From the Department of Biomedical Science, College of Medicine, Florida State University, Tallahassee, Florida 32306 and the § Departments of Medicine, Biochemistry, and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
Received for publication, September 6, 2002, and in revised form, October 31, 2002
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ABSTRACT |
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The 5' stem-loop is a conserved sequence element
found around the translation initiation site of three collagen
mRNAs, Three fibrillar collagen mRNAs, We analyzed previously a regulatory role of the 5' stem-loop in two
experimental systems; quiescent versus activated hepatic stellate cells (HSCs) (6) and fibroblasts cultured in a
three-dimensional matrix (7). Activated HSCs are responsible for
excessive collagen production in liver fibrosis (8, 9). We found that
the 5' stem-loop prevented expression of the reporter genes in
quiescent HSCs, which express low amounts of type I collagen, but
allowed for expression in activated HSCs. This inhibitory effect of the 5' stem-loop was in part mediated by a decreased half-life of the
corresponding mRNAs. Reporter genes with the mutated 5' stem-loop were constitutively expressed to a high level in both cell types. Therefore, expression of the reporter mRNA with the 5' stem-loop resembles expression of endogenous collagen Second, we studied the role of the 5' stem-loop on collagen The mechanism by which the 5' stem-loop targets mRNAs for turnover
in HSCs and fibroblasts grown in a three-dimensional matrix is unknown.
In quiescent HSCs we could not detect any protein binding to the 5'
stem-loop in vitro. In activated HSCs a cytosolic protein
factor(s) of unknown identity binds to the stem-loop and requires a 7mG
cap on the RNA for binding (6). An excess of cap analogue completely
prevents formation of this complex in vitro. The complex is
also found in fibroblasts in postpolysomal cytoplasmic fraction. Its
binding is greatly reduced if the cells are cultured in a
three-dimensional matrix (7). One possibility is that the 5' stem-loop
binding activity may increase the steady-state level of collagen
mRNAs by diverting them from the degradative pathway (11).
Translation and mRNA decay are coupled processes (12). Therefore,
studies on the translation of collagen In this study we investigate the role of the 5' stem-loop in
translation of collagen Constructs--
Plasmid used for in vitro
transcription of PSII mRNA was constructed by cloning of the
double-stranded oligonucleotide with the sequence shown in Fig.
1B into HindIII and NarI sites of the pGL3 vector (Promega). Then, the double-stranded oligonucleotide with
the sequence of the T7 promoter was cloned into
BglII-HindIII sites of the above construct. This
plasmid was linearized with HpaI and transcribed in
vitro with T7 polymerase and Cap-scribe kit (Roche Molecular
Biochemicals) to produce capped PSII mRNA. COLL-START and
COLL-OPTSTART constructs were made by cloning double-stranded oligonucleotides with the sequence shown in Fig. 1B into
SacI-NarI sites of pGL3 and recloning of the
EcoRI-HpaI fragment into
EcoRI-SmaI sites of the pGEM3 vector (Promega).
After linearization with BamHI corresponding capped
mRNAs were synthesized with T7 polymerase and the Cap-scribe kit.
Plasmids for synthesis of the 5' WT-SL and 5' MUT-SL mRNAs were
made by cloning of double-stranded oligonucleotides with the sequence
shown in Fig. 2A into the HindIII-NarI
site of the pGL3 vector and cloning of the T7 promoter as for PSII. Capped mRNAs were made as described for PSII mRNA. The clone
producing competitor A mRNA was made by cloning 75 codons of an
artificial protein into EcoRI-XhoI sites of the
vector pCDNA3 (Stratagene), followed by cloning of an optimal
translation start site (Fig. 1B) to allow expression. This
plasmid was linearized with NotI, and competitor A mRNA
was made as for PSII mRNA. All in vitro produced
mRNAs were gel-purified and analyzed by agarose gel
electrophoresis.
The 5' WT-MH-COLL gene was made by cloning the
BglII-XbaI fragment of mouse genomic DNA clone
containing 220 nt of the promoter and 115 nt of the 5'-UTR (a kind gift
from Dr. M. Breindl) into BglII-XbaI sites of the
pGL3 vector and inserting into the above construct the
XbaI-BamHI fragment of human collagen
A riboprobe for analyzing expression from MH-COLL genes was
made by cloning the XbaI-KpnI fragment of human
cDNA clone into XbaI-KpnI of Bluscript SK
vector (Invitrogen). This plasmid was linearized by NotI and
transcribed by T7 polymerase in the presence of [32P]UTP
as described (20).
In Vitro Translation Reactions--
0.08 pmol of gel-purified
mRNAs was translated in a 50-µl reaction using nuclease-treated
rabbit reticulocyte lysate (Roche Molecular Biochemicals), according to
the manufacturer's instructions. In preliminary experiments 0.08 pmol
of mRNA was found to be a nonsaturation concentration of mRNA
for a 50-µl reaction. Competitor mRNA was added in 10-fold molar
excess (0.8 pmol) to the test mRNA prior to mixing with the lysate.
