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Molecular and Cellular Biology, August 2001, p. 4900-4908, Vol. 21, No. 15
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.15.4900-4908.2001
Linking the 3' Poly(A) Tail to the Subunit Joining
Step of Translation Initiation: Relations of Pab1p, Eukaryotic
Translation Initiation Factor 5B (Fun12p), and Ski2p-Slh1p
Anjanette
Searfoss,1
Thomas E.
Dever,2 and
Reed
Wickner1,*
Laboratory of Biochemistry and Genetics,
National Institute of Diabetes and Digestive and Kidney Diseases,
National Institutes of Health, Bethesda, Maryland
20892-0830,1 and Laboratory of
Eukaryotic Gene Regulation, National Institute of Child Health and
Human Development, National Institutes of Health, Bethesda, Maryland
20892-27162
Received 30 January 2001/Returned for modification 1 March
2001/Accepted 27 April 2001
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ABSTRACT |
The 3' poly(A) structure improves translation of a eukaryotic mRNA
by 50-fold in vivo. This enhancement has been suggested to be due to an
interaction of the poly(A) binding protein, Pab1p, with eukaryotic
translation initiation factor 4G (eIF4G). However, we find that
mutation of eIF4G eliminating its interaction with Pab1p does not
diminish the preference for poly(A)+ mRNA in vivo,
indicating another role for poly(A). We show that either the absence of
Fun12p (eIF5B), or a defect in eIF5, proteins involved in 60S ribosomal
subunit joining, specifically reduces the translation of
poly(A)+ mRNA, suggesting that poly(A) may have a role in
promoting the joining step. Deletion of two nonessential putative RNA
helicases (genes SKI2 and SLH1) makes poly(A)
dispensable for translation. However, in the absence of Fun12p,
eliminating Ski2p and Slh1p shows little enhancement of expression of
non-poly(A) mRNA. This suggests that Ski2p and Slh1p block translation
of non-poly(A) mRNA by an effect on Fun12p, possibly by affecting 60S
subunit joining.
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INTRODUCTION |
The 5' cap (7-methyl-GpppG...)
and 3' poly(A) structures of eukaryotic mRNAs are both critically
important for translation, for mRNA transport from the nucleus, and for
mRNA stability in both the nucleus and the cytoplasm. The in vivo
requirement for translation for the 3' poly(A) structure, estimated by
electroporation of mRNAs into living cells, is about 50-fold, while
that for the 5' cap is about 20-fold (11, 12).
The effects of the 5' cap on translation are mediated by the
cap-binding protein, eukaryotic translation initiation factor 4E
(eIF4E) (Cdc33p), which associates with eIF4G in the eIF4F complex to
promote binding of the mRNA to the 40S ribosomal subunit (through
eIF3). A 43S preinitiation complex consisting of the 40S subunit, eIF3,
and eIF2-GTP-Met-tRNA
is thought to bind to an mRNA
mediated by interaction between eIF3 and eIF4G. This complex scans from
the cap at the 5' end of the mRNA to the first AUG. There, with the
help of eIF5 (Tif5p) and eIF5B (Fun12p), 60S subunit joining occurs and
translation begins (27; reviewed in references 9 and
37). 60S subunit joining requires both eIF5 and eIF5B. While
eIF5 promotes GTP hydrolysis by eIF2, enabling release of the
initiation factors from the 40S subunit, eIF5B has its own GTP binding
activity and hydrolyzes GTP in a ribosome-dependent reaction
(27).
The role of the 3' poly(A) structure in mRNA translation is not yet
completely clear. The poly(A) binding protein (Pab1p) is believed to
mediate many of the effects of the 3' poly(A) structure (reviewed in
reference 34). Because the poly(A) tail apparently has
roles in nuclear processes as well as cytoplasmic events
(22), dissecting these mechanisms is difficult. The
PAB1 gene is essential, indicating that at least one of the
functions of Pab1p is critical to the cell.
