From the Dermatology and Medical Services, Department
of Veterans Affairs Medical Center and Departments of Dermatology and
Medicine, University of California, San Francisco, California 94121 and
¶ Acacia Biosciences, Inc., Richmond, California 94806
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The cDNA sequence of a murine gene whose
expression was up-regulated after epidermal injury was cloned utilizing
differential display. The full-length cDNA was isolated by 3' and
5' rapid amplification of cDNA ends from mouse liver. The predicted
protein is >97% identical to the human sequence for eukaryotic
translation initiation factor (eIF) 6, thus identifying the gene as
murine eIF6. Functional studies of the yeast eIF6 homolog,
YPR016c, were initiated in Saccharomyces
cerevisiae to determine the cellular role(s) of eIF6. Complete
deletion of the YPR016c coding sequence was lethal.
Viability was restored in the presence of either YPR016c or
murine eIF6, when either was expressed as amino-terminal green fluorescent protein fusion protein. Moreover, both fusion proteins localized to nuclear/perinuclear compartments in their respective yeast
strains. When the expression of YPR016c-green fluorescent protein was repressed, there was a dramatic reduction in the 60 S
ribosomal subunit and polysome content and decreased 80S monosome content. Additionally, the YPR016c-depleted cells arrested
in G1. These studies show that YPR016c, which
encodes yeast eIF6, is necessary for maximal polysome formation and
plays an important role in determining free 60 S ribosomal subunit content.
The epidermal permeability barrier is composed of intercellular
lipids in the stratum corneum and protects skin from dessication. After
acute permeability barrier disruption, induced experimentally by either
tape stripping or topical acetone treatment, the barrier is restored by
a coordinate process involving increased lipid synthesis (1, 2),
cytokine and growth factor production (3-5), and DNA replication (6,
7). Whereas many of these repair responses are linked directly to an
increase in transepidermal water loss (a measure of barrier integrity),
others, such as increased pro-inflammatory cytokine production, may be
the result of the attendant epidermal cell injury that accompanies
barrier disruption (3, 8). Utilizing differential display, we have
identified a protein whose mRNA was up-regulated after acute
permeability barrier disruption. The corresponding cDNA was cloned and
sequenced. Here we report that based on its high degree of predicted
amino acid sequence identity (>97%), the cDNA is the murine
ortholog of the recently cloned human translation initiation factor 6 (eIF6)1 for which a yeast
homolog has been identified (9).
The role of eIF6 in translation is not firmly established (10). 80 S
ribosomal subunits, which are released from polysomes after translation
termination, are in equilibrium with 40 S and 60 S subunits. Purified
eIF6 from rabbit reticulocyte lysates binds to the 60 S ribosomal
subunit and exhibits ribosome anti-association activity in an in
vitro assay (11). eIF6 and another translation initiation factor,
eIF3 (a multiprotein complex that also exhibits anti-association
activity via its interaction with the 40 S ribosomal subunit), are
thought to regulate the supply of ribosomal subunits necessary
for translation initiation (10, 12).
Interestingly, in the budding yeast Saccharomyces
cerevisiae, temperature-sensitive mutants of other translation
initiation factors (eIF4E (mRNA cap-binding protein) and eIF3 Materials--
Mouse total RNA from assorted tissues was
purchased from Ambion. Oligonucleotides were purchased from Operon.
Yeast media and chemicals were purchased from Sigma or Fisher. RX Fuji
films or Kodak Biomax MR (Merry X-Ray) films were used for autoradiography.
Permeability Barrier Disruption--
Torsos of hairless mice
(strain HRS/Jhr/+ (Jackson Laboratories) or strain SKH1 (Charles River
Laboratories)) were treated with cellophane tape until transepidermal
water loss reached 6-10 mg/cm2/h (normal rate = 0.2 mg/cm2/h) as measured with an electrolytic water analyzer
(Meeco, Inc.). Controls were untreated animals. Mice were killed, and
the epidermis was isolated as described previously (3).
RNA Fingerprinting--
Total epidermal RNA from isogenic
(HRS/Jhr/+) control and tape-stripped hairless mice was isolated using
the Ultraspec-II RNA isolation kit according to the manufacturer's
instructions (Biotecx Laboratories, Inc.). The Delta RNA Fingerprinting
Kit (CLONTECH) was used to detect differentially
expressed epidermal mRNAs 2 h after tape stripping, according
to the manufacturer's instructions. Briefly, 2 µg of total epidermal
RNA were reversed transcribed into single-stranded cDNA using an
oligodeoxythymidylic acid primer and Moloney murine leukemia virus
reverse transcriptase. The cDNA templates from tape-stripped and
control epidermis were used in the subsequent PCR fingerprinting
reactions in the presence of primers P9 and P3, deoxynucleotide
triphosphates, [ Northern Blot Analyses--
Epidermal poly(A)+ or
total RNA was extracted and prepared for Northern blots as described
previously (3). cDNA probes were labeled with 32P by
the random priming method according to the manufacturer's instructions
(Amersham Corp.). Hybridizations to RNA blots were performed as
described previously (3).
