(Received for publication, January 11, 1996; and in revised form, February 9, 1996)
From the
Poly(A)-binding protein, the most abundant eukaryotic mRNP protein, is known primarily for its association with polyadenylate tails of mRNA. In the yeast, Saccharomyces cerevisiae, this protein (Pabp) was found to be essential for viability and has been implicated in models featuring roles in mRNA stability and as an enhancer of translation initiation. Although the mechanism of action is unknown, it is thought to require an activity to bind poly(A) tails and an additional capacity for an interaction with 60 S ribosomal subunits, perhaps via ribosomal protein L46 (Rpl46). We have found that a significant amount of Pabp in wild-type cells is not associated with polyribosome complexes. The remaining majority, which is found in these complexes, maintains its association even in yeast cells deficient in polyadenylated mRNA and/or Rpl46. These observations suggest that Pabp may not require interaction with poly(A) tails during translation. Further treatment of polyribosome lysates with agents known to differentially disrupt components of polyribosomes indicated that Pabp may require contact with some RNA component of the polyribosome, which could be either non-poly(A)-rich sequences of the translated mRNA or possibly a component of the ribosome. These findings suggest that Pabp may possess the ability to bind to ribosomes independently of its interaction with poly(A). We discuss these conclusions with respect to current models suggesting a multifunctional binding capacity of Pabp.
Eukaryotic mRNA associates with a variety of proteins, both in
the nucleus as heterogeneous nuclear ribonuclear proteins and in the
cytoplasm as messenger ribonuclear proteins. Both of these complexes
have been implicated in various cellular activities including RNA
processing, nuclear transport, mRNA stability, and translation (1, 2, 3) . The most abundant RNA-binding
protein found in a functional messenger ribonuclear proteins complex is
the poly(A)-binding protein (Pabp), ()which can also be
found within nuclei as an heterogeneous nuclear ribonuclear proteins in
some cell types(1) . Early photo-cross-linking and nuclease
protection experiments revealed that Pabp is typically found in
association with polyadenylated mRNA and more specifically with the
poly(A) tail (4, 5, 6, 7) .
Subsequent isolation and disruption of the Pabp gene (PAB1) in Saccharomyces cerevisiae demonstrated that yeast Pabp is
essential for cell viability, suggesting that the critical role of Pabp
involves its binding interaction with mRNA poly(A)
tails(8, 9) . However, the mechanism for conveying the
major function or functions of Pabp is unknown.
Several studies in vitro suggested that Pabp acts in concert with the poly(A) tail to enhance the translational efficiency of mRNA. For example, experiments in rabbit reticulocyte lysates showed that while excess poly(A) can inhibit translation of capped and polyadenylated mRNA, purified Pabp added in trans can reverse the inhibition(10, 11, 12) . Interestingly, these results also led to a model suggesting an interaction between the 5` and 3` termini of mRNA because the effect of added poly(A) and Pabp was cap-dependent(13) .
The function of S. cerevisiae Pabp in vivo was initially assessed by utilizing a temperature-sensitive allele of PAB1 (pab1-f364l), which at the non-permissive temperature resulted in a translation defect in addition to yielding longer than average poly(A) tails(14) . Moreover, the resultant lethality could be suppressed by a mutation (spb2) in ribosomal protein L46 (Rpl46) without restoration of normal poly(A) tail lengths(14) . This genetic result led to the appealing suggestion that the Pabp and the 60 S ribosome (perhaps via Rpl46) interact, thereby implicating a role for Pabp in translation initiation. Taken together, these studies supported a model in which the Pabp facilitates translation by improving the re-initiation efficiency of a terminating ribosome in cis via interaction with the poly(A) tail.
Pabp from Xenopus laevis was also
shown to be associated with translational activity during frog
development(15) . Analyses, beginning with stage VI oocytes and
ending at the tadpole stage, revealed an increased synthesis of Pabp
mRNA and protein that paralleled an increase in poly(A) content, even
though total mRNA synthesis decreased during this time. In addition,
Pabp was preferentially found in tissues with active protein synthesis,
thereby supporting earlier observations that both poly(A) and Pabp are
associated with the translational apparatus. Interestingly, Pabp levels
are extremely low prior to, and during oocyte maturation, a time when
discrimination between poly(A) and poly(A)
mRNAs plays a key role in regulating maternal mRNA translation (15) . The limited amount of Pabp present during this time
could amplify the relative translational advantage of
poly(A)
compared to poly(A)
mRNAs if
Pabp played a key role in initiation.
