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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4023-4030
RAPID COMMUNICATION
Thrombopoietin Production Is Inhibited by a Translational Mechanism
By
Nico Ghilardi,
Adrian Wiestner, and
Radek C. Skoda
From Biozentrum, University of Basel, Basel, Switzerland.
 |
ABSTRACT |
Thrombopoietin (TPO) is a lineage-dominant hematopoietic cytokine
that regulates megakaryopoiesis and platelet production. The major site
of TPO biosynthesis is the liver. Despite easily detectable levels of
liver TPO mRNA, the circulating TPO serum levels are very low. We have
observed that translation of TPO mRNA is inhibited by the presence of
inhibitory elements in the 5 -untranslated region (5-UTR).
Alternative promoter usage and differential splicing generate at least
three TPO mRNA isoforms that differ in the composition of their
5-UTR. Using mutational analysis we show that physiologically
the translation of these TPO mRNA isoforms is strongly inhibited by the
presence of AUG codons, which define several short open reading frames
(ORFs) in the 5-UTR and suppress efficient initiation at the
physiologic start site. The two regularly spliced isoforms, which
account for 98% of TPO mRNA, were almost completely inhibited, whereas a rare splice variant that lacks exon 2 can be more efficiently translated. Thus, inhibition of translation of the TPO mRNA is an
efficient mechanism to prevent overproduction of this highly potent
cytokine.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
THROMBOPOIETIN (TPO) is the primary
regulator of megakaryopoiesis and platelet production.1 The
major site of TPO production is the liver,2 but TPO mRNA is
also found at lower abundance in the kidney,3 the
spleen,4 and in bone marrow (BM) stromal
cells.4,5 The normal serum concentration of TPO is very
low, ranging between 0.5 and 2 pmol/L,6-8
which was one of the factors hindering the purification of the protein
for more than 40 years.9
TPO serum concentrations inversely correlate with the platelet and
megakaryocyte mass.10,11 During thrombocytopenia, TPO production in liver and kidney is not regulated at the transcriptional level.12-14 It remains controversial whether
transcriptional regulation can occur in the BM.4,15 A
simple and elegant model of platelet autoregulation16,17
that involves absorption of TPO by platelets through the TPO receptor,
MPL, is supported by a large amount of experimental
data.1,18 Consistently, mice deficient in mpl showed
dramatically reduced platelet counts and elevated TPO serum
levels.19 Furthermore, TPO-deficient mice displayed
haploinsufficiency,20 indicating that in the heterozygous
state the decrease in platelet mass cannot be compensated by increasing
the TPO production from the remaining wild-type allele. This strongly
argues against a "sensor for platelet mass" that would function
analogous to the "oxygen sensor" for erythropoietin
(EPO).21 However, certain clinical observations are
difficult to explain by the simple version of the autoregulation model.
In particular, some patients with reactive thrombocytosis display TPO
serum concentrations too high for their platelet
count,22,23 suggesting that under certain conditions TPO
production might be upregulated by a mechanism independent of the
platelet mass.
We have previously observed that the wild-type TPO mRNA is
inefficiently translated in vitro and that a splicing mutation affecting the composition of the 5-untranslated region
(5-UTR) results in overproduction of TPO protein, causing
hereditary thrombocythemia in a Dutch family.24 There are
at least two mechanisms by which the 5-UTR might repress
translation.25 Both rely on the fact that the 40 S
ribosomal subunit first binds to the cap structure at the 5-end
of a mRNA and then scans for the first AUG codon. The presence of
stable stem loops between the cap structure and the first AUG can
interfere with ribosomal scanning and has been shown to profoundly
inhibit translation.26,27 Alternatively, the presence of
AUG codons upstream of the actual start site (uAUG) can inhibit
translation by causing premature initiation and thereby preventing the
ribosome from initiating at the physiological start codon.28 The degree of inhibition depends on the sequence
context of the uAUG29 and the length and phase of the
resulting open reading frame (ORF) in respect to the protein coding
sequence.30 The majority (90% to 95%) of eukaryotic mRNAs
do not have uAUG codons in the 5-UTR,29 whereas TPO
belongs to the smaller class of mRNAs with the presence of uAUG in the
5-UTR, which sometimes display translational
regulation.31
Here we studied the translation of three naturally occurring TPO mRNA
isoforms that are generated by alternative promoter usage and
differential splicing and differ in the composition of their
5-UTR, but not in the coding sequence. We performed mutational
analysis and show that translation of these isoforms is inhibited by
upstream AUG codons and not by secondary structure of the 5-UTR.
Furthermore, we found that the least abundant isoform, a splice
variant32 that lacks the noncoding exon 2, was more efficiently translated than the two regularly spliced isoforms, introducing the possibility that regulation of alternative splicing may
serve as an additional control mechanism for TPO production.
| |
MATERIALS AND METHODS |
RNA analysis.