Isolation of Murine eIF6--
A partial cDNA, band clw1 (see
Fig. 1), was obtained using PCR amplification primers P3 and P9
supplied in the Delta RNA Fingerprinting Kit. Band clw1 (which was
actually a mixture of different cDNA molecules) was isolated from
the RNA fingerprinting gel and PCR-amplified using P3 and P9 (according
to the kit instructions) and cloned into the A/T cloning vector pCR2.1
(Invitrogen). The correct clone was identified by differential DNA dot
blot hybridization of the resulting plasmid subclones against
32P-labeled cDNA that was reverse transcribed from
either control or tape-stripped epidermal poly(A)+ RNA
(15). Subclones containing the clw1 insert displayed a darker
hybridization signal using the 32P-labeled cDNA probe
made from tape-stripped epidermal mRNA versus the probe
made from control epidermal RNA. The 150-bp insert, clw1 (and all other
murine cDNA constructs), was sequenced on a ABI 377 or 373 DNA
sequencer (Applied Biosystems) at the Biomolecular Resource Center
(University of California, San Francisco), and all DNA sequences were
analyzed using the Sequence Analysis Consulting Service at the
University of California, San Francisco Adcom Computing Facility (node
name, itsa), the Wisconsin Package Version 9.0 (Genetics Computer
Group, Madison, WI), and DNAstar.
The full-length sequence of murine clw1 (later determined to be murine
eIF6) cDNA was determined for mouse liver from 5' RACE and 3' RACE
cDNA products using the Marathon cDNA amplification kit
(CLONTECH) and the gene-specific primers
5'-GACCACAGAAAGCACACCAATCGT-3' (5' RACE) and
5'-ACGATTGGTGTGCTTTCTGTGGTC-3' (3' RACE), which were designed utilizing
sequence data from the 150-bp partial cDNA clw1. After isolation,
both the 5' and 3' RACE products were subcloned into pCR2.1 according
to the manufacturer's instructions (Invitrogen). Several different
subclones of pCRclw5p and pCRclw3p (the resulting plasmids) containing
inserts of the 5' and 3' RACE PCR products, respectively, were used as
templates to obtain sequence data for full-length clw1 (eIF6) mouse
liver cDNA.
Polymerase Chain Reaction--
PCR reactions were performed
using the Advantage cDNA Polymerase Mix
(CLONTECH Labs, Inc) according to the
manufacturer's instructions. All reactions were performed in a model
TC480 DNA thermal cycler (Perkin Elmer Corp.).
Deletion of the Yeast eIF6 Homolog, YPR016c--
Standard
procedures were employed for all studies in yeast (16).
Saccharomyces cerevisiae strain CBS8/13 (see Table
I), a derivative of S288c (kindly
provided by C. Beh, University of CA, Berkeley), was used for one-step
gene replacement (17) of the yeast eIF6 homolog,
YPR016c. Briefly, the TRP1 gene was PCR-amplified from linearized plasmid pRS404 (18) using primers
5'-GAGTATTTGGAACAAGAGCATAATTCAACTAACTCTAGAAAACAATAT GGCTTAACTATGCGGCATC-3' and 5'-AAACAGACTTGAGGAAGGAGGGG
AATCCCCTCAGGAGTACCTGACATCTGTGCGGTATTTCACACCG-3'. The 1.0kb PCR product,
designed for homologous recombination at the YPR016c locus,
was transformed into yeast by the lithium acetate procedure (19).
Tryptophan prototrophs were isolated from minimal plates (SD). The
YPR016c deletion was confirmed by PCR analysis using primers
which flank the integration site; 5'-CCCATATTCCTTTGTGCAGA-3' and
5'-GTACATAATATACATACAAC-3'. A TRP+ heterozygous
(diploid) colony, which contained the expected 1065-bp wild type
YPR016c PCR product and the 1330-bp PCR product (where the
TRP1 gene had replaced the YPR016c ORF) was
sporulated and subjected to tetrad analysis.