Analysis of Pabp amino acid
sequences from both vertebrate and invertebrate organisms revealed a
highly homologous N terminus containing four RNA binding domains (I,
II, III, and IV), each approximately 90-200 amino acids long and
containing both RNP I- and II-type RNA recognition motifs (reviewed in (16) ). Extensive analyses using truncated Pabp peptides
containing individual and/or combinations of RNA binding domains
revealed that the domains do not harbor redundant functions as shown by
differential binding to homoribopolymers and by the ability for
specific domains to rescue the lethality of a PAB1 chromosomal
deletion(9, 17, 18) . Curiously, individual
domains are more closely related to the corresponding domain among
different organisms than to the remaining domains from the same protein
supporting the notion that each domain has evolved distinct functions.
In yeast, only domain IV can maintain viability, but it has relatively
low specificity for binding to poly(A) (9, 18) ; in
fact, domains I and II, together, generate the greatest specificity and
affinity for poly(A) under physiological salt conditions (100
mM NaCl or KCl), yet are dispensable for cell
growth(14, 18) . Moreover, the temperature-sensitive PAB1 allele, pab1-f364l, actually encodes a
truncated, 66-amino acid peptide containing only a mutated domain IV
and C-terminal residues, and as such lacks the strong poly(A) binding
capability(14) . Suppression of this allelic variant by a 60 S
ribosomal protein (Rpl46) may therefore establish a critical
involvement of Pabp in the translational apparatus, independent of a
role in binding poly(A) tails(14) .
In previous studies, we showed that poly(A)-deficient mRNA was efficiently translated in vivo after inactivation of poly(A) polymerase activity in a temperature-sensitive pap1-1 strain of S. cerevisiae(19, 20) . This suggested that poly(A) tails are not required for efficient translation under these conditions and thereby questioned the necessity for a Pabp-poly(A) tail interaction to facilitate translation. In this report, we present evidence that the Pabp of the yeast S. cerevisiae remains associated with polyribosomes in the presence or absence of polyadenylated mRNA in vivo. Furthermore, the non-lethal deletion of RPL46 does not alter the amount nor ribosomal association of Pabp along polyribosome gradients. Taken together, these findings suggest that the essential function of Pabp might not be exerted via direct interactions with poly(A) tails nor Rpl46, adding further complexity to the mechanism of Pabp action. The apparent association of Pabp with ribosomes suggests an undetermined mechanism whereby Pabp might directly interact with another ribosomal protein, a ribosome-associated protein, or with rRNA.
Twenty 0.6-ml fractions were collected per gradient and
trichloroacetic acid added to 12.5%. Proteins precipitated overnight at
-20 °C, followed by centrifugation. Pellets were washed twice
in ice-cold acetone, dried in vacuo, and resuspended in 70
µl of 1 SDS-polyacrylamide gel loading buffer prior to use.
Isolation of RNA from polyribosome gradient fractions (20 0.6-ml fractions/gradient) and subsequent ribonuclease protection analysis were carried out as described previously(20) .
The current model for Pabp function depicts the protein in a RNP complex, linking mRNA to ribosomes by binding to poly(A) tails and by binding to the 60 S ribosomal subunit through contact with ribosomal protein L46 (Rpl46) (reviewed by Jacobson in (22) ). Such interactions are predicted to enhance the rate of re-initiation of ribosomes in cis, thereby increasing the translational efficiency of a mRNA molecule. To test various features of this model, we considered a series of experiments that measure relative cellular and polyribosome-associated Pabp by monitoring its sedimentation profile in polyribosome gradients from S. cerevisiae deficient in poly(A) and/or Rpl46.