Ribonuclease protection analysis was performed as
described.33 Total RNA from human liver was prepared by the
acid phenol method34 and 15 µg was used for analysis. For
the detection of P1 and P2 TPO transcripts we generated a riboprobe
corresponding to nucleotides 49 to 480 of the TPO cDNA sequence, as
derived from the published TPO genomic sequence.32 This
riboprobe protects a 432-nucleotide (nt) fragment for TPO P1
transcripts and fragments ranging from 142 to 212 nt for P2
transcripts. A second riboprobe corresponding to P1 TPO mRNA that lacks
exon 2 (P1 E2) was constructed using the primers
5-CCGCCCGAAGGATGAAGAC-3 (sense) and
5-AGCAGGCAGCAGGACAGGTG-3 (antisense). This riboprobe
protects a 386-nt fragment for P1E2 and a 336-nt fragment for the
full-length P1 and P2 transcripts. As size markers, RNAs transcribed in
vitro from cDNA constructs corresponding to the different 5-UTR
were used. Radioactive bands were separated on denaturing
polyacrylamide gels and quantitated using a PhosphorImager 425 (Molecular Dynamics Inc, Sunnyvale, CA).
Site-directed mutagenesis of upstream AUG codons.
Upstream AUG codons were mutated by recombinant polymerase chain
reaction (PCR).35 The same antisense primer,
5-CCACGAGTTCCATTCAAGAG-3 (nucleotide 1322), was used in
combination with various individual sense primers in all PCR reactions.
The sense primers for P1 constructs were
5-CGCAGATCTGCCGAAGACTTGTCTTTAAAGCCGACAACG-3
(mutated uAUG 1,2), or
5-CGCAGATCTGATGAAGACTTGTCTTTAAAGATGACAACG-3
(wild-type uAUG 1,2), and the sense primers for P2 constructs were
5-CAGAGATCTGTACGACCTGCTGCTGT-3 (mutated uAUG
5), or 5-CAGAGATCTGTATGACCTGCTGCTGT-3
(wild-type uAUG 5). A unique BglII site for subcloning
(underlined) was introduced into each of the 5 sense primers. To
mutate internal uAUG codons in the 5-UTR by recombinant
PCR,35 we used the following primer combinations: uAUG 3:
5-GTTGCCCGGGTCCAGGAAAAG-3 (sense) and 5-CTTTTCCTGGACCCGGGCAAC-3 (antisense); uAUG 4:
5-CAGGAAAAGCCGGATCCCCC-3 (sense) and
5-GGGGGATCCGGCTTTTCCTG-3 (antisense); uAUG 5:
5-GCAGGCGTACGACCTGCTGC-3 (sense) and
5-GCAGCAGGTCGTACGCCTGC-3 (antisense); uAUG 6:
5-CACCGCCACGCGTCTTCCTA-3 (sense) and
5-TAGGAAGACGCGTGGCGGTG-3 (antisense); uAUG 7:
5-GCCGCCTCCTTGGCCCCAGG-3 (sense) and
5-CCTGGGGCCAAGGAGGCGGC-3 (antisense). To delete the 21-bp
GUG repeat a unique Sac I site (underlined) was introduced in
each of the two primers
5-GAGCCGCGGACCCTGGTCCAGG-3 (sense) and
5-ACACCGCGGAGAAGATTTGGAT-3 (antisense) flanking
the GUG repeat. The PCR products were digested with Sac I and
religated. Combinations of these primers were used in several rounds of
recombinant PCR to generate TPO 5-end fragments carrying
mutations in the 5-UTR (detailed protocol available upon
request). These fragments were digested with BglII and
Pst I (unique endogenous restriction site at position 922 of
TPO cDNA) and ligated as a three-part ligation together with a
Pst I-Xba I fragment representing the 3-portion
of the TPO cDNA and a BamHI-Xba I-digested pcDNA-1/Amp (Invitrogen Corp, San Diego, CA). To prevent artifacts in the translation assays, all junctions were designed so that no new AUG
codons were generated by insert ligation between the T7
promoter and the TPO 5-UTR. All final constructs were sequenced
on an Applied Biosystems 373 DNA sequencer (Perkin Elmer Corp, Foster City, CA).
In vitro transcription and translation.
The constructs in pcDNA1-Amp were linearized with Xba I and 2 µg of linearized DNA was used as templates for in vitro RNA synthesis
for 1 hour at 37°C using T7 RNA polymerase (Stratagene, La Jolla,
CA). The products were digested with DNaseI for 15 minutes at 37°C,
extracted with phenol/chloroform, and ethanol-precipitated. One-half
microgram of each TPO mRNA isoform was translated for 1 hour at
30°C in reticulocyte lysate in the presence of
35S-Methionine according to the instructions of the
manufacturer (Promega Corp, Madison, WI). Radioactive proteins were
separated by 12% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and visualized on a PhosphorImager 425 (Molecular Dynamics Inc).
Cell culture and TPO bioassay.