Plasmid and Strain Constructions--
pLW-1, a yeast expression
plasmid that contains the GAL1 promoter in front of an
amino-terminal fusion of yeast YPR016c and GFP (20), was
constructed as follows. The YPR016c ORF was amplified by
PCR from S288c yeast genomic DNA using
5'-CGCGGCCGGCCGGACTCAATTTGAAAACTCCAATGAA-3' and
5'-CGCGGCCGGCCGAGTAGGTTTCAATCAAAGTATCACG-3'. The resulting PCR product, with FseI sites engineered on both ends, was
cut with FseI and ligated into the FseI site of
pACA1580 to create a GAL1-regulated YPR016c-GFP fusion.
pLW-2 contains 990 bp upstream of the initiating ATG with the
YPR016c ORF to create an amino-terminal fusion of the
YPR016c protein product with GFP under the control of the
YPR016c endogenous promoter and was constructed as follows.
The YPR016c ORF and 990 bp upstream of the initiating ATG
codon were amplified from S288c yeast genomic DNA by PCR using
5'-GGCCGCATGCGTAGTACTGTGAATATCGTCATTGTCG-3' and
5'-CGCGGCCGGCCGAGTAGGTTTCAATCAAAGTATCACG-3'. The resulting PCR
product, containing SphI and FseI sites
engineered at the 5' and 3' ends, respectively, was digested with these
restriction enzymes and ligated into the SphI and
FseI sites of plasmid pJP1.
pCLW-1 was constructed for GAL1-regulated expression of
murine eIF6 as an amino-terminal fusion to GFP in yeast and was
constructed as follows. eIF6 was PCR-amplified from mouse liver
Marathon-Ready cDNA (CLONTECH) using primers
5'-CGCGGCCGGCCGGGCGTCGTTCGAAAACAACTGTGAA-3' and
5'-CGCGGCCGGCCCATGTGAGGCTGTCAATGAGGGAATC-3'. The PCR product with
FseI sites engineered at both the 5' and 3' ends was
digested with FseI and ligated into the FseI site
of pACA1580.
pLW-5 contains a genomic DNA fragment including the YPR016c
gene. This plasmid was isolated from a genomic library (2 µ,
LEU2) by its ability to suppress the lethality of a
ypr016c
To assess the functional interchangeability of murine eIF6 with the
presumptive yeast counterpart Ypr016cp, we carried out a plasmid
shuffling experiment. LWY3, which contains the
ypr016c Fluorescence-activated Cell Sorting--
Yeast were grown in
SGal + amino acid supplements and shook overnight at 30 °C. The next
morning, cells were centrifuged and then resuspended in SD + amino acid
supplements to A600 = 0.05 and incubated at
30 °C for as long as 24 h. Cells were harvested in log phase
(A600 < 1.0) by centrifugation and washed once
with water. Cells were then fixed in 500 µl of 70% EtOH for either 1 h at room temperature or overnight at 4 °C. They were then
washed twice in 20× TE (10 mM Tris, 1 mM EDTA,
pH 8.0) and resuspended in 100 µl of 20× TE + RNase A (1 mg/ml), and
incubation was continued for 4 h at 37 °C. Finally, cells were
washed two times with phosphate-buffered saline, resuspended in 50 µl
of phosphate-buffered saline and 100 µg/ml propidium iodide, and
incubated overnight at 4 °C in the dark. Before FACS analysis, cells
were diluted 10-20-fold in phosphate-buffered saline. A
Becton-Dickinson FACScan benchtop cytometer was utilized at the
Laboratory for Cell Analysis (University of California, San Francisco
Mount Zion Cancer Center, San Francisco, CA).
Polysome/Ribosomal Subunit Profile Analysis--
Analyses of
polysome/ribosomal subunit profiles in strain LWY4 grown either in SGal
(eIF6- induced) or for 16 h in SD (eIF6-depleted) were performed
exactly as described by Zanchin et al. (21), with the
following exceptions. Cell extracts were isolated from 200-ml cultures
grown to mid-exponential phase. Cycloheximide was added (10 mg/ml), and
cells were centrifuged, washed once, and resuspended in breaking buffer
(as described previously) that also contained phenylmethylsulfonyl
fluoride (17 µg/ml). After cell lysis and centrifugation (as
described previously), identical amounts of cell extracts (20 A254 units) were loaded onto 12-ml linear
sucrose gradients (10-50%). After a 3-h centrifugation, the gradients
were fractionated manually into 0.22-ml fractions starting from the
bottom of the tubes. A spectrophotometer was used to monitor absorbance
at 254 nm.