It has recently been observed that eIF4G and Pab1p bind to each other
in the presence of poly(A) (38). This binding would be
expected to circularize the mRNA, since eIF4G is attached to the 5' cap
by its association with eIF4E and to the 3' poly(A) by its binding to
Pab1p. It has been suggested that this is how the poly(A) tail
functions to promote translation of mRNAs. However, mutations of eIF4G
that eliminate the binding to Pab1p do not affect the growth rate of
cells (40), suggesting Pab1p may activate translation by a
different mechanism in vivo.
Biochemical evidence suggested that the role of poly(A) was to promote
joining of 60S subunits to the 40S subunit waiting at the initiator AUG
(23), but these data showed only a twofold effect and
doubts have been raised about this conclusion. A
pab1ts mutant accumulates free 60S subunits at
the nonpermissive temperature (32), just the result
expected if 60S joining is the defective step. However, the
relationship of the action of the joining factors Fun12p and Tif5p to
the poly(A) structure has not been examined.
Although there is a strict requirement for the 3' poly(A) structure for
translation, both in vivo and in vitro, recent work indicates that this
requirement is one imposed by the inhibition of translation of
non-poly(A) mRNAs by two homologous nonessential RNA helicases, Ski2p
and Slh1p (20, 21, 35, 41). Mutations in SKI2
and SLH1, or other nonessential genes involved in this activity (SKI3, SKI7, and SKI8; see also
reference 3), derepress translation of non-poly(A) mRNAs
introduced by electroporation, the naturally poly(A)
mRNAs produced by the L-A and L-BC double-stranded RNA (dsRNA) viruses
of yeast or the poly(A) mRNAs produced from an RNA
polymerase I promoter. Further, ski2
slh1
double mutants treat poly(A)+ and poly(A)
mRNAs the same, translating them at the same rate and for the same
duration (35). Ski2p, Ski3p, and Ski8p are found in a
cytoplasmic complex (4), but their precise role is
unclear. As in yeast, there are two human Ski2p homologues (8,
19, 24, 30).
Here we sought to relate the effects of Ski2p and Slh1p in blocking the
translation of non-poly(A) mRNA to the translation-promoting effects of
Fun12p and Pab1p. Our results support a role of the poly(A) structure
in 60S ribosomal subunit joining promoted by Fun12p (eIF5B) and Tif5p
(eIF5) but argue against a role for the Pab1p-eIF4G interaction in
mediating the poly(A) requirement for translation. We find that the
derepression of translation of non-poly(A) mRNA by the
ski2 slh1 double mutation is abrogated by
deficiency of Fun12p, suggesting a model in which Ski2p and Slh1p
inhibit Fun12p action on mRNAs lacking a 3' poly(A) structure.
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MATERIALS AND METHODS |
Strains.
Strains of S. cerevisiae used are shown
in Table 1.
Electroporation. (i) Preparation of cells.
A total of 50 ml
of cells was grown to an optical density at 600 nm (OD600)
of 0.6. Cells were pelleted, resuspended in 4 ml of spheroplast buffer
A (50 mM Tris Cl, pH 7.5; 1 mM MgCl2; 30 mM dithiothreitol;
15 mM 
-mercaptoethanol; 1 M sorbitol), and 200 µl of a 5-mg/ml
solution of zymolase 20T in spheroplast buffer A was added. Incubation
at 30°C with gentle swirling was continued until spheroplasts were
formed as determined empirically for each strain by diluting the cells
1:100 in sorbitol and in H2O and measuring the decrease in
OD600. Spheroplasts were washed gently in 5 ml of
spheroplast buffer A and pelleted for 5 min at 1,000 rpm in a SS-34
rotor. Cells were resuspended in 1 ml of spheroplast buffer A, added to
9 ml of YPAD (1% yeast extract, 2% peptone, 2% dextrose, 0.04%
adenine sulfate)-1 M sorbitol, and incubated for 90 min at 30°C with
gentle swirling to allow the cells to recover from the zymolase
treatment. Following recovery, cells were gently washed twice using 5 ml of 1 M sorbitol (spinning at 1,000 rpm for 5 min in an SS-34 rotor).
