. Plasmid DNA
was isolated from the ampicillin resistant transformants and introduced into E. coli JM109. Singlestranded phagemid DNA was isolated from the ampicillin resistant JM109 transformants and their base
sequences were determined to confirm the mutations.
Site-directed mutagenesis
Site-directed mutagenesis was performed by using
an in vitro Mutagenesis System kit purchased from
Promega. First, a mutagenic oligonucletide was phos-
DNA sequencing
Sequenase sequencing protocol (Kraft et a!., 1988)
of United States Biochemical was employed to determine DNA base sequences by the dideoxy chain
termination method (Sanger et at., 1977).
5'
~~¢.t1PACATGATACTGACCATAGTACATGTGCAAGTTATTCAAACTAAGAATTACAG
HindIII
- 238
AGCTTCATTTGTCACAGTCACTTCATACTACTATGCTTCTAATGCTTCT~ijta1ITATGCATTGCAGGTTCAACATGCA - 159
Ssp!
ACACTTGGGTTCTCAATGATCTGGCGCTATCATTTGTTACCCGACCGTGCGTAGAAAAAAATGTAAAAATATAGGAATA -80
-54
-40
-28
-7
GCTGTTTTAAGTTATTAAGGTAATAltu.cACGTCGAGCGltUGTAGGTAAA~tiTCTAGAGGCATATCTAGGij~AGA
-1
MetHisValGluArgTrp
MetValGIyLysMetSerArgGlylleSerArgMet
SspJ
ATG AGC AAG TTT TCA CTT AAG TTG GGG AGT AAG ACC TTG AAA AAA iA"tti\Jm TCC AAG AAG
Met Ser Lys Phe Ser Leu Lys Leu Gly Ser Lys Thr Leu Lys Lys Asn lIe Ser Lys Lys
60
20
ACG AAA AAA AAA AAC TCA TTA CAG AAG GCC AAC TTA TTC GAT TGG GAT GAT GCG GAA ACT
Thr Lys Lys Lys Asn Ser Leu GIn Lys Ala Asn Leu Phe Asp Trp Asp Asp Ala Glu Thr
120
40
GCA AGC CTT TCG CAT AAA CCT CAA AGT AAG ATA AAG ATT CAA AGT ATT GAT AAA TTC GAT 180
Ala Ser Leu Ser His Lys Pro GIn Ser Lys lIe Lys lIe GIn Ser lIe Asp Lys Phe Asp 60
CTT GAT GAA GAA TCA TCT TCT AAG AAG AAA TTA GTG ATC AAG CTG AGT .GAA AAT GCG GAT 240
Leu Asp Glu Glu Ser Ser Ser Lys Lys Lys Leu Val lIe Lys Leu Ser Glu Asn Ala Asp 80
.
KpnI
ACA AAA AAA AAC GAT GCT CCT CTG GTG GAA TAT GTC ACC GAA AAA GAA TAT AAT GApt ttlW 300
Thr Lys Lys Asn Asp Ala Pro Leu Val Glu Tyr Val Thr Glu Lys Glu Tyr Asn Glu Val 100
eMf GTC GAG GAA ..... .
Pro Val Glu Glu ..... .
Figure 1. DNA base sequence of the SPP2 gene from S. cerevisiae (Roy et aI., 1995). The amino terminal part of the gene
and its 5' upstream region are shown. Restriction sites and the four ATGs are shadowed. Two short reading frames in the 5'
leader region of the SPP2 gene are indicated.
.
Translational Control of the Yeast SPP2 Gene
618
Measurement of ~-galactosidase activities
Yeast cell extracts were prepared from exponentially grown yeast cells harboring SPP2-lacZ fusion plasmids as described by Roy et al. (1995). Activity of the ~-galactosidase present on the cell extracts was measured by the method of Miller (1972).
A unit was defined as the amount of enzyme required
for the breakdown of 1 nmol of ONPG per min . Protein concentration of the cell extracts was determined
according to the procedure of Bradford (1976).