Fluorescence Microscopy--
GFP and
4',6-diamidine-2-phenylindole-dihydrochloride fluorescence was
evaluated in living yeast using a Nikon Microphot fluorescence microscope. Yeast were incubated in the presence of 1 µg/ml
4',6-diamidine-2-phenylindole-dihydrochloride while being grown in
liquid culture for 1 h before microscopic analysis. The control
yeast strain used in the fluorescence microscopic studies was strain
CBS13 (see Table I) harboring pACA1580.
Isolation of Murine eIF6 cDNA--
RNA fingerprinting was used
to identify epidermal mRNAs that are differentially expressed
2 h after acute permeability barrier disruption by tape stripping.
We chose this time point because previous work showed that mRNAs
encoding proteins involved in permeability barrier repair are
up-regulated within 0.5-4 h after acute barrier disruption (2-5).
Fig. 1 displays the RNA fingerprinting results (for details, see "Experimental Procedures"). The cDNA band labeled clw1 was more abundant in the tape-stripped samples; therefore, it was isolated from the gel and PCR-amplified, and the
purified clw1 subclone (isolation was described under "Experimental Procedures") was used to probe an epidermal RNA blot.
A RNA blot was prepared from the epidermis of outbred hairless mice
that were subjected to acute barrier disruption by tape stripping. A
single 1.3-kb mRNA hybridized with the 150-bp partial clw1 cDNA
probe (Fig. 2). Furthermore, the levels
of this epidermal mRNA (clw1) were increased after tape stripping,
confirming the differential expression of clw1 mRNA in response to
barrier disruption. Subsequent Northern analysis using the partial clw1
cDNA as a probe revealed that a single cross hybridizing 1.3-kb
mRNA was present in a wide variety of mouse
tissues.2
We cloned the full-length clw1 cDNA from mouse liver by 5' and 3'
RACE. Based on the high degree (>97%) of predicted amino acid
sequence identity, it is likely that clw1 cDNA encodes the murine
ortholog of human eIF6 (9). The mouse liver eIF6 cDNA sequence has
been submitted to GenBank (accession number AF047046). Analysis of the
amino acid sequence of murine eIF6 does not reveal any obvious clues as
to its function in translation. However, murine eIF6 and the human,
yeast, and Drosophila presumptive homologs contain a
potential nuclear export signal, LSSLLQVPLVA, which conforms to the
consensus nuclear export signal described as a short, leucine-rich,
hydrophobic sequence (22). Whether this consensus sequence is
functional is not known at the present time.
Functional Studies of a eIF6 Homologous Sequence in S. cerevisiae--
We initiated studies in S. cerevisiae to
delineate the cellular function(s) of eIF6. As a first step, the entire
YPR016c ORF was replaced with the TRP1 gene to
assess the phenotype of YPR016c (eIF6) knockout yeast.
Tetrad dissection of 18 asci from the heterozygous diploid LWY1
(ypr016c
The diploid strain LWY1 (ypr016c
To determine whether murine eIF6 could functionally replace
YPR016c in yeast, ypr016c Localization of murine eIF6-GFP and YPR016c-GFP Fusion Proteins in
Yeast--
We examined live cells by fluorescence microscopy to
localize the eIF6-GFP fusion proteins in strains LWY2, LWY4, and LWY6. In LWY2, we did not detect any GFP fluorescence above the background (data not shown), in agreement with previous findings showing that eIF6
is in very low abundance intracellularly (11). However, upon
examination of nonsynchronous LWY4 and LWY6 cells during log-phase
growth in liquid SGal, a major portion of the GFP fluorescence was
concentrated in a nuclear/perinuclear location that colocalized with
nuclear 4',6-diamidine-2-phenylindole-dihydrochloride staining (Fig.
4 C, D, G, and H).
In contrast, GFP fluorescence in control yeast (grown under the same
conditions) that harbor the parent plasmid was localized uniformly
throughout the yeast cell (Fig. 4A).
Because we only observed GFP fluorescence when the eIF6-GFP fusion
proteins were overexpressed, we next determined whether the repression
of YPR016c-GFP expression by incubating LWY4 cells in decreasing
amounts of galactose would alter the GFP fluorescence pattern in living
yeast. For these determinations, LWY4 and control cells were incubated
during log phase (A600 < 1.0) for 4 h in either SGal or SGal:SRaf (1:2; SRaf contains 6.7 g/liter yeast nitrogen
base without amino acids, 20 g/liter raffinose), conditions that did
not alter growth rates (data not shown), and then examined by
fluorescence microscopy. Again, a distinct nuclear/perinuclear localization of YPR016c-GFP was detected in LWY4 cells grown in SGal:SRaf (Fig. 4E). The localization of GFP fluorescence in
control yeast (harboring pACA1580) grown in SGal:SRaf (1:2) was nearly identical to that shown in Fig. 4A and was therefore omitted.