Reactions were incubated for 30 min at room temperature when a 5-µl
aliquot was analyzed for luciferase activity. Incubations longer than
30 min did not further increase luciferase activity. For the same test
mRNA preparation the reaction was done with and without competitor,
and the ratio of luciferase activity was normalized to that of PSII
mRNA. All experiments were done with two different mRNA
preparations, each done in duplicate.
Transfection of Mov 13 Fibroblasts--
Transient transfections
were done with the calcium phosphate technique using 10 µg of
corresponding MH-COLL plasmids. 24 h after transfection, equal
number of cells were split into two dishes and incubation continued for
an additional 24 h. The cells were then incubated in 0.2% serum
for 24 h and either treated with 4 ng/ml of TGF Western Blots--
50 µg of cellular proteins were run on
7.5% SDS-PAGE gels under reducing or nonreducing conditions, as
indicated. 100 ng of purified rat tail collagen type I (Collaborative
Biomedical Products) was included as control. After transfer, the blots
were probed with 1:1000 dilution of anti-collagen type I antibody
(600-401-103, Rockland) and developed using the ECL system (Amersham
Biosciences). Cellular medium was concentrated on Centricon 100 columns
(Amicon), and equivalent amounts (corresponding to 4 × 105 cells) were analyzed by Western blot as above. For
pepsin digestions, 40 µl of concentrated medium was adjusted to pH
2.5 with acetic acid and digested with 1 µl of 64,000 units/ml of
pepsin (Sigma) for 30 min at room temperature. After neutralization,
the samples were analyzed by Western blot. For collagenase digestion, 1 µl of 4 units/ml of bacterial collagenase (Roche Molecular
Biochemicals) was added to 40 µl of concentrated medium and digested
for 30 min at room temperature.
RNase Protection Assay--
50 µg of total cell RNA was
simultaneously hybridized with collagen-specific riboprobe and
glyceraldehyde-3-phosphate dehydrogenase-specific riboprobe (Ambion),
as previously described (20). The collagen-specific band has an
expected size of 145 nt, and the glyceraldehyde-3-phosphate dehydrogenase-specific band has an expected size of 120 nt.
Translation of Reporter mRNAs with Collagen 5'-UTR
Sequences--
Fig. 1A
shows the sequence of the 5' stem-loop of mouse collagen
Another set of reporter mRNAs was designed to address the role of
the 5' stem-loop in translation. 5' WT-SL reporter contained 63 nt of
the mouse collagen
Next we compared translational efficiency of the 5' WT-SL mRNA and
5' MUT-SL mRNA to PSII mRNA in the presence of 10-fold molar
excess of competitor mRNAs. Three competitor mRNAs were added
in 10-fold molar excess to the reaction, competitor A (described above), Expression of Collagen
To assess the role of the 5' stem-loop in collagen type I synthesis
in vivo we constructed two genes. One gene contained 220 nt
of the promoter of the mouse collagen Synthesis of Collagen Polypeptides from 5' WT-MH-COLL and 5'
MUT-MH-COLL Genes--
First, we measured the intracellular
steady-state levels of collagen polypeptides by Western blot after
transient transfections of 5' WT-MH-COLL and 5' MUT-MH-COLL genes into
Mov 13 cells (Fig. 4A). We
also treated the cells with 4 ng/ml of active TGF
We collected the cell medium and analyzed the equivalent amounts by
Western blot under reducing conditions. Very little of pro- Only the 5' WT-MH-COLL Gene Expresses Properly Folded
Collagen--
Next, we probed the structure of secreted collagen from
Mov 13 fibroblasts by digestion of cellular medium with pepsin and collagenase. We used the medium of cells treated with TGF Unique features of the three fibrillar collagen mRNAs, Various competitor mRNAs inhibit in vitro translation of
a reporter mRNA with collagen Previous reports suggested that N- and C-terminal peptides of type I
collagen inhibit translation of collagen Based on electron microscopy data, assembly of the collagen type I
heterotrimer occurs on the membrane of the endoplasmic reticulum, while
the individual chains are still associated with polysomes or shortly
after their release (14, 17). Lysyl hydroxylase, one of the key enzymes
in collagen modifications, is also associated with the membrane of the
endoplasmic reticulum (37). Membrane association may couple folding
starting from the C terminus of collagen chains, to concomitant
modifications of the selected lysine residues. For collagen type I,
this implies that There are many examples that mRNAs are targeted for translation at
discrete subcellular sites to produce proteins with the concentration
gradient within the cell. Most targeting signals are located in the
3'-UTR of these mRNAs (44). To our knowledge this is the first
example that a RNA element located in the 5'-UTR is involved in
synthesis of a secreted multisubunit protein. This study demonstrates
that the conserved collagen 5' stem-loop has specific functions. In the
absence of RNA-binding proteins, the 5' stem-loop renders the collagen
mRNAs inefficient for translation and therefore susceptible to
regulation, such as by TGF1(I),
2(I), and
1(III). We show here that the 5'
stem-loop of collagen
1(I) mRNA is inhibitory to translation
in vitro. The sequence 5' to the translation initiation
codon, as a part of the 5' stem-loop, is also not efficient in
initiating translation under competitive conditions. This suggests that
collagen
1(I) mRNA may not be a good substrate for translation.