Figure 1:
Amounts
of Pabp and Tcm1p in pap1-1 and rpl46 strains grown
at 25 °C and 35 °C. Strains with the indicated genotype were
grown at 25 °C to similar densities. Half the culture was
harvested, and the remaining half was shifted to 35 °C and
harvested after 1 h. Polyribosome lysates were prepared as described
under ``Experimental Procedures.'' Equal numbers of A units were separated by SDS-polyacrylamide gel
electrophoresis, and the proteins were electrophoretically transferred
to nitrocellulose. Protein levels were determined by immunoblotting
with monoclonal antibodies to each protein and detection with a common
fluorescein-conjugated secondary antibody and quantitated using a
Molecular Dynamics FluorImager 575. Pilot experiments demonstrated that
the levels of protein detected here fall within the linear range of
detection.
We also monitored Pabp levels in a strain containing a deletion of RPL46, since a mutation (spb2) in this non-essential gene suppresses the lethality of a yeast strain containing a mutant Pabp(14) . This suggested that Pabp might functionally interact with Rpl46 and that a chromosomal deletion of RPL46 might alter total steady-state Pabp levels by disrupting an important functional binding interaction. Such disruption might lead to a large pool of dispensable Pabp whose degradation or autoregulation leads to a new steady-state amount of protein. We constructed PAP1 and pap1-1 strains harboring a deletion of RPL46 by replacement of the chromosomal allele with one containing a URA3 disruption (see ``Experimental Procedures''). The deletion results in a cold-sensitive growth phenotype and reduces the normal ratio of 60 S to 40 S ribosomal subunits (see below). Protein lysates from the deletion strains (PAP1, rpl46 and pap1-1, rpl46) were prepared as above and analyzed by Western blot (Fig. 1). Table 1shows the relative amounts of Pabp in RPL46 deletion strains grown at 25 °C and after shifting to 35 °C for 1 h and indicates that Pabp levels do not change significantly, even in the absence of Rpl46. In addition, we also measured relative amounts of the 60 S ribosomal protein, Tcm1p, and found a decrease in its level in RPL46 deletion strains, consistent with a response to a reduction in 60 S ribosomal subunit assembly as observed by deletions of other large subunit ribosomal proteins(24, 25) . Taken together, these findings demonstrate that sudden reductions in poly(A) content or the absence of Rpl46 do not significantly alter total levels of Pabp.
Figure 2:
Polyribosome profiles of pap1-1 and rpl46 strains grown at 25 °C and 35 °C.
Strains with the indicated genotype were grown at 25 °C to similar
densities. Half the culture was harvested, and the remaining half was
shifted to 35 °C and harvested after 1 h. Polyribosome lysates were
prepared as described under ``Experimental Procedures.''
Polyribosomes were separated from 80 S ribosomes and subunits by
ultracentrifugation through 15-50% sucrose gradients. Gradients
were fractionated after continuous monitoring at A, shown below. The position of 80 S, 60 S, and
40 S ribosomes is indicated in each
profile.
Figure 3: Distribution of Pabp in polyribosome gradients from pap1-1 and rpl46 strains grown at 25 °C and 35 °C. Proteins in fractions from the gradients shown in Fig. 2were separated and detected as described in the legend to Fig. 1. The direction of sedimentation is from right to left. Fractions 11 and 12 and fractions 8 and 10 from pap1-1, rpl46 (35 °C) and PAP1, rpl46 (25 °C), respectively, were lost during sample preparation. Corresponding values for Table 3were derived from additional Western blots of these strains.
To determine the impact of a deletion of RPL46 on the levels of polyribosome-associated Pabp, we fractionated polyribosome lysates obtained from PAP1, RPL46; PAP1, rpl46; pap1-1, RPL46; and pap1-1, rpl46 strains grown either at 25 °C or following a 1-h shift to 35 °C. Again, proteins from each fraction were separated by electrophoresis and transferred to nitrocellulose for Western analysis. Fig. 3shows the distribution of Pabp along the polyribosome gradients of each strain. We find that a deletion of RPL46 does not change the relative amount of polyribosome-associated Pabp (Table 2); however, there is shift in Pabp distribution coincident with the redistribution of polyribosomes due to the translation initiation defect seen in PAP1, rpl46 and pap1-1, rpl46 strains at 25 °C. At 35 °C in a pap1-1, rpl46 strain, the Pabp and polyribosome distributions are restored to that observed in strains with a normal RPL46 allele present, paralleling the restoration of a normal ratio of mRNA to ribosomes (Fig. 2). These data indicate that Pabp sediments in association with polyribosomes in the absence of Rpl46.