Because temperature-sensitive mutants of two other translation
initiation factors (cdc33 and cdc63) arrest in
G1 (13, 14, 23), we performed FACS analysis of the
conditional mutant strain LWY4. For these determinations, LWY4 was
grown for up to 24 h in SD, conditions that strongly repress
YPR016c-GFP expression. For comparison, the control LWY5 strain was
analyzed at the same A600 reading after only
5-7 h in SD. These different time points were chosen because 3-4 h
after the switch from SGal to SD, the growth of LWY4 begins to decline
in comparison to that of LWY5. No differences in FACS profiles were
detected when LWY4 cells were grown for 1, 4, or 8 h in SD as
compared with control LWY5 cells.2 However, FACS profiles
showed that the eIF6 conditionally mutant strain LWY4 arrested in
G1 after several generations in SD (after either 16 h2 or 24 h; Fig. 5).
Thus, depletion of eIF6 from S. cerevisiae resulted in the
same cell division cycle phenotype as reported for two other
translation initiation factors, cdc33 and cdc63. Based on this result, we have designated the genetic locus YPR016c as
cdc95. In addition, by forward angle scattering, we also
determined that the relative size of LWY4 cells grown for several
generations in SD is the same as that of LWY5 (control) cells grown to
the same A600 (data not shown).
The effect of eIF6 depletion on translation initiation was determined
by examining the polysome/ribosomal subunit profiles obtained from
strain LWY4 grown in either SGal (eIF6-induced) or SD (eIF6-depleted).
For these studies, LWY4 was grown to mid-exponential phase for 16 h (three to four generations) in either SGal or SD medium, cells were
harvested in the presence of cycloheximide, and sucrose density
gradient analyses were performed using previously published procedures
(21). Fig. 6 displays one set of
representative results obtained from three separate experiments. In all
experiments, there was a drastic reduction in the size of the polysomes
and the total amount of ribosomes present in cells depleted of eIF6. Although more difficult to quantitate, the pool of free 60 S subunits was greatly reduced as well. The pool of free 40 S subunits varied between experiments (increasing in some and decreasing in others) but
remained, on average, roughly constant. The decrease in polysome size
could reflect either a decrease in the cellular content of 60 S
subunits (which would appear as an initiation defect) or a decrease in
ribosomes (40 S and 60 S subunits) if the mRNA content remained the
same. The latter seems to best account for the observed profiles. These
results, when taken together with the nuclear/perinuclear localization
of eIF6-GFP fusion proteins, are consistent with a role for eIF6 in 60 S ribosomal subunit assembly, stability, or nucleo-cytoplasmic
transport.
During initiation, which is generally the rate-limiting step in
translation, a number of soluble protein factors interact transiently
with macromolecular complexes consisting of ribosomes, aminoacyl-tRNAs,
and messenger ribonucleoproteins and form translation initiation
complexes (10). These initiation factors are known to exert control
over total protein synthesis; however, these factors may also play key
roles in the nucleus and/or in the regulation of the cell cycle. The
current model is largely based on reconstitution experiments using
purified factors in conjunction with macromolecular complexes. Whereas
the role of some of these factors is firmly established, many aspects
of the translation initiation model require in vivo
confirmation that can be obtained using S. cerevisiae and
its attendant genetic and molecular biological tools.
In this study, we report the cloning of murine eIF6, a translation
initiation factor that was previously identified in rabbit reticulocyte
lysates by its activity in preventing ribosomal subunit association
(11). The predicted amino acid sequence of murine eIF6 is >97%
identical to eIF6 from human skeletal muscle (9). Several presumptive
eIF6 homologs were identified by BLAST search, and an examination of
their amino acid sequences indicated that this protein is highly
conserved throughout evolution (9). Interestingly, the archaebacterial
S. acidocaldarius eIF6 homolog is transcribed as part of a
polycistronic mRNA that also encodes two large ribosomal subunit
proteins, RL46 and RL31 (24).
Our functional studies of eIF6 in S. cerevisiae revealed new
insights regarding the cellular role(s) of this highly conserved protein. Deletion of the yeast eIF6 homolog YPR016c was
lethal, indicating that YPR016c is an essential gene.
Furthermore, YPR016c was functionally replaced by murine
eIF6 when expressed as an amino-terminal GFP fusion protein, indicating
that the YPR016c locus encodes the yeast eIF6. We found that
a substantial fraction of the murine and yeast eIF6-GFP fusion proteins
was localized in a nuclear/perinuclear compartment. This localization
is consistent with a putative role for eIF6 in the coordination of
nuclear and cytoplasmic events. A nuclear localization has been
reported for two other eIFs, namely, eIF5A (25) and eIF-4E (26).
