From the Department of Biochemistry, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
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ABSTRACT |
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Poly(A)-binding protein (PABP) is an important
regulator of gene expression that has been implicated in control of
translation initiation. Here we report the isolation and the initial
structural and functional characterization of the human PABP gene.
Delineation of the promoter region revealed that it directs the
initiation of transcription at consecutive C residues within a stretch
of pyrimidines. A study of the translational behavior of the
corresponding mRNA demonstrates that it is translationally
repressed upon growth arrest of cultured mouse fibroblasts and
translationally activated in regenerating rat liver. Furthermore,
transfection experiments show that the first 32 nucleotides of PABP
mRNA are sufficient to confer growth-dependent
translational control on a heterologous mRNA. Substitution of the C
residue at the cap site by purines abolishes the translational control
of the chimeric mRNA. These features have established PABP mRNA
as a new member of the terminal oligopyrimidine tract mRNA family.
Members of this family are known to encode for components of the
translational apparatus and to contain an oligopyrimidine tract at the
5' terminus (5'TOP). This motif mediates their translational control in
a growth-dependent manner.
PABP1 is the major
cytoplasmic RNA-binding protein in eukaryotes that exhibits a
preferential affinity for poly(A). This highly conserved protein has
been implicated in regulating the initiation of translation ((1, 2) and
references therein), mRNA stability (3), regulation of poly(A) tail
length during the polyadenylation reaction (4, 5), or poly(A)
shortening (6, 7).
Study of PABP gene expression in various vertebrates has
established the respective mRNA as translationally controlled.
Thus, serum stimulation of quiescent Swiss 3T3 cells seems to
up-regulate the translation of PABP mRNA, as indicated by the
resistance of the induction to actinomycin D treatment (8) and the lack
of change in the level of PABP mRNA (9). Likewise, PABP mRNA is essentially sequestered in messenger ribonucleoprotein in quiescent duck reticulocytes (10), in mouse testis (11), and during early
Xenopus embryogenesis (12).
TOP mRNAs encode for various components of the translational
apparatus, like ribosomal proteins (rp) and elongation factors 1 Growth-dependent translational control of an mRNA
generally correlates with the presence of an oligopyrimidine stretch at its 5' terminus. Yet, the linkage between these functional and structural features is not an absolute one. Thus, human Isolation of Genomic Clones--
A human genomic library from
W138 cell line of lung fibroblasts (Stratagene) was screened with a
32P-labeled probe (1588-bp ScaI-EcoRI
fragment) derived from the human PABP cDNA (24). The initial screen
of 2 × 105 plaques (one genome equivalence) yielded
33 positives, of which three different clones were isolated following a
more stringent wash regime (0.3 M NaCl, 0.03 M
sodium acetate at 67 °C) in the second and third cycles of plaque
purification. Restriction enzyme mapping and sequencing of the
corresponding inserts established that one is pseudogene 1 and the
other two are processed pseudogenes 2 and 3. Processed pseudogene 2 contains the entire sequence of PABP cDNA, whereas the nucleotide
sequence determined in processed pseudogene 3 corresponds to positions
1941 to 2848 in this cDNA. Pseudogene 1 contains a sequence that
corresponds to nucleotides 1357 to 1953 of PABP cDNA and is flanked
by a nonrepetitive sequence.
A second screen of 8 × 105 plaques of the same
library was carried out using replicate Nytran filters (Schleicher & Schuell) with either a 263-bp EcoRI-StuI
5'-terminal region of PABP cDNA or a 200-bp
XhoI-PstI fragment derived from a region flanking the PABP insert in pseudogene 1. Sixteen positive clones were isolated,
and their DNAs were subjected to restriction enzyme digest and Southern
blot hybridization with seven nonoverlapping probes spanning the entire
PABP cDNA. Two overlapping phage clones were selected for further
analysis:
After preliminary mapping with restriction enzymes, selected fragments
were excised and subcloned into plasmid vectors. To determine the
sequence of the promoter as well as exon-intron junctions, all exons
were sequenced using primer walking. Upon identification of each
exon-intron junction, a complementary primer was synthesized and used
to sequence the opposite strand.
DNA Sequencing--
Double-stranded plasmid DNA was sequenced
either manually by the dideoxy method (25) using a Sequenase kit
(U. S. Biochemical Corp., Cleveland, Ohio) or automatically by a
DNA sequencer (ABI 377, Applied Biosystem Inc., Foster City, Ca).