Figure 4:
Effect of EDTA, RNase A, and high salt
treatment on the relative distribution of Pabp, Tcm1p, and TCM1 mRNA in polyribosome gradients from a pap1-1, rpl46 strain grown at 25 °C and shifted to 35 °C. A,
polyribosome lysates from a pap1-1, rpl46 strain were treated
with EDTA, RNase A, or 0.8 M KCl as described under
``Experimental Procedures,'' the polyribosomes separated by
ultracentrifugation through 15-50% sucrose gradients, and the
levels of Pabp and Tcm1p determined as described in the legend to Fig. 1. The positions of Pabp and Tcm1p are indicated at the right of the figure. HS, high salt (0.8 M KCl). B, effect of EDTA on the sedimentation properties
of poly(A)-deficient TCM1 mRNA. Fractions from an EDTA-treated
gradient were subjected to RNase protection analysis with a uniformly P-labeled 226-nucleotide riboprobe complementary to the
last 166 nucleotide of TCM1 mRNA and the first 17 nucleotides
of its poly(A) tail(20) . The products were separated by
electrophoresis and visualized by autoradiography. Lane P contains the probe mixed with DNA molecular size markers (217,
201, 190, 180, 160, and 147 nucleotides). Lane T is RNase
protection products of unfractionated RNA. Under these conditions, the
RNase protection probe detects two species of TCM1 mRNA; TCM1 A
has poly(A) tail lengths between 10
and
20 residues, while TCM1 A
has no poly(A)
tail(20) .
Figure 5: Relative sedimentation properties of Tcm1p, Pabp, and TCM1 mRNA in EDTA-treated polyribosome gradients from a pap1-1, rpl46 strain after a 1-h shift to 35 °C. The graph shows the percent of total Tcm1p, Pabp, and TCM1 mRNA found in each fraction of polyribosome gradients shown in Fig. 4A. Tcm1p and Pabp levels were quantitated by FluorImager analysis of Western blots probed with fluorescein-conjugated secondary antibodies directed against monoclonal antibodies specific to Tcm1p and Pabp. TCM1 mRNA levels were quantitated by storage PhosphorImager analysis of the gel illustrated in Fig. 4B.
About half of the Pabp in EDTA-treated
lysates from poly(A)-deficient cells sediments in the portion of the
gradients containing free 60 S subunits (Fig. 4A and Fig. 5, Table 3), suggesting that Pabp may have an
affinity for ribosomes independent of its ability to bind poly(A).
Alternatively, Pabp may sediment in this portion of the gradient by
virtue of binding to mRNA. We monitored the sedimentation behavior of a
representative mRNA (TCM1, 1200 nucleotides) encoding
ribosomal protein L3 in an effort to differentiate between these two
possibilities. After inactivation of polyadenylation, RNase protection
analysis reveals that this mRNA accumulates as a poly(A)
species (TCM1 A
) and a poly(A)-deficient
species (TCM1 A
) that has poly(A) tails
between about 10 and 20 nucleotides ((20) ; Fig. 4B). The majority of each TCM1 mRNA
species sediments in a peak in fractions 13-16, which partially
overlaps the fractions containing 60 S ribosomes (revealed by Tcm1p; Fig. 4A and Fig. 5). Since the majority of TCM1 mRNA sediments in the portion of the gradient expected of
ribosome-free mRNA after EDTA treatment, we suggest that the Pabp
sedimenting in heavier fractions (fractions 9-13) could be doing
so by virtue of its ability to interact with ribosomes independently of
mRNA. The fact that a significant fraction of Pabp sediments with
poly(A)-deficient polyribosomes is consistent with the possibility that
Pabp may contact ribosomes independently of mRNA. Nevertheless, the
sedimentation behavior of Pabp with ribosomal subunits observed in Fig. 4A could result from binding to small amounts of
mRNA still associated with ribosomes under these conditions.
The poly(A)-binding protein from several organisms has been
the subject of many studies attempting to elucidate the critical
function or functions of this molecule. Its early detection by UV
cross-linking and nuclease protection experiments showed that Pabp
bound to mRNA via the polyadenylate tail and that the protein was
extremely abundant (4, 5, 6, 7) .