Results
S1 mapping of the SPP2 mRNA
Four AUG codons were present in two small open
reading frames composed of 6 and 12 codons, respectively, within 54 bases upstream of the initiation codon of the yeast SPP2 gene (Fig. 1). To see whether
the AUGs were within the SPP2 mRNA, the 5' end
point of the SPP2 transcript was determined by SI
mapping. An SspI restriction fragment spanning - 185
base to +48 base from the SPP2 initiation codon
(Fig. 1) was end-labeled, hybridized with cellular
RNA isolated from yeast cells harboring pJDB207SPP2 plasmid retaining the SPP2 gene (Table 1), dig-
GAT
C
S1
-
Figure 2. An autoradiogram of an end-labeled Ssp I fragment of the SPP2 DNA protected from S1 endonuclease.
The protected fragment indicated by an arrow is 11 2 bases
long. A set of four sequencing ladders was used as size
markers.
Mol. Cells
ested by SI nuclease, and displayed on a denaturing
polyacrylamide gel. As shown in Figure 2, one SIprotected fragment 112 bases long was detected, indicating that transcription of the SPP2 gene begins at
a point 64 bases upstream of the initiation codon .
Thus all four AUG codons are present on the 5' untranslated region of the SPP2 mRNA.
Mutagenesis of the nontranslated AUG codons
We generated a series of mutations with fifteen different combinations in the four AUG codons to see
how AUGs affect SPP2 gene expression. In short, a
HindIII-KpnI fragment of the cloned SPP2 DNA
spanning - 296 bases and + 299 bases from the SPP2
initiation codon was subcloned onto a phagemid
pALTER-l to generate a recombinant phagemid
designated pALTER-l-SPP2 (Fig. 3). Several rounds
of site-directed mutagensis were then performed with
the single-stranded phagemid DNA isloated from
pALTER-l-SPP2, a relevant mutagenic oligonucleotide and an ampicillin resistance repair oligonucleotide as shown in Figure 3 and as described in
Materials and Methods. The mutants were screened
by restriction analyses (data not shown) and DNA
base sequencing (Fig. 4).
Effects of mutations in the nontranslated AUGs on
the SPP2 expression
We investigated how mutations in nontranslated
AUGs affect SPP2 expression by measuring the activities of ~-galactosidase expressed from SPP2-lacZ
genes containing mutations in the non translated
AUGs. First, the HindIII-KpnI fragments of the fifteen different pALTER-l-SPP2' derivatives as well as
wild type pALTER-l-SPP2 were subcloned onto the
multiple cloning site of a yeast-E. coli shuttle vector
YEp357R (Myers et al., 1986) containing the URA3
marker (Fig. 3). In this way, we were able to construct 16 recombinant plasmids designated pKWl11,
pKW122, etc. (Table 2) containing a fused gene of
SPP2-lacZ in frame with a different combination of
the. four AUGs. These plasmids were introduced into
a ura3 yeast strain KY106 (Table 1) and specific activities of ~-galactos idase in crude cell extracts from
the URA3 transformants grown in a uracil-deficient
medium were measured. As shown in Table 2, when
only one of the A TGs was mutated, the ~-galac­
tosidase activity of the SPP2-lacZ fusion increased to
1.8 to 3.6 times of that of the wild type fusion. If
two of the ATGs were changed the activity increased
further 1.7- to 7.0-fold. Mutations in three or four
ATGs resulted in 3.7- to 7.8-fold increases in activity.
Th e mutations are not likely to affect the transcription of the SPP2 gene because no significant difference in the amount of the SPP2-lacZ message was
observed among the mutants (data not shown). These
results indicate that the AUG codons present on the 5'
leader region of the SPP2 transcript are cis-acting regulatory elements for SPP2 gene expression.
619
Hyunsic Choi et al.
Vol. 6 (1996)
H
/
21' \
K
YEp357R-S P P2
- LacZ
LacZ
UR~
oli(f!)
H+K
pALTER-l-SPP2
ssDNA
H.
,f1 SPP2 mutagenic
Amp' ~
primer
primer
l
YEp357R
Ii
u~
H+K
Am~
H
K
Figure 3. A schematic presentation for the construction of YEp3S7R-SPP2'-lacZ by site-directed mutagenesis and subcloning. H, HindIII; K, KpnI.