Primer Extension--
Determination of the transcription start
site in hGH chimeric transcripts was carried out by primer
extension as described previously (16).
Cell Culture and DNA Transfection--
NIH 3T3 mouse fibroblasts
were grown, transfected, and arrested as described (16).
Animals--
Adult (6- to 8- week-old) male Sabra rats (Wistar
origin) were obtained from the Hebrew University breeding center.
Partial hepatectomy, resulting in the removal of 70% of the liver
mass, was performed on male rats as described by Higgins and Anderson (26). Sham-operated rats were laparotomized, and their livers were
manipulated but not excised.
Polysomal Fractionation and RNA Analysis--
Harvesting and
lysis of cells as well as size fractionation of polysomes by
sedimentation through sucrose gradients were performed as described
(27). When polysomal gradients were divided into two fractions
(polysomal and the subpolysomal), RNA was extracted from each fraction
by RNAzol B (Biotecx Laboratories, Houston, Texas), according to the
supplier's instructions, and the poly(A)+ mRNA was
isolated as described (28). In all cases where sucrose gradients were
divided into 12 fractions, RNA was extracted as described (28) and
analyzed without further enrichment through a oligo(dT) column. RNA
(Northern) blot analysis was performed as described (29).
Quantification of the radioactive signals on the blots was carried out
by BioImaging Analyzer (Fujix BAS 1000, Fuji, Japan). To assess the
effectiveness of the growth arrest treatment and the selectivity of the
effect on TOP mRNAs, we compared in each case (even if not shown)
the polysomal association of a chimeric mRNA with that of
endogenous rp mRNA and non-rp mRNA from the same polysomal
gradient. Only experiments in which both these controls exhibited their
typical translational behavior (repressed and unrepressed,
respectively) were included.
Plasmid Constructions--
Standard protocols were used for all
recombinant DNA technology (30).
pPABP-GH1 was constructed through the following steps. (i) An
~1.0-kbp PstI fragment, spanning positions
pPABP-GH2 was constructed through the following steps. (i) An
~2.45-kbp filled-in NotI XbaI fragment,
spanning positions
pPABP-GH3 was constructed by inserting a 266-bp filled-in
AvaI-BamHI fragment (derived from pPABP-GH1),
which spans position
pPABP-GH4 was constructed by inserting a 683-bp
ApaI-SacI (derived from pPABP-GH1) between
filled-in SalI and SacI sites of p0GH. The GH in
the resulting plasmid is preceded by the promoter region (positions
pPABP-GH5 was constructed by inserting a 864-bp
HindII-trimmed (by T4 DNA polymerase)-BglI
fragment (derived from pPABP-5'f (see above)), which spans positions
pPABP-GH6 was constructed through the following steps. (i) A 217-bp
fragment was generated by PCR using oligonucleotide primers hGH-3 (16)
and PABP-41 (GCTCTAGAGTGCGGCGCGGGGTAT; boldface letters correspond to nucleotides
The strategy used for the construction of pPABP-GH7 was similar to that
described for pPABP-GH6 except for the usage of oligonucleotide PABP-42
(GCTCTAGAGCTCTTTCCTCCTGTT; boldface letters
correspond to nucleotides
pPABP-GH8 was constructed through the following steps. (i) An ~440-bp
fragment was generated by PCR using oligonucleotide primers pUC-1 (16)
and PABP-43 (CGGGATCCAAGCACCGCCTCCTGCA; boldface
letters correspond to nucleotides
pPABP-GH11s and pPABP-GH11(as) were constructed by annealing two
complementary oligonucleotides: PABP-47
(GATCCTTCTCCCCGGCGGTTAGTGCTGAGAGTGC; boldface
letters correspond to nucleotides +3 to +33 of PABP gene) and PABP-48
(GATCGCACTCTCAGCACTAACCGCCGGGGAGAAGG; boldface
letters correspond to nucleotides +34 to +4 of PABP gene. The
underlined nucleotides correspond to the protruding ends of BamHI site). The resulting double-stranded oligonucleotide
was inserted into the BamHI site of PABP-GH8 in sense and
antisense orientations, respectively. The structure of all constructs
described here was confirmed by DNA sequencing.
Quantitative Analysis of Human Growth Hormone--
The amount of
growth hormone secreted by cells to the medium during the last 3 h
of incubation was determined in an aliquot of the medium by
radioimmunoassay with a commercial kit (Nichols Institute, San Juan
Capistrano, CA).