Cells were resuspended in 1 ml of 1 M sorbitol at 2 × 108 cells/ml and used for electroporation. (see references
11 and 12).
(ii) Preparation of mRNAs.
For the measurement of
translation in vivo, we used reporter luciferase mRNAs described first
by Gallie (12). The T7 promoter-based luciferase
constructs were linearized as follows: LUC A0 is linearized with
SmaI, and LUC A50 is linearized with DraI. LUC
A50 produces an mRNA with a poly(A)50 tail. The capped
poly(A)+ (C+A+) and poly(A) (C+A
) mRNAs
were synthesized with Ambion's mMessage mMachine kit. The uncapped
mRNAs (CA+ and CA) were synthesized with the MegaScript kit
(Ambion). A total of 2 µg of RNA was used per electroporation.
(iii) Electroporation procedure.
Prior to electroporation,
the electroporation cuvettes (0.2-cm electrode gap; Bio-Rad) were kept
on ice. Then, 2 µg of each reporter mRNA and 180 µl of yeast
spheroplasts were added to a cuvette and pulsed immediately (800 V, 25 F, 1,000 
). This results in a pulse that ranges from 20 to 25 ms in
duration. Immediately following the electroporation pulse, 1.2 ml of
ice-cold YPAD-1 M sorbitol was added to the cuvettes, which were kept
on ice.
(iv) Measurement of reporter activity.
The spheroplasts were
transferred to ice-cold tubes (Falcon 2059) and placed at room
temperature (or at 37°C for the temperature-sensitive mutants) with
gentle swirling. Samples were collected at 0, 5, 10, 20, 40, 60, and
120 min. Then, 150 to 200 µl of cells were removed at each time
point, the cells were spun down, and the cell pellets were frozen
immediately in an ethanol-dry ice bath. To measure luciferase activity,
we used the Luciferase Assay Reporter system (Promega). The cell
pellets were resuspended in 50 to 100 µl of 1 × Reporter Lysis
Buffer (Promega) and vortexed vigorously for 30 s to break open
the spheroplasts. Next, 20 µl of the cell lysate was combined with
200 µl of reconstituted Luciferase Assay Substrate (Promega), and the
activity was measured immediately in an LKB Wallac 1250 Luminometer.
Introducing the RNAs into the same preparation of spheroplasts results
in a 5 to 15% variability in luciferase activity measured
per
microgram of protein. The light output is proportional to
the
luciferase concentration with 1.0 light unit corresponding
to 165 femtograms of luciferase enzyme (as measured in the Wallac
1250 Luminometer). The activity was normalized to the amount of
total
protein (measured by Bradford
assays).
(v) Kinetics.
Variations in the rate of luciferase
expression generally reflect differences in translation rate, while the
duration of expression reflects stability of the mRNA (11,
12). The maximum luciferase translation rate (<0.4 U/µg of
cell protein/min) is about a 106 part of the total
protein synthesis in these cells.
Measurement of mRNA turnover.
To measure the effect of
ski2 slh1 on mRNA decay, 200 ml of mutant
and wild-type cells was grown to an OD600 of 0.6. Transcription was inhibited by the addition of thiolutin (the kind gift
of Edmund Hafner, Pfizer) as described previously (26a), and aliquots
of cells were removed at intervals. RNA was extracted using the RNeasy Mini RNA extraction kit (Qiagen) after breaking the cells with glass
beads. Extracted RNA was analyzed by the Northern blot method. The
membranes were hybridized with either 5'-32P-labeled
oligonucleotide probes complementary to regions of the coding sequences
of STE2 and URA5 or to an RNA probe complementary to 18S rRNA.
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RESULTS |
Effect of mutations in eIF3 and eIF2 on translation of
electroporated mRNAs.
Electroporation faithfully reflected the
known requirements of translation in that it requires the 5' cap and 3'
poly(A) structures. In wild-type cells, the 3' poly(A) structure
increased translation rate by more than 80-fold, while the 5' cap
structure enhanced the translation rate about 8-fold (Fig.