COS cells were transiently transfected by the diethyl
aminoethyl (DEAE)-Dextran method36 with equal
amounts of cDNA expression constructs for the different TPO
5-UTR mutants. After 48 hours, the supernatants were obtained
and TPO concentration was measured using a bioassay.12
| |
RESULTS |
Because our previous work suggested that the 5-UTR of the TPO
mRNA contains elements that inhibit translation,24 we
examined whether regulation of TPO translation might be a general
mechanism for controlling TPO protein levels. The TPO 5-UTR is
encoded by exons 1, 2, and a large part of exon 3.32 Usage
of alternative promoters (P1 and P2) and differential splicing generate
TPO mRNAs that differ in the length and composition of their
5-UTR (Fig 1A through C). To assess
the relative contribution of TPO mRNA isoforms to the overall
production of TPO protein, we first determined their relative abundance
in human liver RNA by a ribonuclease (RNase) protection assay. Using a
riboprobe that can distinguish between P1 and P2 transcripts
(Fig 2A), we found a weak full-length protected band corresponding to P1 transcripts, and several shorter fragments corresponding to the multiple transcriptional start sites
described for P237-39 (Fig 2A). Quantitation of these
radioactive bands showed that in human liver RNA approximately 10% of
all transcripts are synthesized from P1, whereas 90% originate from P2. The same result was obtained with RNA from three independent livers
(not shown). The majority of P1 transcripts contained exons 1 through 3 (P1-wt). An alternatively spliced P1 transcript that lacks exon 2 (P1E2) was detectable using a second riboprobe (Fig 2B). The
identity of this band was confirmed by reverse transcriptase (RT)-PCR
and sequencing (not shown). The majority of P2 transcripts contain
exons 2 through 5 (Fig 2A and B). Quantitation of the bands in Fig 2A
and B showed that the P2-wt transcripts accounted for approximately
90%, P1-wt for 8%, and P1E2 for 2% of total TPO mRNA.
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| Fig 1.
(A) Structure of the human TPO gene. Exons are drawn as
boxes and the protein coding region is shaded. Arrows mark the start
sites for promoter 1 and 2 (P1 and P2). Solid lines connecting exons
indicate normal splice events, dashed lines alternative splice events.
 E2, exon 2 skipping. (B) Exon composition of the TPO mRNA splice
variants. P1-wt and P2-wt, the full-length TPO mRNA transcribed from P1
or P2, respectively; P1E2, alternatively spliced TPO mRNA with exon
2 skipping. (C) Position of uAUG in the TPO 5-UTR. The
full-length mRNA sequence of the TPO 5-UTR beginning with the P1
start site, as determined by Chang et al,32 is shown.
Upstream AUG codons are boxed and numbered in the order as they appear.
Translation of wild-type TPO protein starts at the eighth AUG (AUG 8).
The deduced amino acid sequence is shown in the one-letter code. A
stretch of GUG triplets is underlined. Triangles and Roman numerals
mark the locations of introns.32 Filled arrowhead, P1 start
site mapped by Chang et al32; gray arrowhead, P2 start site
determined by Sohma et al37; open arrowheads, P2 start
sites mapped by Kamura et al38 and Gurney et
al.39
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| Fig 2.
Differential promoter usage and alternative splicing of
TPO pre-mRNA. (A) Ribonuclease protection analysis of human liver RNA.
(Top) Lanes 1 and 2, undigested riboprobe and tRNA control. P1
transcripts (P1-wt) account for approximately 10% of TPO mRNA. Note
that P2 transcripts (P2-wt) initiate at multiple start sites. In vitro
transcribed sense mRNAs corresponding to different 5-UTR were
used as RNA size markers (lanes 4 through 6). Numbers at the right
indicate length of the RNA size markers in nucleotides. (Bottom) Length
and position of the riboprobe (thick line) and the protected fragments
(arrows) with respect to the TPO mRNA 5-end. (B) Assessment of
exon 2 skipping (P1E2) by ribonuclease protection. Annotation as in
(A).
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One mechanism by which the 5-UTR might inhibit translation is
aberrant initiation at uAUG codons.28 The 5-UTR
sequence of the longest TPO mRNA transcript (P1-wt) contains seven uAUG codons (Fig 1C), whereas the alternative TPO isoforms display a reduced
number and different composition of uAUG codons. An alternative
mechanism of how the efficiency of translation might be reduced is the
presence of stem loops in the 5-UTR, which might interfere with
ribosomal scanning.26,27 Using the Zuker algorithm,40 we found that none of the TPO mRNA isoforms is predicted to form stem loops sufficiently stable to inhibit ribosomal scanning.26,27
To more directly test whether the presence of uAUG codons in the
5-UTR of the TPO mRNA isoforms is responsible for the inhibition of translation, we introduced point mutations in individual uAUG codons
by site-directed mutagenesis (Fig 3).