Molecular Probes--
The isolated fragment probes used in the
Northern blot analysis were a 1.15-kb PstI fragment
containing mouse Organization of the Human PABP Gene--
A human genomic library
in
The gene consists of 15 exons totaling 2.86 kb and 14 introns covering
about 22 kb. The sequences flanking the exon-intron junctions conform
to the consensus for 5' and 3' splice sites (Table
I).
The entire exonic sequence is 12 nucleotides longer than that of the
published cDNA sequence (24). This dissimilarity is part of several
differences summarized in Table II, of
which the major one is an insertion of 9 nucleotides leading to the
addition of 3 amino acids (Lys-Phe-Gly) following amino acid residue
212 in the published sequence (24). The sequence and the relative location of these three residues is identical to that found in mouse
PABP protein and is similar to the corresponding sequence in
Xenopus laevis protein (Table II). The isolated human gene encodes for a protein that is identical in size to that deduced from
the mouse cDNA and differ by only three conservative replacements: Lys to Arg, Glu to Asp, and Ser to Thr at positions 176, 259, and 576, respectively (35). In light of the apparent evolutionary conservation
of PABP amino acid sequence, it is conceivable that the missing three
residues in the original report (24) is because of a sequencing error,
whereas other differences might simply reflect polymorphism in the
human population.
Interestingly, the PABP genes from Xenopus and human not
only show a high degree of conservation of the amino acid sequence (93% of identical residues) but also an identical distribution of the
four RNA binding domains among the different exons. Each of these
functional domains are divided between two or three exons, which are
the same exons in both species (Fig. 1a and Ref. 36).
5' and 3' Boundaries of the PABP Gene--
Primer extension
analysis of human PABP mRNA was carried out twice using
lymphoblastoid cells. The extended DNA was electrophoresed on a
sequencing gel alongside with sequencing reaction of either the
cDNA clone or the pPABP-GH2. In both cases, they comigrated with
nucleotides corresponding to position
The 3'-end of the PABP gene has been determined to reside at the
nucleotide immediately preceding the poly(A) tail in both the PABP
cDNA and the processed pseudogene 3 (Fig. 2).
It should be noted that scanning of the promoter and the intronic as
well as the 3' flanking sequences of the human PABP gene against the
EMBL data base has revealed significant homology with two ESTs. Thus, a
sequence of the antisense strand within the promoter region (positions
+98 to Functional Characterization of the PABP Promoter--
Conceivably,
the 5'TOP identified in the PABP gene might explain the previous
observation concerning growth-dependent translational control of PABP mRNA. However, examining this possibility is based on assessment of the translational efficiency of chimeric mRNAs containing the 5'TOP of PABP mRNA. Formation of an mRNA with an authentic PABP 5'TOP requires the construction of a chimeric gene containing PABP promoter and the first transcribed nucleotides of PABP
gene followed by the sequence of a reporter gene. Previous such
analyses of several other TOP genes have disclosed critical transcriptional regulatory elements within the first exon or even the
first intron (21). Hence, a prerequisite for functional analysis of the
5'TOP element is a functional characterization of the promoter region.
To this end, we first sequenced the promoter region from position
Fig. 4 shows that the promoter activity
was not affected when the 5'-flanking sequence was shortened from 378 to 234 nt (PABP-GH3). It was decreased about 2-fold when further
shortened to 156 nt (PABP-GH7), dropped abruptly when it included just
72 nt (PABP-GH4), and was completely abolished when only 43 nt were
left (PABP-GH6). The complete loss of the promoter activity apparent in
the last construct might reflect the omission of sequences perfectly
matching the binding sites of ATF and SP1, two ubiquitous transcription factors (Fig. 3). Sequential deletion of sequences within the 5'-UTR
led to 1.5-fold decline in the abundance of hGH mRNA and the
synthesis rate of hGH, when this region decreased from 486 nt
(PABP-GH5) to 32 nt (PABP-GH1). A further decrease by 3- to 5-fold was
detected upon complete deletion of transcribed PABP sequence (compare
PABP-GH1 and PABP-GH8 in Fig. 4). It appears that a regulatory element
residing within the first 32 nt of the transcribed region modulates the
abundance of the GH transcript in an orientation-dependent
manner, as its reinsertion in opposite orientation into pPABP-GH8 does
not resume its activity (compare PABP-GH11(as) and pPABP-GH8 in Fig.