Since the 5' stem-loop binds protein factors in collagen-producing
cells, this binding may regulate its translation in vivo.
We studied in vivo translation of collagen
1(I) mRNA
after transfecting collagen
1(I) genes with and without the 5'
stem-loop into Mov 13 fibroblasts. The mRNA with the
1(I) 5'
stem-loop was translated into pepsin-resistant collagen, which was
secreted into the cellular medium. This mRNA also produced more
disulfide-bonded high molecular weight collagen found intracellularly.
The mRNA in which the 5' stem-loop was mutated, but without
affecting the coding region of the gene, was translated into
pepsin-sensitive collagen and produced only trace amounts of
disulfide-bonded collagen. This suggests that the 5' stem-loop is
required for proper folding or stabilization of the collagen triple
helix. To our knowledge this is the first example that an RNA element
located in the 5'-untranslated region is involved in synthesis of a
secreted multisubunit protein. We suggest that 5' stem-loop, with its
cognate binding proteins, targets collagen mRNAs for coordinate
translation and couples translation apparatus to the rest of the
collagen biosynthetic pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISSCUSSION
REFERENCES
1(I),
2(I), and
1(III), are coordinately regulated in fibrotic processes of various
organs (1, 2). In the 5'-UTR1
of these mRNAs there is a stem loop structure (5' stem-loop) encompassing the translation initiation codon (3). The 5' stem-loop is
located about 75 nt from the cap and has a stability of
G = 25-30 kcal/mol. The 5' stem-loop is well
conserved in evolution, differing by only two nucleotides in
Xenopus and human collagen mRNAs (4). The sequence
flanking the 5' stem-loop is not conserved. A similar stem-loop
structure is also found around the translation start codon of the sea
urchin collagen gene (5). Evolutionary conservation of this sequence
suggests an important function.
1(I) mRNA in HSCs; it is low in quiescent HSCs and elevated in activated HSCs and regulated by a post-transcriptional mechanism (6).
1(I)
mRNA expression in fibroblasts cultured in a three-dimensional matrix where the fibroblasts revert from an activated phenotype to a
more quiescent phenotype. This is accompanied by destabilization of
endogenous collagen
1(I) mRNA (10). The reporter collagen
1(I) mRNA with the intact 5' stem-loop was less stable than the identical mRNA with the mutated 5' stem-loop in the cells grown in
the matrix. Thus, the 5' stem-loop is required for accelerated decay of
collagen
1(I) mRNA in cells that down-regulate collagen synthesis (7).
1(I) mRNA are required to
provide insight into the mechanism of stabilization of this mRNA.
All collagen
1(I) mRNA is associated with membrane-bound polysomes and is not found on free polysomes or in postpolysomal supernatant.2 It is not known
if this association is because of targeting of the mRNA or
targeting by the leader peptide after initiation of translation.
Collagens are secreted proteins, and their translation is coupled to
export of the peptides into the endoplasmic reticulum (13). There is
substantial evidence that all three peptides initiate folding into the
heterotrimer while still associated with polysomes on the endoplasmic
reticulum (14-17). When folding is initiated, the collagen trimer is
released in the lumen of the endoplasmic reticulum. In human disease
osteogenesis imperfecta (OI) certain mutations of
1(I) chain
decrease the rate of assembly of collagen type I. Unassembled OI
1(I) chains are hypermodified on proline and lysine residues and
degraded (18, 19). This suggests that modification and assembly
processes are in a kinetic equilibrium. It is possible that the 5'
stem-loop binding activity may target collagen mRNAs to translation
at such sites.
1(I) mRNA in vitro and
in vivo. We found that the 5' stem-loop inhibits translation
in vitro and when more than one mRNA is competing for a
limited amount of translational apparatus. In fibroblasts in
vivo, the 5' stem-loop is necessary for efficient folding or
synthesis of stable triple helical collagen. To our knowledge this is
the first example of an RNA element that affects protein folding.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISSCUSSION
REFERENCES
-globin mRNA and tobacco mosaic virus (TMV)
mRNA were prepared from the Roche Molecular Biochemicals in
vitro translation kit.