Several of these uAUGs are in a favorable sequence context for
initiation, as defined by Kozak.29 To assure that the
mutations will prevent the ribosome from initiating, two bases of the
uAUG codon were altered in most positions (Fig 3).
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| Fig 3.
Summary of the site directed mutagenesis of uAUG codons.
(Top) The Kozak consensus sequence favorable for ribosomal
initiation.29 The most critical residues, positions  3
and +4, are typed in bold characters. Left column: sequence context
of TPO uAUG codons. Residues in positions 3 and +4 that match the
Kozak consensus are typed in boldface. Right column: mutations in uAUG
sequences designed to inactivate ribosomal initiation.
|
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We analyzed each isoform individually. First, we examined mRNAs that
originate from P2 and represent the majority of TPO transcripts. Transcription initiation occurs at multiple start sites in exon 2, resulting in mRNA molecules with a variable length of the 5-UTR and different numbers of uAUG codons. The longest P2
transcripts37 retain uAUG 5, 6, and 7, whereas the shortest
contain only uAUG 7 (see Fig 1C). The three uAUG codons of P2-wt mRNA
define two ORFs upstream of the eighth AUG (AUG 8), the physiologic TPO
initiation codon (Fig 4A). To
include all possible uORFs, we designed our cDNA constructs to start
upstream of uAUG 5. Together, these ORFs strongly inhibited wild-type
TPO protein production, as measured by an in vitro
transcription-translation assay (Fig 4B, lane 2). Consistently, no TPO
bioactivity was detectable in supernatants of COS cells transfected
with P2-wt cDNA (Fig 4C, lane 2). Deletion of all but the last seven
nucleotides upstream of AUG 8 (UTR) improved translation and
resulted in secretion of TPO bioactivity into COS supernatants (Fig 4B
and C, lanes 1). Removal of uAUG 5 and 6 did not enhance translation
(lanes 3), showing that uAUG 7 is sufficient to inhibit TPO production.
In contrast, the absence of uAUG 7 improved translation in reticulocyte
lysate to levels similar to the UTR mRNA. TPO production in COS
cells was also improved (Fig 4B and C, lanes 4), although not to the
same degree as for the UTR mRNA. This discrepancy was not due to a
difference in transfection efficiency, because equal amounts of human
TPO mRNA were detected by Northern blot analysis (not shown). Possible explanations might be that there are differences between COS cells and
reticulocyte lysates in the efficiency of initiation at uAUG 5 and 6 and/or the re-initiation at AUG 8. Finally, inactivation of all
uAUGs resulted in more efficient in vitro translation and COS TPO
production than UTR (Fig 4B and C, lanes 5). This result was
obtained in two independent transfections and at present we do not
understand why the addition of a 258-nt long 5-UTR without uAUGs
improved TPO production to levels higher than UTR. Nevertheless, inhibition of translation is primarily mediated by the presence of uAUG
codons and is not due to formation of RNA secondary structures.
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| Fig 4.
Analysis of the translational efficiency of TPO
P2-wt transcripts in vitro and in transfected COS cells. (A) Exon
composition of the TPO mRNA. The TPO protein coding region is shown in
gray. The uAUG triplets (filled circles) are numbered in the order they
appear in the P1-wt transcript and the resulting ORFs (horizontal solid
lines) are placed in the three possible reading frames (Roman
numerals). The thick solid line with arrowhead represents the ORF
encoding TPO protein. (B) In vitro transcription translation analysis.
Equal amounts of in vitro transcribed TPO mRNA variants (top) were
translated in vitro in reticulocyte lysate in the presence of
35S-methionine (bottom). UTR, mRNA with deletion of the
entire 5-UTR; nm, mRNA with no mutations in the 5-UTR;
numbers above individual lanes indicate the position of mutated uAUGs.
The protein bands in the bottom panel were identified as: wt TPO,
initiation at the physiological start site (AUG 8); asterisk, cryptic
non-AUG initiation within exon 3. (C) TPO production and secretion by
transfected COS cells. Presence of TPO in COS cell supernatants was
determined by bioassay. Mock transfected COS cells were set as
background and cells transfected with a UTR expression construct as
100%. Bars indicate the median ± SEM of triplicates.
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The results for the longer transcripts initiating at P1 are summarized
in Fig 5A through C. The seven uAUG codons
of P1-wt define five ORFs upstream of AUG 8 (Fig 5A). Together, these
ORFs almost completely inhibited wild-type TPO protein production in vitro (Fig 5B, lane 2). A strong, slower migrating band corresponding to initiation at uAUG 4 was observed. Using the algorithm by Nielsen et
al,41 the stretched amino terminus of this protein was
predicted to be nonfunctional as a signal peptide. Consistently, no TPO bioactivity was detectable in supernatants of COS cells transfected with P1-wt cDNA (Fig 5C, lane 2). Individual mutations in uAUG 4 or
uAUG 7 slightly improved translation without producing measurable TPO
in COS supernatants (lanes 3 and 4), suggesting that each of these ORFs
is sufficient to repress TPO production. The double mutation in uAUG 4 and uAUG 7 further relieved repression of translation, but resulted in
TPO bioactivity only slightly above background (lane 5). A similar
difference between the reticulocyte lysate and COS cell assay systems
was already observed for the P2-wt construct also lacking uAUG 7 (Fig
4). When we simultaneously mutated all seven uAUGs, we observed
improved translation and secretion of TPO protein (lanes 6). The
efficiency of translation and secretion was further improved to levels
comparable to the UTR construct when we deleted a stretch of four
in-frame GUG codons located just 3 of uAUG 4 (lanes 7), which
appear to function as cryptic ribosomal initiation sites.42
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| Fig 5.