These primary observations established the notion that Pabp exerts an
important function in association with the poly(A) tail, and early
studies suggested that the function was to control 3`-exonucleolytic
activity by establishing a physical barrier to such enzymes, thereby
regulating stability of the mRNA(27, 28) . This
proposed function was consistent with models generated for poly(A) tail
function which feature a role in enhancing mRNA stability and
implicated the Pabp as the mediator of this effect. Recent findings
showing that Pabp defects cause premature decapping and decay of
poly(A) mRNA in yeast also indicate an important role
for this protein in mRNA decay(29) .
Pabp was assigned an
additional role as a stimulatory factor for translation initiation of
poly(A) mRNA. This was determined by genetic
strategies where a temperature-sensitive allele (pab1-f364l)
of the S. cerevisiae Papb gene (PAB1) was expressed
at non-permissive temperatures resulting in an apparent translation
defect as determined by examination of polyribosome profiles 14).
Moreover, one suppressor (spb2) of this temperature-sensitive
allele was a mutant form of the 60 S ribosomal protein L46 (Rpl46),
suggesting an interaction between Pabp and the 60 S
subunit(14) . Other studies have also implicated Pabp as a
translation enhancing factor, since inclusion of this protein in
mammalian translation extracts containing synthetic mRNAs facilitated
the recovery of poly(A)
rather than
poly(A)
mRNA translation initially repressed by
addition of poly(A). Here, again, the translational role of Pabp is
apparently exerted through the poly(A)
tail(10, 11, 12) . More recently, Tarun and
Sachs (30) provided evidence that Pabp may play a role in
enhancing the formation of 48 S preinitiation complexes in yeast.
In this report, we present evidence that the polyribosome binding function of S. cerevisiae Pabp apparently does not require mRNA with normal length poly(A) tails. In addition, we propose that a fraction of Pabp may associate with ribosomes independently of mRNA and that this association may involve an RNA or protein component of the ribosome. Our experiments demonstrated comparable distributions of Pabp along polyribosome gradients from both PAP1 and pap1-1 strains shifted to 35 °C, suggesting that Pabp may not require poly(A) tails for its association with polyribosomes. We also determined that Pabp does not require Rpl46 for its ribosomal association, since deletion of the RPL46 gene in either PAP1 or pap1-1 backgrounds does not interfere with the co-sedimentation of Pabp and polyribosomes. From these results, we conclude that Pabp may not require poly(A) tails or Rpl46 for its essential function or functions.
The Pabps from several organisms ranging from invertebrates to mammals display many similarities and extensive homology in the N-terminal portion of the protein, which contains four RNA binding domains (RBDs) approximately 100 amino acids long, each containing the conserved RNP I (8 amino acids, ``octamer'') and II (6 amino acids) motifs, as well as additional conserved hydrophobic residues found throughout each domain. The N terminus was therefore predicted to harbor the activity necessary for binding to, for example, the poly(A) tails of mRNA. Structure/function studies using deletion mutants of yeast and amphibian Pabp have revealed that each RNA binding domain is not functionally equivalent within a single protein but may have comparable activity to the analogous domain in Pabps from different organisms(17, 18) . The Pabp from X. laevis binds specifically to poly(A) and with significant affinity to poly(G), while having a lesser capacity to bind poly(U) in vitro. Truncated Pabps from X. laevis revealed that Domains I and II are necessary for specific RNA binding to poly(A)(17) . Domain IV, alone, can bind poly(A) and poly(U) but with little discrimination; in fact, this domain binds better to poly(U) and more poorly to poly(A) compared to the full-length Pabp. Domains II and III together were found to have normal discrimination and binding efficiencies whereas domain I alone could not bind RNA.
In S. cerevisiae, domain IV joined with C-terminal residues
was sufficient to maintain viability in a strain lacking a functional
chromosomal copy of Pabp, suggesting that the critical function lies
within this sequence(9, 18) . Interestingly, domain IV
is the most conserved RNA binding domain among Pabps from S.
cerevisiae, Schizosaccharomyces pombe, X.