1A). To further assess the fidelity of
this method, we tested the effects of prt1-1, with an
altered eIF3 subunit (13, 16, 28), and
gcd11-508, with an altered eIF2 
subunit
(14). In each case, the translation of electroporated
messages was substantially reduced, regardless of the presence of 5'
cap or 3' poly(A) (Fig. 1). This result is as expected, since eIF2 and
eIF3 are necessary for bringing the initiator Met-tRNA and the 40S
ribosomal subunit, respectively, into the initiation complex,
regardless of the presence or absence of cap or poly(A).
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FIG. 1.
(A) Isogenic strains H117 (wild type) and H272
(gcd11-508) were electroporated (1.3 × 108
cells) with 2 µg of RNA. Cells were maintained at 25°C and assayed
for luciferase activity at the indicated timepoints as described
previously (21). Protein concentrations of all lysates
were measured by the Bio-Rad protein assay kit. The results shown are
an average of three experiments. The maximum luciferase translation
rate (<0.4 U/µg of cell protein/min) is about a 106
part of the total protein synthesis in these cells. C+ or C, mRNA
with or without 5' cap structure; A+ or A, mRNA with or without 3'
poly(A) structure. (B) Isogenic strains H2783 (wild type) and H1676
(prt1-1) were treated as in panel A except the cells were
maintained at 37°C throughout the indicated time course.
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Pab1p is required for the poly(A) effect.
Although the
pab1 mutation is lethal, it is suppressed by mutation of
many of the genes needed for 60S ribosome biogenesis, such as
spb2-1, a mutation in ribosomal protein L46
(33). So while it is impossible to compare wild-type and
pab1 strains, one can compare spb2-1 strains
with pab1 spb2-1 cells. We found that the
pab1 mutation selectively lowers translation of
poly(A)+ mRNAs, with little effect on
poly(A) mRNAs (Fig. 2).
This in vivo result was similar to previously reported in vitro results
of Tarun and Sachs (39). This further supports the view
that Pab1p is a mediator of the poly(A) effect on translation.
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FIG. 2.
Isogenic strains YAS43 (wild type), YAS216
(spb2-1), and YAS227 (spb2-1 pab1) were
electroporated with 2 µg of RNA. Cells were maintained at 25°C and
assayed for luciferase activity at the times indicated as described in
Materials and Methods.
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The spb2-1 suppression of pab1 does not
bypass the requirement for poly(A).
Mutation in any of several
genes that reduce the level of free 60S ribosomal subunits makes the
PAB1 gene dispensable for growth. It is suggested that the
excess of free 40S ribosomal subunits facilitates cap-mediated
initiation so that the requirement for the poly(A) (and thus Pab1p) is
diminished (39). This hypothesis would predict that
deficiency of 60S subunits would by the same mechanism diminish the
requirement for the 3' poly(A) structure. We examined the effect on the
poly(A) requirement of the spb2-1 mutation and found that
there is a modest general decrease in efficiency of translation of all
mRNAs, regardless of the presence of cap or poly(A) structure (Fig. 2).
Translation of non-poly(A) mRNA did not increase as predicted in the
spb2-1 mutant. In this mutant, poly(A)+ mRNA was
still translated 22 times better than was non-poly(A) mRNA.
Pab1p-eIF4G interaction is not necessary for the poly(A)
effect.
Pab1p interacts with eIF4G in vitro in the presence of
poly(A) (38), and this interaction is proposed to be the
basis of the requirement for poly(A) for translation (40).
This model predicts that a mutation preventing this interaction should
reduce or eliminate the requirement for the 3' poly(A) structure in the presence of competing poly(A)+ mRNAs. We used a strain in
which both genes for eIF4G (TIF4631 and TIF4632)
are deleted and mutant or wild-type eIF4G is supplied from a plasmid
(40). The tif4632-233 mutant eliminates the
interaction of eIF4G and Pab1p (40), but we found that
this mutation has no effect on the preference for poly(A)+
mRNAs in vivo (Fig. 3). The ratios of
translation rate for C+A+ to C+A mRNA were 46 in the wild type and 47 in the tif4632-233 mutant. The ratios for CA+ to CA
were 42 for the wild type and 47 for the mutant. These results suggest
that, while Pab1p is a mediator of the poly(A) effect on translation,
it must have an action in addition to its interaction with eIF4G.