Analysis of the translational efficiency of TPO P1
transcripts in vitro and in transfected COS cells. Annotation as in Fig
4. (A through C) Full-length P1 transcript (P1-wt); (D through F)
transcripts with exon 2 skipping (P1E2). (A and D) Exon composition
of the TPO mRNA isoforms. Open rectangle, GUG repeat. (B and E) In
vitro transcription translation analysis. (Top) GUG, mRNA with
deletion of the cryptic GUG initiation sites. (Bottom) uAUG 4 and GUG,
proteins with extended amino terminus through in-frame initiation at
the fourth uAUG or the GUG repeat, respectively. (C and F) TPO
production and secretion by transfected COS cells.
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Given the high degree of translational repression of TPO P1 mRNA, it is
conceivable that low abundant splice variants that lack one or several
uAUG codons might overproportionally contribute to TPO protein
production. Therefore, we investigated P1E2 transcripts, which only
retain uAUG 1, 2, and 7 in their 5-UTR (Fig 5D). P1E2 mRNA is
translated more efficiently into TPO protein than P1-wt mRNA (compare
lanes 2 of Fig 5B and E), although not as efficient as UTR mRNA (Fig
5E, lanes 1 and 2). Removal of uAUG 1 and 2 did not improve TPO
production (lanes 3), but removal of uAUG 7 enhanced translation as
efficiently as removal of the entire UTR (lanes 4) or a construct with
mutations in all uAUGs (lanes 5). For all P1E2 mutants, the in vitro
translation results correlate well with the corresponding TPO secretion
by transfected COS cells (Fig 5F).
| |
DISCUSSION |
Our results show that the translation of TPO mRNA is physiologically
almost completely inhibited by the presence of uAUG codons in the
5-UTR. According to the ribosomal scanning model,28 these uAUGs lead to premature ribosomal initiation followed by translation of a short peptide and partial dissociation of the ribosome
from the mRNA when a stop codon is encountered
(Fig 6). Removal of all uAUG codons by
point mutations improved translation to the same degree as deletion of
the entire 5-UTR, indicating that the formation of RNA secondary
structures does not play a major role in repressing TPO mRNA
translation (Figs 4 and 5). Mutation of individual uAUG codons showed
that uAUG 7 is a potent inhibitor of translation, most likely because
the corresponding uORF extends past the physiological start site, AUG
8. Interestingly, this uORF is conserved between human,2
mouse,43 and rat.44 Additional strong
inhibition is conferred by uAUG 4 in P1-wt and by uAUG 5 and 6 in P2-wt
transcripts, whereas uAUGs 1, 2, and 3 had only minor effects. Thus,
the strong inhibition of translation by multiple uAUGs in the
5-UTR of the TPO mRNA constitutes a reliable mechanism for
preventing overproduction of this highly potent cytokine by the liver.
Indeed, loss of this inhibition has been shown to cause thrombocytosis
in a family with hereditary thrombocythemia,24 and it will
be interesting to determine whether the same mechanism is involved in
the pathogenesis of other families with thrombocythemia. Moreover, this
translational inhibition appears to be used in other members of the
helical cytokine family, eg, interleukin-7 (IL-7) and
IL-15.45,46
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| Fig 6.
Effect of upstream ORFs on TPO mRNA translation. (Top)
Simplified drawing of TPO mRNA with only one uORF in the 5-UTR
followed by the TPO coding region (open boxes). According to the
ribosomal scanning model, the 40 S ribosomal subunit will bind the cap
structure at the 5 end of the mRNA and scan the mRNA until it
encounters the first AUG, where a functional ribosome is assembled. The
ribosome will initiate translation and synthesize a short peptide until
a stop codon is reached. Here the ribosome dissociates from the mRNA.
This will prevent the ribosome from reaching the physiological TPO
start codon. However, a minor proportion of 40 S subunits may remain
associated with the mRNA and continue scanning for a downstream AUG.