1(I)
cDNA (a kind gift from F. Ramirez). This restores the 5' stem-loop, which is identical in mouse and human collagen
1(I) mRNA and includes the entire coding region and 3'-UTR of human collagen
1(I)
cDNA. The 5' MUT-MH-COLL gene was made identically
except that a substitution of 18 nt, shown in Fig. 2A, was
introduced into the mouse genomic clone before reconstituting the
full-size construct.
1 (R&D Systems)
or left untreated for an additional 24 h. Cells and cellular
medium were collected and analyzed by RNase protection assay or Western
blot. Stably transfected Mov 13 fibroblasts were developed by
transfection of MH-COLL genes and pCDNA3 vector in a
ratio of 10:1 and selection with G418 for 3 weeks. G418-resistant cells
were pooled and processed as above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISSCUSSION
REFERENCES
1(I) mRNA. To the right is shown the consensus 5' stem-loop sequence, which can be derived from
1(I),
2(I), and
1(III) mRNAs of evolutionary distant organisms (ranging from fish to humans). Since the sequence around the collagen
1(I) mRNA start codon, as a part of the 5' stem-loop, does not match the consensus sequence derived by Kozak (22, 23), one set of mRNAs was
constructed to investigate how the sequence of collagen
1(I)
mRNA immediately 5' to the start codon affects translation.
Therefore, we constructed a reporter mRNA containing only the last
25 nt of the mouse collagen
1(I) 5' stem-loop linked in-frame with a
luciferase mRNA (COLL START, Fig. 1B). In
this construct the 5' stem-loop cannot form because its 5'-region was
deleted, but it contains the collagen
1(I) start codon in its
natural sequence context. Control mRNAs had a short 5'-UTR of
35-36 nt without any structural elements or short upstream open
reading frames (uORF) (PSII and COLL-OPTSTART, Fig. 1B). The COLL-OPTSTART differs from the COLL-START
mRNA by 9 nt preceding the start codon, which were optimized to
conform to the Kozak rules, while in COLL-START they were from mouse
collagen
1(I) mRNA. PSII mRNA had a 5'-UTR derived from the
pGL3 vector, which is optimized for efficient translation and was used
as control. 3' to the start codon all constructs had the rest of the
sequence of the 5' stem-loop (underlined in Fig.
1B), followed by the luciferase ORF. The mRNAs were made
in vitro with 7mG cap, and their integrity was analyzed by
agarose gel electrophoresis (Fig. 1C). These mRNAs did
not contain a poly(A) tail, because of the small effect that the
poly(A) tail has on translation in vitro (21). These test mRNAs were translated in rabbit reticulocyte lysate with or without of 10-fold molar excess of a competitor mRNA. The competitor
mRNA had the optimal start site followed by an ORF of 75 amino
acids (competitor A). The sequence of the 5'-UTR of competitor A is shown in Fig. 1B (COMP A). Without competitor A
all three test mRNAs yielded similar amounts of the luciferase
protein. However, in the presence of a 10-fold amount of competitor A
the efficiency of translation was reduced 5-fold for PSII mRNA,
19-fold for COLL-START mRNA, and 8.4-fold for COLL-OPTSTART
mRNA. In Fig. 1D this result is shown normalized to the
inhibition of PSII mRNA. Because the highest inhibition was
observed when the collagen
1(I) sequence preceded the start codon
(COLL-START), we concluded that this sequence is suboptimal in
promoting translation initiation when competing with another mRNA
for the translation machinery.
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Fig. 1.
Suboptimal translation initiation site of
collagen 1(I) mRNA. A,
conserved stem-loop structure (5' stem-loop) found around translation
initiation codon of fibrillar collagen mRNAs. The sequence of the
mouse collagen
1(I) 5' stem-loop is shown to the left and
a consensus sequence found in
1(I),
2(I), and
1(III) mRNAs
of various organisms is shown to the right. Translation
initiation codon is in bold. B, test mRNAs
used to assess the functionality of the translation initiation site of
collagen
1(I) mRNA. Sequence of the 5'-UTR of test mRNAs is
shown. This sequence is followed in frame by the luciferase open
reading frame starting from the codon 13. The start codons are in
bold letters, and the sequence derived from mouse collagen
1(I) mRNA is underlined. Sequence of the 5'-UTR of
competitor A (COMP A) is also shown. C, mRNAs
shown in A were transcribed in vitro in the
presence of the cap analog 7mGpppG, gel-purified, and analyzed on
agarose gel for integrity. 0.08 pmol of RNA was loaded on the gel. Comp
A is the competitor mRNA made as above and added in 10-fold molar
excess to in vitro translation reactions. D,
in vitro translation of the mRNAs shown in B
under competitive conditions. 0.08 pmol of test mRNAs was
translated in vitro or was mixed with 10-fold molar excess
of competitor A mRNA and than translated. After 30 min of
incubation luciferase activity was determined. The ratio of luciferase activity with
and without competitor mRNA was calculated and arbitrarily set as 1 for PSII mRNA. The result shown is from two independent experiments
each performed in duplicate. The error bar ± S.D. is
shown.