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FIG. 3.
Isogenic strains 4G1 (wild type) and 4G2
(tif4632-233) were electroporated with 2 µg of RNA. Cells
were maintained at 25°C and assayed for luciferase activity at the
indicated times as described in the text.
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3' poly(A) and the 60S joining step.
Both eIF5B (Fun12p) and
eIF5 (Tif5p) are known to be involved in the 60S ribosomal subunit
joining step (5, 27). We found that deletion of
FUN12 specifically diminished the translation of
poly(A)+ mRNA and had far less effect on non-poly(A) mRNA
expression (Fig. 4). The
fun12 mutation decreased the rate of translation of C+A+ mRNA by >10- fold but reduced the rate of C+A mRNA by only 1.3-fold. This result suggests that the 3' poly(A) structure is important for the
Fun12p-dependent step, namely, 60S ribosomal subunit joining. In the
absence of Fun12p, poly(A) had only a small effect on translation efficiency. The contrast of fun12 and
gcd11-508 is shown in Fig. 5.
While fun12 affected primarily poly(A)+ mRNA,
gcd11-508 (eIF2) affected translation of
poly(A)+ and poly(A) mRNAs to a
comparable extent.
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FIG. 4.
Isogenic strains (A) J115 (wild type) and J116
(fun12) or (B) isogenic strains KAY71 (wild type) and
KAY73 (ssu2-1/tif5) were electroporated with 2 µg of RNA.
Cells were maintained at 25°C and assayed for luciferase activity at
the indicated times as described in the text.
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FIG. 5.
Comparison of effects of fun12 and
gcd11-508 mutations on translation rates for
poly(A)+ and poly(A) mRNAs. The comparison is
for C+A+ and C+A mRNAs.
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We obtained similar results with a
tif5 (
ssu2-1)
mutation affecting eIF5 (Fig.
4). In this case we saw a decrease of
2.5-fold
for C+A+ mRNA. The effect was not as great as for the
fun12 strain,
perhaps because, unlike
FUN12,
TIF5 is an essential gene and cannot
be completely inactivated for
the
assay.
Ski2p, Fun12p, and the poly(A) requirement.
In a
FUN12+ strain, a ski2 deletion
resulted in a dramatic increase in the efficiency of expression of
non-poly(A) mRNA (21). Like Ski2p, Slh1p is a nonessential
RNA helicase that represses dsRNA virus copy number (20).
In the ski2 slh1 double mutant poly(A)+ and non-poly(A) mRNAs were translated with equal
efficiency for the same duration (35) (Fig.
6). We found that deletion of
FUN12 almost completely eliminated this effect (Fig. 6).
Most of the increased translation efficiency of a C+A mRNA seen in a
ski2 slh1 double mutant was lost in the
fun12 ski2 slh1 strain. This
result is consistent with a model in which Ski2p and Slh1p act through
Fun12p.
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FIG. 6.
Strains 3221 (wild type), 4107 (ski2
slh1), J116 (fun12), and AMS3
(ski2 slh1 fun12) were
electroporated with 2 µg of RNA. Cells were maintained at 25°C and
assayed for luciferase activity at the indicated times as described in
Materials and Methods. Symbols:  , wild type;  , ski2
slh1;  , fun12;  , ski2
slh1 fun12.
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Expression of LUC mRNAs and mRNA turnover.
In addition to
translation rates, mRNA turnover can also affect the expression of LUC
mRNAs, and both cap and 3'poly(A) affect each process. We used several
approaches to distinguish these effects. 32P-labeled LUC
mRNAs were electroporated, and their degradation was measured over time
by extraction, analysis on gels, and autoradiography. It was found that
C+A+, C+A, CA+, and CA LUC mRNAs all had similar half-lives in
the wild type and in ski2 and ski6 mutants (2, 21). Thus, this system did not reflect the known
greater instability of mRNAs lacking cap or poly(A), but this result
does argue that differences measured in our experiments are due to differences in translation rather than mRNA turnover. Similar experiments comparing ski2 slh1 double
mutants with an isogenic wild-type strain gave similar results (data
not shown).