4). These results suggest that PABP promoter extends into the
transcribed region, as has been previously shown for several other TOP
genes (21, 39). Yet, we cannot formally exclude the possibility that
the first 32 nucleotides of PABP mRNAs play a role in stabilization
of the transcript. Whatever the mechanism, based on these observations, we used in subsequent experiments a promoter region spanning positions The Translational cis-Regulatory Element of PABP mRNA Resides
within the First 32 Nucleotides and Is
5'TOP-dependent--
Analysis of the polysomal
distribution of PABP mRNA in NIH 3T3 cells reveals that this
mRNA is subject to translation control in a
growth-dependent manner. Thus, PABP mRNA is mostly
associated with polysomes in growing cells and sequestered in
subpolysomal fraction (messenger ribonucleoprotein particles) upon
growth arrest (Fig. 5, endogenous PABP).
Previous attempts to delimit the translational cis-regulatory element
of various TOP mRNAs have shown that it is confined to within their
first 30 nt (16, 18, 23). However, the 5'-UTR of PABP mRNA (505 nt
long) is considerably larger than those of other TOP mRNAs (an
average of 40 nt). Hence, to delineate the corresponding region in PABP
mRNA, we set out to examine the translational behavior of two
mRNAs containing the first 265 nt (PABP-GH2) or just 32 nt
(PABP-GH1) of PABP mRNA. A prerequisite for such an experiment with
chimeric TOP mRNAs is the establishment of the transcription start
site by primer extension analysis. Fig. 6
shows indeed that the major cap sites of the mRNA encoded by
PABP-GH1 are at two consecutive C residues, coinciding with positions
+2 and +3 in the human PABP gene. Likewise, the same start sites were
identified for the PABP-GH2 construct, although they are embedded
within a much larger PABP sequence and their identification required
the use of a different primer (Fig. 6).
Analysis of the polysomal distribution of the mRNAs encoding
PABP-GH1 and PABP-GH2 demonstrates that they are both translationally repressed upon growth arrest of NIH 3T3, as do endogenous mRNAs encoding PABP and EF1 PABP mRNA Is Translationally Regulated during Liver
Regeneration--
To study the translational behavior of PABP mRNA
in whole animals, we exploited the fact that when the liver reaches the
adult stage it becomes quiescent, yet it retains the capacity to resume proliferation after partial hepatectomy. Hence, polysomes from sham-operated and regenerating liver were size-fractionated by sucrose
gradient centrifugation, and the polysomal association of various
mRNAs was assessed by Northern blot analysis of each gradient
fraction (Fig. 7). Our results
demonstrate that the proportion of PABP mRNA associated with
polysomes increased from 37% in sham-operated liver to 72% within
15 h after partial hepatectomy (Fig. 7). A similar recruitment
into polysomes (from 40% in control to 88% in regenerating liver) is
apparent also for EF1 Structural analysis of the human PABP gene as well as of chimeric
genes driven by PABP promoter revealed that the transcription starts
mainly at one or two C residues within an oligopyrimidine stretch. The
presence of 12-nucleotide long 5'TOP appears to provide the structural
basis for its growth-dependent translational control in
fibroblasts (Fig. 5 and Refs. 8 and 9) or adult liver (Fig. 7), and
thus, has established PABP mRNA as a new member of the TOP mRNA
family. Interestingly, PABP 5'TOP sequence is unique among the TOP
mRNAs, as it contains an exceptionally high ratio of C to T (3 to
1). In comparison, the average ratio in 31 vertebrate rp mRNAs is
1.03 ± 0.08, and in five non-rp TOP mRNAs, it is 0.76 ± 0.19 (13). Whether this high C content has an additional regulatory
role is yet to be determined. Nevertheless, shifting the first C of
PABP 5'TOP into position +3 completely abolished the translational
response to growth arrest (PABP-GH11s, Fig. 5). This is consistent with
our previous report that the pyrimidine tract fails to exert its effect
even when preceded by a single A residue (16). Formally, we cannot rule
out the possibility that the loss of translational control is because of the concomitant shortening of the pyrimidine stretch from 12 to 11 residues. However, the latter explanation is less likely, as most 5'TOP
motifs contain eight pyrimidines. Moreover, several 5'TOPs with just
five pyrimidines have been shown to suffice the translational control
mechanism (16-18).