1(I) 5'-UTR with the 5' stem-loop in-frame with
LUC, while the 5' MUT-SL reporter had substitutions in the 5' stem-loop
to abolish its formation (Fig.
2A). Translation of these
reporters was compared in vitro to the PSII mRNA
(described above) without competitor mRNA or under competitive
conditions. The integrity of the mRNAs is shown in Fig.
2B. Without competitor, the 5' WT-SL mRNA was translated
about 3-fold less efficiently than PSII (arbitrarily set as 1) and 5'
SL-MUT mRNAs. The latter two were translated with comparative
efficiency (Fig. 2C). This was not due to preferential
degradation of the 5' WT-SL mRNA in the lysate, because extraction
of the RNAs from the lysate after a 1-h incubation and retranslation in
fresh lysate yielded the same result (not shown).
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Fig. 2.
Inhibitory effect of the 5' stem-loop on
translation in vitro. A, test
mRNAs used to assess the effect of the 5' stem-loop on translation
in vitro. The 5'-UTRs are shown. This sequence is followed
in-frame by the luciferase open reading frame starting from the codon
13. The start codons are in bold letters. Sequence derived
from mouse collagen 1(I) mRNA is underlined with
sequence of the 5' stem-loop overlined. B,
mRNAs shown in A were transcribed in vitro in
the presence of cap analog 7mGpppG, gel-purified, and analyzed on
agarose gel for integrity. 0.08 fmol of RNA was loaded on the gel.
C, in vitro translation of the mRNAs shown in
B. 0.08 fmol of test mRNAs was translated in
vitro without competitor mRNA. After 30 min of incubation,
luciferase activity was determined. The luciferase activity produced by
PSII mRNA was arbitrarily set as 1. The result shown is from two
independent experiments each performed in duplicate. The error
bar ± S.D. is shown. D, in vitro
translation of the mRNAs shown in B under competitive
conditions with competitor A. The experiment was performed as in Fig.
1. The result shown is from two independent experiments each performed
in duplicate. The error bar ± S.D. is shown.
E, same experiment as in D, but the competitor
was
-globin mRNA. F, same experiment as in
D, but the competitor was tobacco mosaic virus mRNA
(TMV).
-globin mRNA, and TMV. With competitor A 5' WT-SL
reporter mRNA was translated about 30-fold less efficiently, when
compared with PSII mRNA. 5' MUT-SL mRNA was translated only
5.5-fold less efficiently relative to PSII mRNA (Fig.
2D). When
-globin mRNA was used as competitor the
respective ratios were 4.6- and 3.6-fold (Fig. 2E). When
viral RNA was used as a competitor the translation of 5' WT-SL and PSII
mRNAs was not affected, while translation of the 5'MUT-SL mRNA
was increased 2-fold (Fig. 2F). Based on the results in Fig.
2, we concluded that the 5' stem-loop is inhibitory for translation
in vitro in the absence of a competitor mRNA, while
various competitor mRNAs have either a strong inhibitory effect or
show only a small effect. We analyzed if reticulocyte lysates contain
the 5' stem-loop binding activity by performing gel mobility shift
analysis using capped 5' stem-loop RNA or inverted 5' stem-loop RNA as
probes, as described (6). We could not detect specific binding to the
5' stem-loop (data not shown).
1(I) Reporter Genes in Mov 13 Fibroblasts--
Mov 13 fibroblasts were derived from mice in which
insertion of a retrovirus into the first intron of collagen
1(I)
gene had inactivated the transcription of this gene (24). Mov 13 fibroblasts transcribe the
2(I) gene and thus provide a unique opportunity to study translation of collagen type I mRNAs and assembly of the collagen trimer when the
1(I) mRNA is encoded by
various transgenes. Fig. 3 shows
characterization of Mov 13 fibroblasts. No collagen
1(I)
polypeptides can be detected by Western blot among cellular proteins of
Mov 13 fibroblasts (Fig. 3A, lane 1). For
comparison, cellular proteins of NIH 3T3 fibroblasts and purified
collagen from rat tail were analyzed in lanes 3 and 2, respectively. Pro-
1(I) (about 175 kDa) and
1(I)
(about 120 kDa) peptides were seen in the NIH 3T3 sample. The antibody
used did not detect the
2(I) chain. The
1(I) monomer and higher
molecular weight cross-links of type I collagen are seen in the rat
tail sample, which served as markers.
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Fig. 3.
Expression of collagen
1(I) genes in Mov 13 fibroblasts.
A, characterization of collagen expression by Western blot.