As a second approach to examine the role of mRNA turnover in our
results, we examined directly the effect of the
ski2
slh1 double mutation on turnover of endogenous mRNAs. We
found no effect
on the turnover of
URA5 or
STE2
mRNAs of the double mutation (Fig.
7),
nor was the stability of
ACT1 or
PGK1 mRNAs
affected (
35).
This confirmed earlier results indicating
that unless the major
Xrn1p 5'
3' mRNA degradation system is blocked,
the
ski2 mutation
has no effect on mRNA turnover
(
18). We further checked that
the electroporation
procedure does not inactivate the Xrn1p-catalyzed
mRNA degradation
system (data not shown).
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FIG. 7.
The ski2 slh1 double
mutation does not affect turnover of endogenous STE2 or
URA5 mRNAs. Transcription was arrested by the addition of
thiolutin (gift of Edmund Hafner, Pfizer, Groton, Conn.), and the RNA
was extracted and analyzed by Northern hybridization (26a).
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A third criterion for distinguishing the effects on mRNA turnover from
those on translation are the kinetics of luciferase
synthesis.
Differences in expression at the earliest times are
most likely due to
translation rates and, in cases where the expression
is proceeding
linearly, rates can be clearly distinguished from
yields. Functional
half-lives of electroporated C+A+ and C+A
LUC mRNAs varied little
between wild-type cells and
ski2 slh1 double mutants (
35). Moreover, the half-life in wild-type
cells
of electroporated C+A+ mRNA (62 min) was only about twice that
of
C+A
mRNA (36 min), a difference insufficient to account for
the
>40-fold difference in the expression of these two species
(
35). Thus, the bulk of the effects we report here are due
to
changes in translation
rates.
Specifically, in the
fun12 strain, expression from C+A
LUC mRNA has not plateaued before 60 min (Fig.
4A, right), so rates
are
being measured. The same is true of the expression from C+A
mRNA in
the
gcd11-508 mutant (Fig.
1A, right). The rate on C+A
mRNA is lowered fivefold by
gcd11-508 but is not
significantly
changed by
fun12. This result shows again
that translation of
C+A
mRNA is affected by the
gcd11
mutation but not by the
fun12 mutation. The mRNA
stability is not the process affected. The
data for the
ssu2-1 (
tif5) also shows an effect only on
poly(A)
+ mRNAs (Fig.
4), but enzyme accumulation plateaus
early in the
time course, so it remains possible that an effect on
translation
is obscured by rapid degradation of the non-poly(A)
mRNA.
The
pab1 mutation substantially affects translation of
poly(A)
+ mRNA (Fig.
2). On C+A
mRNA, expression ceases
quickly in both
the
spb2-1 and
spb2-1 pab1
mutants, but the double mutant expresses
as much luciferase as does the
single mutant, and the kinetics
are identical, suggesting that there is
little effect of
pab1 on translation of the C+A
mRNA.
However, it is possible in this
case that an effect on translation is
obscured by an opposite
effect on mRNA turnover. Likewise, the
spb2-1 mutant shows no
increase in expression of C+A
mRNA,
contrary to the prediction
of the 40S abundance model, but an increase
in expression might
be obscured here by a simultaneous decrease in
stability due to
the
mutant.
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DISCUSSION |
mRNA electroporation accurately reflects in vivo translation.
The translation of electroporated mRNAs faithfully reflects the in vivo
process because it takes place in living viable cells and because it
shows the expected requirement for the 5' cap and 3' poly(A) structure
(12). We further show here that mutations in eIF3
(prt1-1) and eIF2 (gcd11-508) result in the
expected diminished translation of all species of electroporated mRNA.