RNA-protein binding experiments have recently provided some clues
concerning putative specific trans-acting factors that might be
involved in the translational control of TOP mRNAs (41-46). Nevertheless, the relevance of proteins that specifically bind to the
5'TOP or the adjacent downstream sequences is still unclear, as the
binding activity remains unchanged under various growth conditions, at
which the translational efficiency of rp mRNAs is repressed or
derepressed. In contrast, a recent report has demonstrated that direct
interaction of repressor molecules with pyrimidine-rich sequences
within the 3' UTR of 15-lipoxygenase mRNA mediates its
translational silencing (47).
Another putative component of the translational control mechanism,
which has recently attracted much attention, is p70 S6 kinase
(p70s6k). Numerous studies have shown that mitogenic or
hormonal stimulations induce phosphorylation of rpS6 by this enzyme
with a concomitant derepression of the translation of TOP mRNAs
(48). Furthermore, inhibition of p70s6k by the
immunosuppressant rapamycin or by expression of a dominant-negative p70s6k mutant selectively repressed the translation of this
class of mRNAs (49, 50). These correlations have led to the
assumption that growth-dependent translational control of
TOP mRNAs involves a p70s6k-mediated signal transduction pathway.
It is tempting to speculate that the growth-dependent
translational control of PABP mRNA in fibroblasts and liver, as
well as of those encoding rps and EF1 The 5'-UTR of PABP mRNA (505 nt) is substantially larger than the
average length (44 ± 4) of this region in other 36 vertebrate TOP
mRNAs rigorously analyzed (13). An intriguing possibility is that
sequences downstream of the 5'TOP serve an additional regulatory
function(s). Indeed, human PABP mRNA contains an A-rich region
spanning position 73 to 123, which is evolutionarily conserved from
yeast (Ref. 51 and references therein). The identification of this
motif led to the hypothesis that PABP mRNA is autogenously regulated at the translational level through binding of the resulting protein to the 5'-UTR (52). In vitro experiments have shown that in the addition of PABP to a cell-free translation system selectively inhibits the translation of PABP mRNA and that this repression is mediated through the A-rich region (51, 53). Nevertheless, inactivation of poly(A) polymerase in yeast, which is
followed by the loss of poly(A) and consequently to an increase in the
ratio of PABP to poly(A), had only a minor effect on the total level of
PABP (54). The regulatory role, if any, of this conserved motif in
mammalian cells has yet to be determined.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and
2 (EF1
and EF2). These mRNAs are candidates for
growth-dependent translational control mediated through a
translational cis-regulatory element. This approximately
30-nucleotide-long element is composed of a cytidine residue at the cap
site followed by an uninterrupted stretch of up to 13 pyrimidines
(13-15) and sequences immediately downstream (16, 17).
-tubulin mRNA is refractory to growth arrest in all of the examined cell lines, although it contains a bona fide translational cis-regulatory element including a 5'TOP, which is able to confer translational control on heterologous mRNA (18). In contrast, the 6.0-kb
transcript of insulin-like growth factor II is translationally
regulated in a growth-dependent manner (19), yet it does
not contain a 5'TOP (20). In light of these exceptions, the structural
basis for the translational control of PABP mRNA could not be
unequivocally predicted. Moreover, the cumulative experience in
assessing the regulatory role of 5'TOP motifs in the expression of
various TOP genes has underscored the prerequisite to delimit the
corresponding promoter regions (16, 21-23). Hence, to establish the
structural base for the translational behavior of PABP mRNA, we set
out to clone and characterize the corresponding human gene. Our
structural and functional analyses have established the transcription
start site of PABP gene within a stretch of pyrimidine leading to the production of a TOP mRNA with an exceptionally long 5'-UTR. The translational efficiency of PABP mRNA tightly correlates with growth conditions of cultured fibroblasts as well as rat liver. The
translational cis-regulatory element has been delimited to reside
within the first 32 nucleotides of this mRNA and involves the 5'TOP motif.
EXPERIMENTAL PROCEDURE
Top
Abstract
Introduction
Procedures
Results
Discussion
References
E, which hybridized with 5'-end and mid-region probes, and
I, which hybridized with mid-region and 3'-end probes.