Western blot with 50 µg of cellular proteins of Mov 13 fibroblasts
(lane 1) and NIH 3T3 fibroblasts (lane 3) and 100 ng of purified rat tail type I collagen (lane 2). Migration
of individual collagen
1(I) chains and high molecular weight
cross-linked collagen (HMW COLL) found only in rat tail is
indicated to the right. The antibody used does not recognize
2(I) chain. B, hybrid mouse-human collagen reporter
genes. Mouse collagen
1(I) promoter (220 nt) is shown as thin
line, transcription start site is indicated by arrow,
mouse collagen 5'-UTR and the 5' stem-loop is shown as thick
lines, and human collagen
1(I) cDNA as an interrupted
thick line (5' WT-MH-COLL). The 5'
MUT-MH-COLL gene is identical except it has a substitution
of 18 nt within the 5' stem-loop, which abolishes its formation. This
mutation is the same as that shown in Fig. 2. C, expression
of mouse-human collagen genes in Mov 13 fibroblasts. RNase protection
assay done with 50 µg of total RNA from Mov 13 fibroblasts
transiently transfected with 5' WT-MH-COLL (WT)
and 5' MUT-MH-COLL (MUT) genes. Untransfected Mov
13 fibroblasts (Mov 13) are shown as controls. Riboprobe
specific for the human collagen
1(I) gene was hybridized
simultaneously with riboprobe specific for the mouse
glyceraldehyde-3-phosphate dehydrogenase gene, as internal control.
Migration of the respective protected bands is indicated to the
right.
1(I) gene followed by the
mouse collagen 5'-UTR including the 5' stem-loop, ligated to the
full-size human collagen
1(I) cDNA (5' WT-MH-COLL,
Fig. 3B). The gene has an open reading frame encoding a
full-size human collagen pro-
1(I) polypeptide. The other gene is
identical, except it has an 18-nt mutation within the 5' stem-loop,
which destroys its formation (5' MUT-MH-COLL). This mutation does not
affect the coding region of the gene, it encodes for the identical
polypeptide as the 5' WT-MH-COLL gene. The genes were
transiently transfected into Mov 13 fibroblasts and mRNA analyzed
by RNase protection assay (Fig. 3C). Untransfected Mov 13 fibroblasts show no expression of collagen
1(I) mRNA.
Transfected cells show high level of mRNAs transcribed from both
transgenes, enabling us to study how collagen mRNAs with the 5'
stem-loop and without it are translated in vivo.
1, to assess how
this profibrogenic cytokine (25, 26) would affect the expression. The
blot was done under nonreducing conditions to assess the synthesis of
disulfide-bonded collagen species. The major collagen detected was the
pro-
1(I) chain, and both genes synthesized a similar level of the
peptide. Its steady-state level was unaffected by TGF
. However, the
5' WT-MH-COLL gene yielded some of the disulfide-linked
higher molecular weight collagen species (HMW COLL), while the 5'
MUT-MH-COLL gene did not (Fig. 4, compare lanes 1 and 2 to lanes 3 and 4). We could not
distinguish, with our antibody, whether these species were homo or
hetero multimers of type I collagen, although these collagen moieties
comigrated with the collagen species found in rat tail type I collagen
(lane 5). The result suggested that the 5' stem-loop,
although an RNA element, is involved in more efficient formation of
disulfide-bonded collagen monomers, suggesting better registration of
collagen chains. This prompted us to investigate if the collagen
synthesized by 5' WT-MH-COLL mRNA is more efficiently secreted into
the cellular medium.
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Fig. 4.
Synthesis of collagen
1(I) from 5' WT-MH-COLL and 5'
MUT-MH-COLL genes. A, Western blot
under nonreducing conditions with 50 µg of cellular proteins of
transiently transfected Mov 13 fibroblasts as in Fig. 3. 48 h
after transfections the cells were serum-starved for 24 h and
either left unstimulated (
) or stimulated with 4 ng/ml of TGF
1 (+)
for an additional 24 h. As a control rat tail collagen was run in
lane 5. Migration of high molecular weight collagen
(HMW COLL) and individual
1(I) chains is shown to the
right. B, secretion into cellular medium of
collagen synthesized from 5' WT-MH-COLL and 5'
MUT-MH-COLL genes. Western blot under reducing conditions
with equivalent amounts of cellular medium from Mov 13 fibroblasts
transiently transfected as in A. Lane 1 is rat
tail collagen as size marker. Migration of high molecular weight
collagen (HMW COLL) and individual
1(I) chains is shown
to the left. C, collagen secretion by stably
transfected Mov 13 fibroblasts. After 3 weeks of selection for stably
transfected cells, pools of cells were combined and analyzed as in
B.