Discussion
There are four AUG co dons in two short open reading frames within 54 bases upstream of the irlitiation
codon of the SPP2 gene (Fig. 1). All of the AUGs
are within the 5' untranslated region of the SPP2
mRNA, because transcription of the SPP2 gene begins at a point 64 bases upstream of the initiation codon (Fig. 2). In E. coli, binding of a ribosome to a ri-
bosome-binding site in an mRNA is prereqUIsIte for
an efficient translation of the mRNA (Shine and Dalgarno, 1974). Instead of using a ribosome-binding
site for irlitiation, the 40 S small subunit of the eukaryotic ribosomes bind to the 5' cap and scan for the
first AUG where the 60 S large subunit and other initiation factors are assembled to initiate the translation
(Kozak, 1989). However, transcripts of a few eukaryotic genes contain one or few AUGs upstream of
Mol. Cells
Translational Control of the Yeast SPP2 Gene
620
(A)
, - - - - - - - SPP2 -/acZ
-64
-54
I
-7
-28
-40
tl
TAAGGTAATAATGCACGTCGAGCGATGGTAGGTAAAATGTCTAGAGGCAT..ITCTAGGATGl·AGAATG
1-
(B)
WT
T
!
T
C •
-54{ I) -40(II)
ll+N
ill+N
C
-28(ill)
------ll+ill
!
C
l+ll+ill
-7(N)
--
I+ll+N
I+ll
I +ill
I+N
GATC
GATC
GATC
I +ill+N
II +ill+N
I+ll+ill+N
---
-Figure 4. (A) DNA base sequence of the 5' untranslated leader of the SPP2 gene. Locations of four substitution mutations
are indicated by vertica l arrows. (B) Autoradiograms showing altered base sequences of the fifteen mutations retaining diffe rent combinations of the fo ur ATGs. Four horizontal arrows indicate locati ons of the mutations.
the real initiation codons (Hinnebu sch and Liebman,
1991). A s an explan ati on fo r this, it w as proposed
th at some 40 S subunits reached to the upstream
AUGs assemble with 60 S subunits and synth esize
short leader peptides, and that remaining 40 S subunits pass th e upstream AUG s and ride furth er to the
real initiation codon (Kozak, 1989). This proposal indicates that AUGs upstream of the initi ation codon are
cis-acting regul atory elements for gene expression.
Th e fo ur AUG codons might then act as titrators of
scanning ribosomes to inhibit the translation of the
SPP2 message. In fact, w hen the AUGs were mutated
to other codons, elevated expressions of SPP2-lacZ fusion were observed (T able 2). Similar findings have
been reported from other yeast genes such as CYC1
(Sherm an and Stewart, 1982) and GCN4 (Hinnebusch
and Liebman, 1991). Each of the four AUGs show ed
a di ffe rent effect on gene expression, suggesting that
the A UG codon as well as its surrounding sequence
context affects SPP2 expression (Bairn et aI. , 1985; Ci-
Vol. 6 (1996)
Hyunsic Choi et al.
Table 2. Relative ~-galactosidase activities expressed in
yeast with SPP2-lacZ genes containing mutations in the
upstream AUG codons of the SPP2. The experiments were
repeated three times and standard deviations of the relative
specific activities were calculated.
Relative ~-galac­
Upstream ATG mutations
tosidase activity
- 7
- 54 - 40 - 28
pKWlll -ATG--ATG--ATG--ATG-pKW122
pKW127
pKW142
pKW147
1.00
---ATG--ATG--ATG--ATG
ATG--ATG--ATG-ATG
ATG--ATG-ATG--ATGi-----
1.84± 0.27
2.12± 0.31
1.92±0.31
3.62± 0.22
pKW152
ATG--ATG-pKW157
ATG
ATG-pKW162
ATG--ATGi----pKW167 -ATG
ATG-pKWI72 -ATG
ATGi----pKWI77 -ATG-ATGr-------
2.26±0.59
2.28±0.91
5.18 ± 0.95
1.65 ± 0.09
5.13 ± 0.79
6.95 ± 2.11
pKW182 - - - - - - - - A T G - pKW187
ATG
pKWI92
ATG
pKW197 -ATG
5.40 ±
4.47 ±
4.49 ±
3.71 ±
pKW199
7.80 ± 2.71
2.40
1.37
1.16
0.67
gan and Donahue, 1987; Hamilton et a!., 1987; Pelletier and Sonenberg, 1985). The AUGs appear to act
as cis-acting regulatory elements for SPP2 gene expression on a translational level, because no significant difference in the amount of the SPP2-lacZ
message was observed among the mutants.
Acknowledgment
This work was supported by a Genetic Engineering
Research Grant from the Ministry of Education of the
Republic of Korea in 1994.
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