378 to +635
of human PABP gene, was inserted into PstI site of pUC18 to
yield pPABP-5'f. (ii) A 450-bp fragment was generated by PCR (31) using
oligonucleotide primers pUC-1 (16) and PABP-8
(CGGGATCCCACTCTCAGGACTAACC; boldface letters
correspond to nucleotides +32 to +15 of PABP gene and are preceded by
an underlined BamHI recognition site) and pPABP-5'f as a
template. (iii) The PCR-generated fragment was cleaved by
HindIII and BamHI. The resulting 410-bp fragment was cloned between the HindIII and BamHI sites of
the promoter-less plasmid p0GH, which contains the hGH gene
(32). The resulting construct contains the hGH gene preceded by a human
PABP sequence spanning positions
378 to +32.
378 to +2070 of human PABP gene, was inserted
between filled-in SalI site and SmaI site of
pUC18 to yield pPABP-5'. (ii) pPABP-5' was cut by StuI and
EcoRI, leaving a PABP sequence spanning positions
378 to
+265 linked to the plasmid. (iii) A 2.1-kbp BamHI
(filled-in) EcoRI fragment containing the hGH gene from p0GH
was inserted between StuI and EcoRI sites of
pPABP-5'.
234 to +32 of the human PABP gene, inbetween
filled-in SalI and BamHI sites of p0GH.
72 to +32) of human PABP gene.
378 to +486 of the human PABP gene, inbetween HindII site
and filled in SalI site of p0GH.
43 to
28 of PABP gene and are
preceded by an underlined XbaI recognition site) and
pPABP-GH1 as a template. (ii) The PCR-generated fragment was made
blunt-ended and then cleaved by BamHI, and the resulting
80-bp fragment was cloned between the HincII and
BamHI sites of p0GH. The resulting construct contains the
hGH gene preceded by a human PABP sequence spanning positions
43 to
+32.
156 to
141 of PABP gene and are preceded
by an underlined XbaI recognition site) instead of PABP-41.
The resulting construct contains the hGH gene preceded by a human PABP
sequence, spanning positions
156 to +32.
1 to
17 of PABP gene and are
preceded by an underlined BamHI recognition site) and pPABP-GH1 as a template. (ii) The PCR-generated fragment was then cleaved by BamHI and HindIII, and the resulting
383-bp fragment was cloned between the HindIII and
BamHI sites of p0GH. The resulting construct contains the
hGH gene preceded by a human PABP sequence spanning positions
378 to
1.
-actin cDNA (33), a 1.56-kb
SacI-EcoRI fragment containing the 3' half of human PABP cDNA (24), a 1.2-kb HindIII fragment
containing rat albumin cDNA (34), a 1.8-kb BglI fragment
containing mouse EF1
cDNA (kindly provided by L. I. Slobin), and a 0.8-kb HindIII fragment containing hGH
cDNA (kindly provided by T. Fogel, Bio-Technology General).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Fix vector was screened as described under "Experimental
Procedures." We isolated two overlapping clones,
E and
I (Fig.
1a), which collectively span
an ~25-kb region that encompasses the PABP gene. Restriction fragment
mapping and Southern blot analysis of the phages and various subclones together with DNA sequencing of selected segments disclosed the complete organization of the PABP gene (Fig. 1b).
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Fig. 1.
Structure of human PABP gene. a,
alignment of the overlapping human PABP genomic clones E and
I
and the PABP gene is shown. The location of restriction enzymes used
for subcloning are indicated: E, EcoRI;
H, HindIII; N, NotI;
P, PstI; S, SacI;
St, StuI; X, XbaI. Exons
are depicted as boxes, intron and flanking sequences are
depicted as thin lines. b, an expanded map of the
exons and adjacent intronic sequences are shown. Exons containing 5'-
and 3'-UTRs as well as RNA binding domains (RBD 1 to 4) are
marked.
Organization of human PABP gene
Sequence diversity between human PABP cDNA and gene
2 and
1 with respect to the
published sequence of human PABP cDNA (data not shown). Circumstantial evidence concerning the transcription start site has
been obtained from sequence analysis of PABP processed pseudogene 2. This gene is flanked on both ends by direct repeats. It has been
previously shown that in many cases the 5' repeat is separated by one
nucleotide from the established cap site (37, 38). Indeed, the 5'
repeat is located just three nucleotides upstream of the 5'-end of the
cDNA (Fig. 2). Taken together, we
have concluded that the transcription of PABP gene initiates at two
consecutive C residues, and the resulting mRNA contains a
5'-terminal oligopyrimidine tract of 12 or 11 residues (Fig. 2). Like
most other TOP mRNAs, the distal cap site is preceded by a few
pyrimidine residues, and the entire pyrimidine tract is flanked by
GC-rich sequences (13). It appears, therefore, that PABP mRNA is a
bona fide TOP mRNA.