1(I)
polypeptide (175 kDa) was secreted out of nonstimulated cells and in
the same amount for 5' WT-MH-COLL and 5'
MUT-MH-COLL genes (Fig. 4B, lanes 2 and 4). Only tracing amounts were processed to mature
1(I) chain of about 120 kDa. With TGF
1 stimulation a much higher
amount of pro-
1(I) chain was found in the cellular medium, but both
genes produced similar amount of the protein (lanes 3 and
5). Since there was no change in mRNA level with TGF
1
stimulation (not shown), we concluded that TGF
1-stimulated translation or secretion of type I collagen independent of the 5'
stem-loop. Alternatively, TGF
may have decreased extracellular degradation of collagen. In stably transfected Mov 13 fibroblasts TGF
1 also stimulated extracellular accumulation of collagen, but no
difference between the 5' WT-MH-COLL and 5'
MUT-MH-COLL genes was seen (Fig. 4C).
1, because it contained the higher amount of collagen that facilitated the analysis. The medium was subjected to digestion with pepsin and collagenase as described under "Material and Methods" and analyzed by Western blot. In undigested medium under reducing conditions, the
predominant collagen species was the pro-
1(I) chain (Fig. 5A, lane 1). When
the medium of cells expressing the 5' WT-MH-COLL gene was
digested with pepsin the molecular mass of this chain was reduced to
about 120 kDa (lane 2). This suggests cleavage of the
globular domains, but folding of the core domain into pepsin-resistant triple helix. When the medium was digested with bacterial collagenase, no collagen peptides could be detected, suggesting the specificity of
the bands (lane 3). Thus, mRNA with the 5' stem-loop
directs synthesis of triple helical collagen, which accumulated in the cell medium. When the medium of 5' MUT-MH-COLL-expressing cells was
digested with pepsin no pepsin-resistant fragment was obtained (lane 5), although the amount of collagen secreted was
comparable to that of the 5' WT-MN-COLL gene (lane
4). Thus, mRNA without the 5' stem-loop directs synthesis of
structurally aberrant collagen that could not resist limited pepsin
digestion. Digestion with collagenase served as the specificity control
(lane 6). The result obtained with the medium from stably
transfected Mov 13 cells is shown in Fig. 5B. Again, the 5'
WT-MH-COLL gene yielded secreted triple helical collagen
(lane 2), while the 5' MUT-MH-COLL gene produced
a pepsin-sensitive collagen (lane 5). We concluded from these experiments that the 5' stem-loop is necessary for productive collagen synthesis and, although it was mutated without affecting the
coding region of the mRNA, has a profound effect on collagen protein folding or stability.
View larger version (53K):
[in a new window]
Fig. 5.
Pepsin-resistant collagen was produced by the
5' WT-MH-COLL gene. A, Mov 13 fibroblasts were transiently transfected with 5' WT-MH-COLL
(WT, lanes 1-3) and 5' MUT-MH-COLL
(MUT, lanes 4-6). After treatment with TGF ,
cellular medium was analyzed by Western blot under reducing conditions.
Lanes 1 and 4 are undigested medium, lanes
2 and 5 are medium digested with 1000 units of pepsin
and lanes 3 and 6 are medium digested with 4 units of collagenase. Migration of pro-
1(I) chain and triple helical
core (THC) of type I collagen is indicated to the
left. B, identical experiment as in A,
but performed with the medium of stably transfected Mov 13 fibroblasts.
DISSCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISSCUSSION
REFERENCES
1(I)
mRNA,
2(I) mRNA, and
1(III) mRNA, is the 5' stem-loop
structure that encompasses the start codon (3). The 5' stem-loop has an
important role in regulating
1(I) mRNA stability (3, 6, 7) and
is conserved in collagen mRNAs of evolutionary distant species (4,
5). Since the start codon is part of this stem-loop, the sequence
constraints required to maintain the 5' stem-loop dictate the sequence
around translation initiation. Therefore, the start codon in collagen
mRNAs is not in the sequence context necessary for optimal
translation initiation (22, 23). We have shown here that it is not
efficiently recognized in vitro if more than one mRNA
species are competing for the translation machinery. When the start
codon was optimized we could increase translation 4-fold under
competitive conditions (Fig. 1C). The 5' stem-loop structure
has a stability of 25-30 kcal/mol. This is insufficient to block
scanning ribosomes to reach the start codon, because stem-loop
structures of about 70 kcal/mol are needed (27, 28). Nevertheless,
reporter mRNA with the 5' stem-loop (5' WT-SL) was translated
3-fold less efficiently, even in the absence of competitor, than
similar mRNA in which the stem-loop was mutated (5'
MUT-SL, Fig. 2C). It seems that the structure of
the 5' stem-loop together with its suboptimal translation start site is
responsible for this effect.