Depletion of yeast eIF6-GFP in the conditionally mutant strain LWY4
resulted in decreased polysome content, a dramatic reduction in 60 S
ribosomal subunit content, and diminished 80 S monoribosome content.
When taken together with the nuclear/perinuclear localization of
eIF6-GFP fusion proteins reported herein, these results suggest that
eIF6 plays a key role in 60 S subunit assembly, stability, or
nucleo-cytoplasmic transport. Other yeast mutants in nonribosomal protein genes that are defective in 60 S subunit assembly have been
reported previously (21, 27, 28). Whether eIF6 plays a direct role in
the initiation phase of translation or functions primarily to provide
physiologic quantities of stable 60 S ribosomal subunits remains to be determined.
Depletion of eIF6-GFP also caused G1 arrest, as has been
reported for temperature-sensitive alleles of two other yeast eIFs, cdc63 (which encodes the In yeast, as in mammalian cells, critical growth requirements in
G1 must be met for the cells to enter S phase and replicate their DNA (31). However, experiments that used protein synthesis inhibitors such as cycloheximide demonstrated that protein synthesis is
required for progression through all stages of the yeast cell cycle
(32, 33). Why then, would depletion of three different eIFs in the cell
cause a specific arrest in G1? Each of the three eIFs in
question is thought to function by providing critical components of
translation initiation complexes. One role ascribed to eIF3 is the
dissociation of 80 S ribosomes into 40 S and 60 S subunits (12, 34).
Our results in yeast show that eIF6 plays a key role in determining
free 60 S ribosomal subunit content, whereas eIF4E plays a role in the
recruitment of capped mRNAs to the translation initiation complex
(10). It has been shown that when alterations such as mutations in eIF3
in cdc63 cells (35) cause translation initiation complexes
to become rate limiting, inefficiently translated mRNAs are
affected preferentially (36). One such mRNA encodes the
G1-specific cyclin Cln3p, which is necessary for the
G1-S-phase transition.
Polymenis and Schmidt (35) recently demonstrated that the translational
regulation of CLN3 provides a mechanism to link cell growth
and cell division. They showed that a translational control element (an
upstream ORF) in the 5' leader of CLN3 mRNA functions to
diminish CLN3 expression in either cdc63 cells or poor growth media, conditions in which translation initiation components are low or rate-limiting. Because the results from our
gradient analyses indicate that eIF6 functions to provide free 60 S
ribosomal subunits for translation, we predict that CLN3
mRNA would also be inefficiently translated in cells in which YPR016c expression is conditionally repressed. Translational
regulation of another cell cycle-regulatory molecule, the
cyclin-dependent kinase inhibitor p27KIP1, has
also been reported (37, 38).
The mechanism(s) responsible for the relatively rapid increase of eIF6
mRNA levels in murine epidermis after tissue injury is(are) yet to
be determined. It is tempting to speculate that cytokines may be the
mediators, because these molecules are known to be produced rapidly in
response to cutaneous barrier disruption (3, 4, 39, 40). It will be of
interest to determine whether other types of genotoxic stress, such as
UV irradiation and DNA-damaging agents, also affect eIF6 expression in
yeast and mammalian tissues. It is also important to determine the
downstream consequences of increased cellular eIF6 levels in relation
to protein synthesis, in particular.
Recent studies have shown additional roles for known eIFs. For example,
eIF5A is believed to mediate the nucleo-cytoplasmic transport of
specific cellular RNAs and ribonucleoproteins through the nuclear pores
(25, 41, 42). It will therefore be of interest to identify proteins
that interact with eIF6 to clarify the role(s) of this essential
protein. The yeast two-hybrid assay may be a useful approach.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(a
component of eIF3)) arrest in G1 and are classified as cell
division cycle (cdc) mutants cdc33 and
cdc63, respectively (13, 14). In addition to isolating the
full-length cDNA sequence of murine eIF6 and reporting the
up-regulation of murine eIF6 mRNA levels in response to epidermal
injury, we initiated studies using S. cerevisiae as a model
to begin to understand the role(s) of eIF6 in the cell. We show here
that complete deletion of the gene for the yeast eIF6 homolog
YPR016c from S. cerevisiae is lethal, indicating that YPR016c is essential. Furthermore, expression of either
YPR016c-GFP or murine eIF6-GFP fusion proteins restored
viability, suggesting that the YPR016c gene product is
indeed yeast eIF6. A nuclear/perinuclear localization of the eIF6-GFP
fusion proteins in yeast is also reported. Finally, we show that
depletion of eIF6 results in reduced polysome size and dramatically
reduces free 60 S ribosomal subunit content. Taken together, these
results suggest that eIF6 plays a role in 60 S ribosomal subunit
assembly, stability, or nucleo-cytoplasmic transport.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-33P]dATP, and KlenTaq Polymerase Mix
(CLONTECH). The resulting PCR reaction products
were applied to a 6% polyacrylamide/8 M urea gel in
Tris-borate EDTA buffer and resolved after electrophoresis by
autoradiography using Kodak Biomax MR film.