laevis, and humans (18) . In addition, this truncated
peptide was shown to bind poly(A) in vitro, but in the absence
of competitor RNA(9) . In contrast, binding studies of all four
RNA binding domains from S. cerevisiae demonstrated a lack of
poly(A) binding specificity for individual domains, while only domains
I and II together displayed the strongest and most specific poly(A)
binding activity at physiological salt concentrations (0.1 M KCl or 0.1 M NaCl) in the presence of
heparin(18) . Under these conditions, domain IV bound better to
poly(U) and poly(G) than to poly(A). Finally, this study also confirmed
that domains I and II are not necessary for cell viability. The SPB (suppressor of poly(A)-binding protein) mutations, including rpl46 (spb2), were selected to suppress the defective
function of a truncated Pabp containing only the essential domain IV
and C-terminal sequences lacking the strong poly(A) binding
activity(14) . Moreover, the allele of PAB1 used for
suppression analysis contained an additional point mutation in domain
IV, suggesting that the suppressors may bypass a non-poly(A) binding
function of Pabp.
A recent study of HeLa cell Pabp showed that the
protein was extremely abundant in the cytoplasm with a concentration of
approximately 4 µM and with only 30% of Pabp binding with
high affinity (K
7 nM) to available
poly(A) tails (31) . This suggested that the majority of Pabp
in HeLa cells could bind to alternative, non-poly(A), RNA sequences
having lower affinities (K
> 0.5
µM) for Pabp. Evidence for such a conclusion was obtained
from a ``Selex''-type experiment predicting Pabp binding to a
non-poly(A)-rich heptameric sequence. In addition, a previous study
demonstrated that HeLa cell Pabp can be photo-cross-linked to RNA in
cells labeled with either [
H]adenosine or
[
H]uridine, suggesting that the protein can bind
RNA sequences in addition to the poly(A) tail (32) . Finally,
Drawbridge et al.(33) reported that at least 90% of
sea urchin Pabp was uncomplexed to poly(A). Taken together, these
findings support the possibility that the essential function of HeLa
cell Pabp might be carried out by RNA contacts other than the poly(A)
tail.
The data presented here support the possibility that Pabp may have multiple functions that do not require a high affinity for mRNA poly(A) tails. An interaction with poly(A) may facilitate some function of Pabp, but lack of this sequence apparently does not inhibit the vital cellular role of Pabp. We demonstrated that Pabp remains associated with polyribosomes in the absence of poly(A) tails. This association may result from binding to a non-poly(A) RNA sequence, which might be a component of the ribosome, or it may result from a protein-protein interaction such as an undetermined ribosomal protein or associated factor. We have also shown by quantitation of Western blots derived from fractionated polyribosome gradients that a large fraction (approximately 40%; see Table 2) of Pabp does not sediment with polyribosomes, arguing that, similar to the situation found in HeLa cells and sea urchins, Pabp exists in excess of poly(A) binding substrate(31, 33) . These results support the idea that the mechanism through which Pabp may enhance translation initiation is more complicated than an interaction with ribosomes via the poly(A) tail and offers the intriguing possibility of other potential Pabp contacts in the polyribosome RNP.
We have also provided evidence that polyribosome-associated Pabp does not require the presence of Rpl46 in the 60 S ribosomal subunit. This finding is somewhat surprising, since a mutant of Rpl46 (spb2) suppresses the lethality of a temperature-sensitive allele of Pabp (pab1-f364l) containing only the essential RBD IV and C-terminal sequence(14) . In this case, suppression may be through an indirect interaction with Pabp or perhaps a bypass mechanism. Consistent with a bypass mechanism, suppression of pab1-f364l by rpl46 mutations does not lead to restoration of abnormally long cytoplasmic poly(A) tails found in cells at non-permissive temperature(14) , and structure/function studies demonstrate that the RBD IV and C-terminal sequence comprising Pabp-f364l lack an efficient poly(A) binding capacity(18) . These considerations imply that if Pabp's ribosomal association conveys a critical role in translation initiation without the need for poly(A) binding, then we might also expect that the poly(A) status of an mRNA may not significantly alter this function of Pabp.
Finally, some RNA-binding proteins containing multiple RNP I and II motifs bind simultaneously to more than one RNA. For example, U1A binds pre-mRNA through RBD domain II and U1 small nuclear RNA through domain I, supporting the idea that the individual RNA binding domains have evolved distinct functions(26) . The same may hold true for Pabp, whereby domains I and II bind to poly(A) tails while domain IV engages in crucial contacts with other RNA sequences associated with ribosomes.