(Bottom) TPO 5-UTR with point mutations in all uAUG codons will
allow the 40 S subunit to reach the physiological start site more
efficiently and initiate translation of the TPO protein.
|
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TPO mRNA is transcribed from two promoters resulting in two major
mRNAs, P1-wt and P2-wt, and an alternatively spliced isoform, P1E2.32,37,38 We confirmed that P2 transcripts initiate
at multiple transcriptional start sites.37-39 In contrast
to a previous report by Kamura et al,38 who concluded that
P1 transcripts were barely detectable in the adult liver, we found that
approximately 10% of liver TPO mRNA originated from P1 (Fig 2). Thus,
P1 transcripts might significantly contribute to the TPO protein
production. Analysis of translational efficiency showed that the TPO
mRNA isoforms were strongly inhibited by uORFs. However, the rare
splice variant P1E2 produced more TPO than the P1-wt and P2-wt
transcripts (Figs 4 and 5). This opens the possibility that modulating
the proportion of P1E2 transcripts by differential splicing might constitute a means to augment TPO production in situations of increased
platelet demand. Because alternative splicing would not change the
abundance of total TPO mRNA, such a mechanism would be compatible with
the observation that TPO mRNA in liver, kidney, and spleen is not
regulated at the transcriptional level.12-14
Preferential usage of alternatively spliced mRNA variants in response
to interferon- and lipopolysaccharide have been reported for
IL-1547 and in response to phorbol esters for
CD44.48 It is conceivable that selection of translationally
more efficient TPO mRNA isoforms in response to proinflammatory
cytokines or other extracellular signals might account for the high TPO
serum levels in many patients with reactive
thrombocytosis23 and, thus, constitute an additional
mechanism in the regulation of platelet production. However, testing
this hypothesis in human patients will be difficult because of limited
availability of these tissues.
| |
ACKNOWLEDGMENT |
We thank David C. Seldin and Michael Altmann for helpful comments on
the manuscript.
| |
FOOTNOTES |
Submitted June 16, 1998;
accepted September 10, 1998.
Supported by grants from the Swiss National Science Foundation
(32-35503.92, 31-46857.96) and Schweizerische Krebsliga (KFS287-2-1996) to R.C.S., and from the Swiss National Science Foundation
(3135-040025.94) and the Roche Research Foundation (96-240) to
A.W.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Radek C. Skoda, MD, Biozentrum, University
of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland; e-mail:
skoda{at}ubaclu.unibas.ch.
| |
REFERENCES |
1.
Kaushansky K:
Thrombopoietin: Understanding and manipulating platelet production.
Annu Rev Med
48:1, 1997[Medline]
[Order article via Infotrieve]
2.
de Sauvage FJ, Hass PE, Spencer SD, Malloy BE, Gurney AL, Spencer SA, Darbonne WC, Henzel WJ, Wong SC, Kuang WJ, Oles KJ, Hultgren B, Solberg LA, Goeddel DV, Eaton DL:
Stimulation of megakaryocytopoiesis and thrombopoiesis by the c-mpl ligand.
Nature
369:533, 1994[Medline]
[Order article via Infotrieve]
3.
Foster DC, Sprecher CA, Grant FJ, Kramer JM, Kuijper JL, Holly RD, Whitmore TE, Heipel MD, Bell LA, Ching A, Mcgrane V, Hart C, Ohara PJ, Lok S:
Human thrombopoietin: Gene structure, cDNA sequence, expression, and chromosomal localization.
Proc Natl Acad Sci USA
91:13023, 1994[Abstract/Free Full Text]
4.
Sungaran R, Markovic B, Chong BH:
Localization and regulation of thrombopoietin mRNA expression in human kidney, liver, bone marrow, and spleen using in situ hybridization.
Blood
89:101, 1997[Abstract/Free Full Text]
5.
Guerriero A, Worford L, Holland HK, Guo GR, Sheehan K, Waller EK:
Thrombopoietin is synthesized by bone marrow stromal cells.
Blood
90:3444, 1997[Abstract/Free Full Text]
6.
Kosugi S, Kurata Y, Tomiyama Y, Tahara T, Kato T, Tadokoro S, Shiraga M, Honda S, Kanakura Y, Matsuzawa Y:
Circulating thrombopoietin level in chronic immune thrombocytopenic purpura.
Br J Haematol
93:704, 1996[Medline]
[Order article via Infotrieve]
7.
Tahara T, Usuki K, Sato H, Ohashi H, Morita H, Tsumura H, Matsumoto A, Miyazaki H, Urabe A, Kato T:
A sensitive sandwich ELISA for measuring thrombopoietin in human serum: Serum thrombopoietin levels in healthy volunteers and in patients with haemopoietic disorders.
Br J Haematol
93:783, 1996[Medline]
[Order article via Infotrieve]
8.
Emmons RVB, Reid DM, Cohen RL, Meng G, Young NS, Dunbar CE, Shulman NR:
Human thrombopoietin levels are high when thrombocytopenia is due to megakaryocyte deficiency and low when due to increased platelet destruction.
Blood
87:4068, 1996[Abstract/Free Full Text]
9.
Kelemen E:
Specific thrombopoietin cloned and sequenced With personal retrospect and clinical prospects.