We have used this method to dissect the role of the 3' poly(A)
structure in the translation process, relating it to the poly(A)
binding protein (Pab1p), the initiation factor eIF4G, the factors eIF5
(Tif5p) and eIF5B (Fun12p) involved in 60S subunit joining, and the RNA helicases Ski2p and Slh1p that regulate the poly(A) requirement for translation.
Interaction of eIF4G and Pab1p does not affect the requirement for
3' poly(A) for translation.
In the presence of RNA, the poly(A)
binding protein (Pab1p) binds to a region of eIF4G included in residues
201 to 317 of eIF4G2 or residues 188 to 300 of eIF4G1 (38,
40). Deletion of this region completely abrogates the binding
reaction, as does substitution of residues 233 to 236 of eIF4G2
(RLRK AVAA) or residues 213 to 216 of eIF4G1 (KLRK
AAAA) (40). This interaction has been proposed
to be the mechanism of the poly(A) requirement for efficient
translation of mRNAs. Such an interaction is proposed to enhance
recruitment of 40S subunits through the eIF4G-eIF3 interaction, as well
as promote the recycling of ribosomes that have completed a polypeptide
(reviewed in reference 31). However, elimination of the
Pab1p-eIF4G interaction by mutation of the interacting domain of eIF4G
does not affect the growth rate of yeast cells, suggesting that this
model in its simplest form is unlikely. Furthermore, the effect of
eliminating this interaction on the in vitro system is seen only with
uncapped mRNAs (40). Except for the yeast RNA virus mRNAs,
all yeast mRNAs are believed to be capped, and the requirement for a
poly(A) for translation is nearly absolute.
One alternative to the simple model described above relies on the fact
that if many or most mRNAs lack a poly(A) structure,
as in a
mutant in poly(A) polymerase, non-poly(A) mRNAs may be
found on
polysomes (
17,
29). Removing the competition with
poly(A)
+ mRNAs allows non-poly(A) mRNAs to be utilized. If
the eIF4G-Pab1p
interaction is disrupted and if this is the (or a)
mediator of
the function of the poly(A), this model would suggest that
all
messages would be translated well because none would have the
poly(A) function. A strong prediction of this model is that in
such a
strain, translation of a non-poly(A) mRNA would be as efficient
as
would translation of a poly(A)
+ mRNA.
However, we have found that elimination of the Pab1p-eIF4G interaction,
by substitution of the interacting region of eIF4G,
does not affect the
requirement for poly(A) for translation. Even
the magnitude of the
requirement is unaltered by this change.
This indicates that the
Pab1p-eIF4G interaction is dispensable
for translation and is not the
reason why the 3' poly(A) is essential
for
translation.
spb2-1 suppression of pab1 is not by
reducing the requirement for poly(A).
Although pab1
is lethal, it is well suppressed by any of a number of mutations
producing a deficiency of 60S subunits, including mutations in genes
encoding 60S ribosomal subunit proteins and other factors needed for
ribosome biogenesis (33). All such mutants have an excess
of free 40S subunits, and it has been suggested that these facilitate
40S joining so that the poly(A) structure is no longer as essential for
initiation (39). This model predicts that non-poly(A) mRNA
should be better translated in the mutant than in the wild-type cells.
We tested this hypothesis by examining the effect of spb2-1
on the requirement for poly(A) and found that non-poly(A) mRNA was
actually less efficiently translated and that there remained a 22-fold
stimulation of translation by the presence of the 3' poly(A) structure.
Similar results have also been reported with mutants in the
mak21 gene, which are also involved in 60S subunit
biogenesis (10). This indicates that another mechanism
must be invoked to explain the viability of spb2-1 pab1 strains.
A role for poly(A) in subunit joining.