Yeast strains
::TRP1 deletion in a multicopy
suppression screen.
::TRP1 deletion and is viable by virtue
of the presence of pLW-5 (YPR016c, LEU2), was
transformed with either pLW-1 (GAL1-YPR016c-GFP,
URA3) or pCLW-1 (GAL1-murine eIF6-GFP,
URA3). The two transformants, LWY5 and LWY7 (also described
in Table I), were grown under nonselective conditions in the presence
of leucine and galactose, and the yeast uracil prototrophs LWY4 and
LWY6, which maintained pLW-1 and pCLW-1, respectively, were isolated.
LWY5 was used as a control strain for FACS analysis of LWY4.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (43K):
[in a new window]
Fig. 1.
The partial cDNA clw1 was discovered in
mouse epidermis by the RNA fingerprinting technique. An
autoradiograph of RNA fingerprinting gel is shown. Total RNA (2 µg)
from control or tape-stripped epidermis was converted to cDNA, and
PCR fingerprinting reactions were performed (see "Experimental
Procedures"). Samples were run on a 6% polyacrylamide/8
M urea gel, and the gel was dried and exposed to film.
Lane 1, control epidermal cDNA; lane 2,
control epidermal cDNA (1:10 dilution); lane 3,
tape-stripped epidermal cDNA; lane 4, tape-stripped
epidermal cDNA (1:10 dilution); lane 5, water control.
Arrow, clw1 band.
View larger version (26K):
[in a new window]
Fig. 2.
Partial clw1 cDNA hybridized to 1.3-kb
epidermal mRNA that is up-regulated after acute permeability
barrier disruption by tape stripping. Autoradiographs of Northern
blot are shown. Mouse epidermis was tape-stripped, epidermal
poly(A)+ mRNA was isolated, and Northern blot analyses
were performed as described under "Experimental Procedures" using
either clw1 or rat cyclophilin cDNA as probes. Each lane was loaded
with 8 µg of epidermal poly(A)+ mRNA.
::TRP1/ YPR016c) resulted in a
segregation pattern of two live spores and two dead spores. None of the
viable segregants were TRP+, indicating that
YPR016c is required for viability. Microscopic examination
of the meiotic segregants revealed that the
ypr016c
::TRP1 cells had arrested between the
second and fourth cell divisions.2
::TRP1/YPR016c)
was transformed with pLW-2 and sporulated. pLW-2 expresses a
YPR016c-GFP fusion protein under the control of the
endogenous YPR016c promoter. Tetrad analysis of LWY1
harboring pLW-2 produced four viable segregants (two TRP+
segregants and two TRP
segregants), indicating that the
YPR016c-GFP fusion protein was functional and could
substitute for the endogenous YPR016c protein product.
::TRP1
strains that contained either YPR016c (LWY4) or murine eIF6
(LWY6) expressed as GAL1-regulated GFP fusion proteins were
constructed. The growth characteristics of LWY4 and LWY6 on SGal,
conditions that up-regulate GAL1 promoter activity, were
compared with a control strain (CBS13 harboring GAL1-GFP) in
Fig. 3. As shown in Fig. 3 (left
side), the growth rates of LWY4 (harboring
GAL1-YPR016c-GFP) and LWY6 (harboring GAL1-murine eIF6-GFP) versus control appeared
somewhat slower on SGal, indicating that overexpression of the eIF6-GFP
fusion proteins had no major effects on cell growth. This result also indicated that the murine eIF6-GFP fusion protein functionally replaced
YPR016c gene function. When the growth rates of these strains were monitored in liquid SGal for three to four generations, both LWY4 and control yeast exhibited a doubling time of 4 h, whereas the doubling time for LWY6 was 6 h.2
Importantly, when LWY4 and LWY6 were grown on SD (Fig. 3, right side), conditions that repress GAL1 promoter activity,
cell growth was arrested. This result demonstrated that the cells were
dependent upon GAL1-regulated expression of either the
YPR016c-GFP (in LWY4) or the murine eIF6-GFP (in LWY6)
fusion proteins for viability.