Leukemia
9:1, 1995[Medline]
[Order article via Infotrieve]
10.
Kuter DJ, Rosenberg RD:
Regulation of megakaryocyte ploidy in vivo in the rat.
Blood
75:74, 1990[Abstract/Free Full Text]
11.
Wendling F, Maraskovsky E, Debili N, Florindo C, Teepe M, Titeux M, Methia N, Bretongorius J, Cosman D, Vainchenker W:
C-mpl ligand is a humoral regulator of megakaryocytopoiesis.
Nature
369:571, 1994[Medline]
[Order article via Infotrieve]
12.
Stoffel R, Wiestner A, Skoda RC:
Thrombopoietin in thrombocytopenic mice: Evidence against regulation at the mRNA level and for a direct regulatory role of platelets.
Blood
87:567, 1996[Abstract/Free Full Text]
13.
Fielder PJ, Gurney AL, Stefanich E, Marian M, Moore MW, Carver-Moore K, de Sauvage FJ:
Regulation of thrombopoietin levels by c-mpl-mediated binding to platelets.
Blood
87:2154, 1996[Abstract/Free Full Text]
14.
Cohen-Solal K, Villeval JL, Titeux M, Lok S, Vainchenker W, Wendling F:
Constitutive expression of Mpl ligand transcripts during thrombocytopenia or thrombocytosis.
Blood
88:2578, 1996[Abstract/Free Full Text]
15.
McCarty JM, Sprugel KH, Fox NE, Sabath DE, Kaushansky K:
Murine thrombopoietin mRNA levels are modulated by platelet count.
Blood
86:3668, 1995[Abstract/Free Full Text]
16.
De Gabriele G, Penington DG:
Regulation of platelet production: "Hypersplenism" in the experimental animal.
Br J Haematol
13:384, 1967[Medline]
[Order article via Infotrieve]
17.
Kuter DJ, Rosenberg RD:
The reciprocal relationship of thrombopoietin (c-mpl ligand) to changes in the platelet mass during busulfan-induced thrombocytopenia in the rabbit.
Blood
85:2720, 1995[Abstract/Free Full Text]
18.
Eaton DL, de Sauvage FJ:
Thrombopoietin: The primary regulator of megakaryocytopoiesis and thrombopoiesis.
Exp Hematol
25:1, 1997[Medline]
[Order article via Infotrieve]
19.
Gurney AL, Carver-Moore K, de Sauvage FJ, Moore MW:
Thrombocytopenia in c-mpl-deficient mice.
Science
265:1445, 1994[Abstract/Free Full Text]
20.
de Sauvage FJ, Carver-Moore K, Luoh SM, Ryan A, Dowd M, Eaton DL, Moore MW:
Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin.
J Exp Med
183:651, 1996[Abstract/Free Full Text]
21.
Bunn H, Gu J, Huang L, Park JW:
Erythropoietin: A model system for studying oxygen-dependent gene regulation.
J Exp Biol
201:1197, 1998[Abstract]
22.
Pitcher L, Taylor K, Nichol J, Selsi D, Rodwell R, Marty J, Taylor D, Wright S, Moore D, Kelly C, Rentoul A:
Thrombopoietin measurement in thrombocytosis: Dysregulation and lack of feedback inhibition in essential thrombocythaemia.
Br J Haematol
99:929, 1997[Medline]
[Order article via Infotrieve]
23.
Cerutti A, Custodi P, Duranti M, Noris P, Balduini CL:
Thrombopoietin levels in patients with primary and reactive thrombocytosis.
Br J Haematol
99:281, 1997[Medline]
[Order article via Infotrieve]
24.
Wiestner A, Schlemper RJ, van der Maas AP, Skoda RC:
An activating splice donor mutation in the thrombopoietin gene causes hereditary thrombocythaemia.
Nat Genet
18:49, 1998[Medline]
[Order article via Infotrieve]
25.
Kozak M:
Structural features in eukaryotic mRNAs that modulate the initiation of translation.
J Biol Chem
266:19867, 1991[Free Full Text]
26.
Kozak M:
Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs.
Mol Cell Biol
9:5134, 1989[Abstract/Free Full Text]
27.
Kozak M:
Influences of mRNA secondary structure on initiation by eukaryotic ribosomes.
Proc Natl Acad Sci USA
83:2850, 1986[Abstract/Free Full Text]
28.
Kozak M:
The scanning model for translation: An update.
J Cell Biol
108:229, 1989[Abstract/Free Full Text]
29.
Kozak M:
An analysis of 5-noncoding sequences from 699 vertebrate messenger RNAs.
Nucleic Acids Res
15:8125, 1987[Abstract/Free Full Text]
30.
Luukkonen BG, Tan W, Schwartz S:
Efficiency of reinitiation of translation on human immunodeficiency virus type 1 mRNAs is determined by the length of the upstream open reading frame and by intercistronic distance.
J Virol
69:4086, 1995[Abstract]
31.