1(I) 5' stem-loop (5'WT-SL) to a
different degree. Competitor A inhibited translation of 5' WT-SL
mRNA 30-fold relative to a control mRNA (PSII) and 6-fold
relative to the identical mRNA without the stem-loop (5'
MUT-SL) (Fig. 2D).
-globin mRNA had a
smaller effect. Viral mRNA showed no inhibition on 5' WT-SL mRNA, while 5' MUT-SL mRNA is translated better. The reason for this is unclear, but TMV mRNA with its Omega sequence may titrate an inhibitor of translation (29). The result with competitors corroborates the finding that the 5' stem-loop may compromise translation of collagen
1(I) mRNA, which may be a weak substrate for translation in the absence of its RNA-binding proteins. Since the
5' stem-loop binds protein factors in collagen-producing cells, it is
possible that binding of these factors regulates translation of
collagen
1(I) mRNA. Cloning of these proteins and their addition to the in vitro translation reaction will address this question.
1(I) mRNA (30, 31). We
did not see any inhibition when these recombinant peptides were added
to the in vitro translation reaction (data not shown). Also,
the
1(I),
2(I), and
1(III) collagen mRNAs contain two short uORF preceding the start codon. We did not see any change in
translation of our reporter mRNAs in vitro when these
uORF were abolished (data not shown). Although it is known that uORFs can regulate translation in yeast (32, 33), there are only a few
examples of their role in translational regulation in higher organisms
(34-36).
1(I) and
2(I) chains may be synthesized by
ribosomes positioned in close proximity on the endoplasmic reticulum
membrane. Such coordinated translation would greatly increase local
concentration of the chains. We hypothesized that the sequence elements
that modulate loading of ribosomes on collagen
1(I) mRNA may be
involved in targeting for such coordinated translation. Therefore, we
mutated the 5' stem-loop and analyzed production of collagen trimers
in vivo from the hybrid mouse-human collagen genes (5'
WT-MH-COLL and 5' MUT-MH-COLL) (Fig. 5). The
human collagen
1(I) gene could rescue the phenotype of Mov 13 mice,
proving that human collagen
1(I) polypeptide is functional in mouse
(38, 39). The 5' WT-MH-COLL gene produced triple helical
collagen in Mov 13 fibroblasts; however, the 5' MUT-MH-COLL
gene produced collagen, which was sensitive to digestion with pepsin
(Fig. 5). The structurally aberrant collagen was produced although the
5' MUT-MH-COLL gene had the identical coding region. This
pepsin-sensitive collagen may represent individual
1(I) chains,
which were not efficiently folded into triple helix and were secreted
as monomers, or alternatively, the monomers were not properly modified
and an unstable triple helix was secreted. The 5' MUT-MH-COLL chains
had identical electrophoretic mobility to the 5' WT-MH-COLL chains,
excluding a major difference in post-translational modification,
although subtle differences may remain undetected. If the chains were
not efficiently folded, the 5' stem-loop may be required to increase
their local concentration, which would facilitate the chain
registration. The result shown in Fig. 4 where the 5'
WT-MH-COLL gene produced more disulfide-linked high
molecular weight collagen suggests that this may be the case. If the
chains were not properly modified, the 5' stem-loop may target collagen
mRNAs for translation to discrete regions of the endoplasmic
reticulum where there is optimal concentration of collagen-specific
modifying enzymes and molecular chaperones (40). Although we do not
have direct evidence for this, we think that the 5' stem-loop also
couples the translational machinery to the rest of the collagen
biosynthetic pathway, because collagen biosynthesis requires coordinate
action of translational apparatus, modifying enzymes, and molecular
chaperones (41). In patients with OI, where folding of collagen type I
chains is impaired, the mutant chains are hypermodified and subjected
to degradation (19, 42). Interestingly, one patient with OI type I was
described who had a mutation in the 5' stem-loop in the absence of any
other mutation of the collagen
1(I) gene (43).
. In collagen-producing cells, the 5'
stem-loop has a novel function of directing the post-translational
modification of collagen to produce mature triple helices. The 5'
stem-loop almost certainly acts through its cognate RNA-binding
proteins. Cloning of these protein factors will help us elucidate the
complex biosynthesis of type I collagen.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant 1R01DK59466-01A1 (to B. S.).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.
To whom correspondence should be addressed: Dept. of Biomedical
Science Florida State University College of Medicine, Tallahassee, FL
32306. Tel.: 850-644-7600; Fax: 850-644-8924; E-mail:
branko.stefanovic@med.fsu.edu.
Published, JBC Papers in Press, November 4, 2002, DOI 10.1074/jbc.M209175200
2 B. Stefanovic and D. Brenner, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: UTR, untranslated region; nt, nucleotide; WT, wild type; uORF, upstream open reading frame; HSC, hepatic stellate cells; LUC, luciferase; TMV, tobacco mosaic virus; TGF, transforming growth factor; OI, osteogenesis imperfecta.
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