View larger version (69K):
[in a new window]
Fig. 3.
Growth characteristics of the conditional
mutants LWY4 ( ypr016c + pLW-1) and LWY6
(
ypr016c + pCLW-1). Strains LWY4 and LWY6 are
deleted at the YPR016c locus and harbor plasmids that encode
the S. cerevisiae eIF6 homolog YPR016c and murine
eIF6, respectively, as GFP fusion proteins, under the control of the
GAL1 promoter. The control strain is CBS13 (see Table I),
which harbors the parental GAL1-GFP plasmid.
View larger version (83K):
[in a new window]
Fig. 4.
Fluorescence microscopic localization of
murine and yeast eIF6-GFP fusion proteins in yeast. GFP and
4',6-diamidine-2-phenylindole-dihydrochloride fluorescence was examined
in live yeast as described under "Experimental Procedures."
A and B, control yeast, strain CBS13
(GAL1-GFP, URA3) grown in SGal; C and
D, strain LWY4 ( ypr016c + GAL1-YPR016c-GFP, URA3) grown in SGal;
E and F, strain LWY4 grown in SGal:SRaf (1:2);
G and H, strain LWY6 (
ypr016c
+ GAL1-murine eIF6-GFP, URA3) grown in SGal.
Bar, 20 µm. Insets, utilizing Adobe Photoshop,
cells were selected, and the image size was increased approximately
100%.
View larger version (14K):
[in a new window]
Fig. 5.
Fluorescence-activated cell sorting analysis
of LWY4 and LWY5 after growth in SD media. Yeast were grown
overnight in SGal and then shifted to SD media and prepared for FACS
analysis as described under "Experimental Procedures." LWY4
( ypr016c + GAL1-YPR016c-GFP,
URA3) cells were harvested after 24 h in SD. For
comparison, control strain LWY5 was harvested after 7 h in SD at
the same A600 as LWY4
(A600 = 0.9).
View larger version (13K):
[in a new window]
Fig. 6.
Polysome/ribosomal subunit profiles from
strain LWY4 grown in SGal versus SD. The eIF6
conditionally mutant strain LWY4 ( ypr016c + GAL1-YPR016c-GFP, URA3) was grown either in
SGal or SD (16 h), and polysome/ribosomal subunit analyses were
performed as described under "Experimental Procedures." The results
shown are representative of three separate experiments. Peaks
representing polysomes, monosomes, and ribosomal subunits are labeled
as follows: s, small ribosomal subunit (40 S); l,
large ribosomal subunit (60 S); m, monoribosomes;
p, polysomes.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit of eIF3; Refs. 14 and
23) and cdc33 (which encodes eIF4E or mRNA cap-binding
protein; Ref. 13). Furthermore, temperature-sensitive mutants in two
other protein components of the eIF3 complex also arrest in
G1 (29, 30). Because depletion of yeast eIF6 caused a cell
division cycle type of arrest, we designated the YPR016c
locus as cdc95.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Debra Crumrine for expert assistance with photographic techniques. We also thank Dr. A. Farrell (University of Hamburg, Hamburg, Germany) and Dr. Charles Boone (Queen's University, Kingston, Ontario, Canada) for insightful scientific discussions.
![]() |
Noted Added in Proof |
---|
During the preparation of this manuscript we became aware of the publication of the cDNA sequence for murine mast cell elF6, also referred to as, imc-415 (Cho, S. H., Cho, J.-J., Kim, I. S., Vliagoftis, H., Metcalfe, D. D., and Oh, C. K. (1998) Biochem. Biophys. Res. Commun. 252, 123-127).
![]() |
FOOTNOTES |
---|
* This work was supported by a grant from the Research Service of the Department of Veterans Affairs Medical Center and National Institutes of Health Grant AR 39639.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF047046.
§ To whom correspondence should be addressed: Dept. of Veterans Affairs Medical Center, 4150 Clement St., 111F, San Francisco, CA 94121. Tel.: 415-750-2005; Fax: 415-750-6927; E-mail:walkman{at}itsa.ucsf.edu.
2 L. C. Wood, C. Grunfeld, and K. R. Feingold, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: eIF, eukaryotic translation initiation factor; RACE, rapid amplification of cDNA ends; cdc, cell division cycle; GFP, green fluorescent protein; PCR, polymerase chain reaction; ORF, open reading frame; FACS, fluorescence-activated cell-sorting; bp, base pair(s); SGal, 6.7 g/liter yeast nitrogen base, without amino acids, 20 g/liter galactose; SD, 6.7 g/liter yeast nitrogen base, without amino acids, 20 g/liter dextrose.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|