Kozak M:
An analysis of vertebrate mRNA sequences: Intimations of translational control.
J Cell Biol
115:887, 1991[Abstract/Free Full Text]
32.
Chang MS, McNinch J, Basu R, Shutter J, Hsu RY, Perkins C, Mar V, Suggs S, Welcher A, Li L, Lu H, Bartley T, Hunt P, Martin F, Samal B, Bogenberger J:
Cloning and characterization of the human megakaryocyte growth and development factor (mgdf) gene.
J Biol Chem
270:511, 1995[Abstract/Free Full Text]
33.
Krieg PA, Melton DA:
In vitro RNA synthesis with SP6 RNA polymerase.
Methods Enzymol
155:397, 1987[Medline]
[Order article via Infotrieve]
34.
Chomczynski P, Sachhi N:
Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:156, 1987[Medline]
[Order article via Infotrieve]
35.
Ho SN, Hunt HD, Horton RM, Pullen JK, Pease LR:
Site-directed mutagenesis by overlap extension using the polymerase chain reaction.
Gene
77:51, 1989[Medline]
[Order article via Infotrieve]
36.
Seed B, Aruffo A:
Molecular cloning of the CD2 antigen, the T-cell erythrocyte receptor, by a rapid immunoselection procedure.
Proc Natl Acad Sci USA
84:3365, 1987[Abstract/Free Full Text]
37.
Sohma Y, Akahori H, Seki N, Hori T, Ogami K, Kato T, Shimada Y, Kawamura K, Miyazaki H:
Molecular cloning and chromosomal localization of the human thrombopoietin gene.
FEBS Lett
353:57, 1994[Medline]
[Order article via Infotrieve]
38.
Kamura T, Handa H, Hamasaki N, Kitajima S:
Characterization of the human thrombopoietin gene promoterA possible role of an Ets transcription factor, E4TF1/GABP.
J Biol Chem
272:11361, 1997[Abstract/Free Full Text]
39.
Gurney AL, Kuang WJ, Xie MH, Malloy BE, Eaton DL, de Sauvage FJ:
Genomic structure, chromosomal localization, and conserved alternative splice forms of thrombopoietin.
Blood
85:981, 1995[Abstract/Free Full Text]
40.
Jaeger JA, Turner DH, Zuker M:
Improved predictions of secondary structures for RNA.
Proc Natl Acad Sci USA
86:7706, 1989[Abstract/Free Full Text]
41.
Nielsen H, Engelbrecht E, Brunak S, von Heijne G:
Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites.
Protein Eng
10:1, 1997[Abstract/Free Full Text]
42.
Peabody DS:
Translation initiation at non-AUG triplets in mammalian cells.
J Biol Chem
264:5031, 1989[Abstract/Free Full Text]
43.
Lok S, Kaushansky K, Holly RD, Kuijper JL, Loftonday CE, Oort PJ, Grant FJ, Heipel MD, Burkhead SK, Kramer JM, Bell LA, Sprecher CA, Blumberg H, Johnson R, Prunkard D, Ching A, Mathewes SL, Bailey MC, Forstrom JW, Buddle MM, Osborn SG, Evans SJ, Sheppard PO, Presnell SR, Ohara PJ, Foster DC:
Cloning and expression of murine thrombopoietin cDNA and stimulation of platelet production in vivo.
Nature
369:565, 1994[Medline]
[Order article via Infotrieve]
44.
Ogami K, Shimada Y, Sohma Y, Akahori H, Kato T, Kawamura K, Miyazaki H:
The sequence of a rat cDNA encoding thrombopoietin.
Gene
158:309, 1995[Medline]
[Order article via Infotrieve]
45.
Bamford RN, Battiata AP, Burton JD, Sharma H, Waldmann TA:
Interleukin (IL) 15/IL-T production by the adult T-cell leukemia cell line HuT-102 is associated with a human T-cell lymphotrophic virus type I region /IL-15 fusion message that lacks many upstream AUGs that normally attenuates IL-15 mRNA translation.
Proc Natl Acad Sci USA
93:2897, 1996[Abstract/Free Full Text]
46.
Namen AE, Lupton S, Hjerrild K, Wignall J, Mochizuki DY, Schmierer A, Mosley B, March CJ, Urdal D, Gillis S:
Stimulation of B-cell progenitors by cloned murine interleukin-7.
Nature
333:571, 1988[Medline]
[Order article via Infotrieve]
47.
Nishimura H, Washizu J, Nakamura N, Enomoto A, Yoshikai Y:
Translational efficiency is up-regulated by alternative exon in murine IL-15 mRNA.
J Immunol
160:936, 1998[Abstract/Free Full Text]
48.
Konig H, Ponta H, Herrlich P:
Coupling of signal transduction to alternative pre-mRNA splicing by a composite splice regulator.
EMBO J
17:2904, 1998[Medline]
[Order article via Infotrieve]
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Abstract
Introduction