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Fig. 2.
The 3' and 5' boundaries of PABP gene.
Sequences at the 5' and 3' termini of human PABP cDNA were aligned
with the corresponding genomic sequences of the active PABP gene,
processed pseudogene 2, and processed pseudogene 3. White
letters represent conserved nucleotides. Dots depict
sequences not included in this alignment, and boxed
sequences are repetitive elements delimiting the processed
pseudogene. The 5'-end was determined by primer extension analysis of
PABP mRNA, and the 3'-end was determined by the nucleotide
preceding the poly(A) sequence in both PABP cDNA and processed
pseudogene 3.
205) show 94.3% homology with a rat ovary mRNA of unknown
function (accession number AI176738). Similarly, a sequence of the
antisense strand spanning the first 108 nucleotides of exon 15 and the
last 132 nucleotides of intron 14 exhibits 96.6% homology with a human
testis mRNA of unknown function (accession number AI140680).
Conceivably, these sequences result from reverse transcription of PABP
mRNA molecules derived by the rare usage of an alternative upstream
transcription start site (AI176738) or of a splicing intermediate
(AI140680).
378
(Fig. 3). PABP promoter includes a
canonical TATAA box spanning positions
30 to
26. In that respect it
is similar to the promoters of EF1
and EF2 genes but differs from those of most rp genes (13). Next, we constructed various chimeras harboring the hGH gene preceded by sequences containing portions of
various lengths of the 5'-flanking sequences and 5'-UTR of the PABP
gene. These constructs were transiently transfected into HeLa cells,
and their expression was assessed by monitoring both the relative
abundance of the resulting transcripts and the amount of hGH secreted
into the medium.
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Fig. 3.
Nucleotide sequence of the promoter and the
first exon of PABP gene. The canonical TATA element is
underlined, and the consensus recognition sequence for
transcription factors SP1 and ATF are overlined. The
transcription start sites are marked by arrows, and the
initiation codon is boxed. The sequence of exon 1 is framed,
and the first nucleotides of intron 1 are presented in lowercase
letters. Left and right brackets delimit
sequences included in PABP-GH (PG) clones, and the numbers refer to the
specific chimeric clones schematically depicted in Fig. 5.
378 to +32 or further downstream.
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Fig. 4.
Delimitation of the PABP gene promoter.
HeLa cells were transiently transfected with 4 µg of various PABP-GH
constructs and incubated for 21 h, and then the medium was
replaced. The amount of hGH secreted during the next 3 h was
measured in an aliquot of the medium. The relative abundance of hGH
mRNA was assessed by Northern blot hybridization of mRNAs
extracted from the cells immediately after aliquoting the medium.
The radioactive signals were quantified by phosphorimaging. In the
schematic presentations of PABP-GH constructs, the 5'-flanking
sequence was denoted as a thin line, and exon 1 was denoted
as a dotted box. The relative promoter activity of the
chimeric genes was expressed either as the amount of secreted hGH or as
the abundance of the corresponding mRNA. The results were
normalized to those obtained for PABP-GH5 and are presented as average
±S.E. of the number of measurements indicated in
parenthesis.
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Fig. 5.
The first 32 nucleotides of PABP mRNA are
sufficient, and the integrity of the 5'TOP is critical for the
translational control. NIH 3T3 cells were stably transfected with
the indicated chimeric GH constructs. Cytoplasmic extracts were
prepared from growing (G) or nongrowing cells
(NG) because of 24 h aphidicolin (5 µg/ml) treatment.
These extracts were centrifuged through sucrose gradients and separated
into polysomal (P) and subpolysomal (S)
fractions. RNA from equivalent aliquots of these fractions was analyzed
by Northern blot hybridization with hGH cDNA for detection of the
chimeric transcripts and the cDNAs for PABP, actin, and EF1 for
the corresponding endogenous mRNAs. The sequences around the
transcription start sites (designated by arrows) of the
human PABP gene or of the respective chimeric construct are indicated
at the left of the autoradiograms.
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Fig. 6.