An earlier model for
the role of poly(A) in translation, based on in vitro data, centered on
a role for poly(A) in promoting the 60S subunit joining reaction
(23). The description of two factors involved in 60S
subunit joining, eIF5 encoded by TIF5 (5) and
eIF5B encoded by FUN12 (6, 27), enabled a new test of this model. We find that defects in either of these factors result in decreased translation of poly(A)+ mRNA but little
change in the translation of non-poly(A) mRNA. This supports the model
in which poly(A) promotes 60S subunit joining by promoting the activity
of Fun12p and Tif5p. Our data support a role of Pab1p in mediating the
action of poly(A). Thus, Pab1p-poly(A) may influence 60S joining with
the 40S subunits at the initiator AUG, thereby increasing the
efficiency of translation of poly(A)+ mRNAs.
Role of Ski2p-Slh1p in the action of poly(A).
In the absence
of the related RNA helicases Ski2p and Slh1p, there is no difference in
either the rate or the duration of translation of poly(A)+
and poly(A) mRNAs (35). This implies that
Ski2p and Slh1p block the translation of non-poly(A) mRNAs. Blocking
the Xrn1p-catalyzed 5'3' degradation of mRNAs reveals an effect of
Ski2p on 3'5' mRNA degradation (18). However, the
experiments reported here and previously use cells and mRNAs not
blocked for the action of the Xrn1p system and, under these conditions,
there is no effect of Ski2p on mRNA turnover. We have further shown
that mRNA turnover is unaffected by the ski2
slh1 double mutation (35), and
electroporated cells have an active Xrn1 degradation system (A. Searfoss and R. Wickner, unpublished data). These and other
considerations (35) indicate that the effects observed
here are on translation.
It was previously hypothesized that the Ski proteins act by affecting
60S ribosome biogenesis to produce a requirement for
the 3' poly(A) for
subunit joining (
21,
25). Indeed,
ski6 mutants
show both alterations of 60S subunit structure and relative
poly(A)
independence of translation, supporting this mechanism
(
2).
Our finding that most of the increased translation of non-poly(A) mRNA
seen in a
ski2 slh1 double mutant is
prevented by
a further
fun12 mutation is consistent with
a model in which
Ski2p and Slh1p block translation by blocking Fun12p
action. An
heuristic model that summarizes these results is as follows:
3'poly(A)-Pab1p
|Ski2p-Slh1p
|Fun12p-Tif5p
60S subunit
joining. This model
qualitatively explains the requirement for poly(A)
in wild-type,
but not in cells defective for downstream components
Ski2p-Slh1p
or Fun12p (Fig.
8). It
further explains why the
ski2 slh1 strains
have elevated translation of poly(A)
mRNAs, whereas the
fun12 strains have depressed translation
specifically of
poly(A)
+ mRNAs. Finally, it explains why the
fun12 mutation is epistatic
to
ski2
slh1.
View larger version (10K):
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|
FIG. 8.
Model of the action of poly(A) to promote 60S ribosomal
subunit joining. Poly(A)-Pab1p inhibits Ski2p-Slh1p which inhibits
Fun12p-Tif5p which promotes 60S ribosomal subunit joining.
|
|
While a molecular explanation will await biochemical studies, one could
speculate that the schematic interactions shown above
reflect actual
protein-protein interactions, with Pab1p-poly(A)
binding to
Ski2p-Slh1p, which in turn bind to Fun12p and/or Tif5p.
Recent
biochemical evidence suggests the 3' end of an mRNA can
affect subunit
joining. Binding of a protein to a 3'UTR site of
lipoxygenase mRNA
inhibits 60S subunit joining in extracts of
erythroid precursor cells
(
26). Other molecular mechanisms could
underlie the
functional pathway above; however, our results do
implicate the 60S
joining step as both a target and a regulated
mediator of
poly(A)-dependent
translation.
| |
ACKNOWLEDGMENTS |
We thank Thomas F. Donahue for allowing us to use the
ssu2-1 mutant prior to publication, and we are grateful to
Alan Hinnebusch, Herman Edskes, and Dan Masison for critical reading of
the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Bldg. 8, Room
225, NIH, 8 Center Dr. MSC 0830, Bethesda, MD 20892-0830. Phone: (301) 496-3452. Fax: (301) 402-0240. E-mail:
wickner{at}helix.nih.gov.
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Abstract
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