Determination of the transcription start site
of PABP-GH transcripts by primer extension. 5 µg of
poly(A)+ mRNA from NIH 3T3 stably transfected with the
indicated chimeric PABP-GH constructs were annealed to 5'-end-labeled
synthetic oligonucleotides complementary to nucleotides +31 to +12 of
hGH gene (PABP-GH1 and PABP-GH11s) or to nucleotides +48 to +29 of
human PABP gene (PABP-GH2). The primers were extended with avian
myeloblastosis virus reverse transcriptase, and the extended products
(P) were analyzed on a 6% acrylamide-urea gel alongside a
dideoxy sequencing reaction (A, C, G, T) in which the same primer
(unlabeled) was used. Large and small asterisks
indicate major and minor transcription start sites, respectively.
(Fig. 5). These results indicate that the first 32 nucleotides of PABP mRNA include all the regulatory
elements required for conferring growth-dependent
translational control on a heterologous mRNA. Our primer extension
analyses have demonstrated that endogenous PABP mRNA and mRNAs
encoding PABP-GH1 and PABP-GH2 do not start at the same nucleotide.
However, in all cases, the major transcription start sites are at one
or two C residues within a stretch of four consecutive pyrimidines. To
examine whether substitution of the C residue at the cap site by
purines affects the translational control, we constructed the
PABP-GH11s gene. This gene is similar to that of PABP-GH1, except for
the replacement of the C residues at positions +1 and +2 by the
tetranucleotide GGAT. This change leads to the selection of the new A
residue as the major transcription start site (Fig. 6). The fact that the A at the cap site is followed by a stretch of 11 pyrimidines is not
sufficient to render this mRNA translationally regulated (Fig. 5).
It appears, therefore, that translational control of PABP mRNA is
strictly dependent on the location of the oligopyrimidine tract at the
very 5'-end.
mRNA, another TOP mRNA. In contrast,
albumin mRNA was efficiently translated under both these
conditions, despite a slight unloading from polysomes following the
operation (from 94% in polysomes to 83%). It should be noted that the
translational behavior of albumin mRNA is not exceptional, as any
of the other four non-TOP mRNAs previously examined in the
regenerating liver exhibited no translational activation (40).
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Fig. 7.
PABP mRNA is translationally activated
during liver regeneration. Polysomal (1 to 8) and subpolysomal (9 to 12) fractions from sham-operated (C) and regenerating
(PH) liver (15 h postoperative) are shown. RNA isolated from
these fractions was applied to Northern blot analysis and hybridized
with labeled cDNAs encoding PABP, EF1 , and albumin. For each
treatment, the same RNA preparations were hybridized with the different
probes. The autoradiographic signals were quantified by
phosphorimaging, and the relative amounts of the mRNAs in each
fraction are graphically depicted in the right panels.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, reflects corresponding
variations in the amounts or the activities of the pyrimidine-binding
protein and/or p70s6k.
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ACKNOWLEDGEMENTS |
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We are grateful to Thierry Grange for the human PABP cDNA and to Evelyne Segall and the late Elias Froimovitch for the synthesis of oligonucleotides.
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FOOTNOTES |
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* This work was supported from United State-Israel Binational Science Foundation Grant BSF-93-00032 and in part by Grant 3599 from the Chief Scientist's office of Ministry of Health, Israel.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.
A recipient of awards from the Foulkes Foundation (London) and
from the Kornfeld Foundation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U68093-68105 for the promoter and the 15 exons of human PABP gene, U60801 and U64661 for processed pseudogene 2 and 3, respectively, and U664662 for pseudogenes 1.
§ Present address: Dept. of Biochemistry, University of Cambridge, Tennis Court Rd., Cambridge CB2 1QW, UK.
¶ Present address: Bruce Rappaport Faculty of Medicine and Research Unit and Rambam Medical Center, Technion-Israel Institute of Technology, Haifa 31096, Israel.
Present address: Dana-Farber Cancer Institute and Harvard
Medical School, Boston, MA 02115.
** To whom correspondence should be addressed: Dept. of Biochemistry, The Hebrew University-Hadassah Medical School, P. O. Box 12272, Jerusalem 91120, Israel. Tel.: 972-2-6758290; Fax: 972-2-6757379. E-mail: meyuhas{at}cc.huji.ac.il.
The abbreviations used are: PABP, poly(A)-binding protein; hGH, human growth hormone; PCR, polymerase chain reaction; rp, ribosomal protein; TOP, terminal oligopyrimidine tract; UTR, untranslated region: EF, elongation factor; kb, kilobase(s); kbp, kilobase pair; bp, base pair; nt, nucleotides.
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REFERENCES |
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