From the Division of Neuropathology, Department of Pathology and
Laboratory Medicine, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104
The tetracycline-responsive expression system of
Bujard was used to compare rates of decay of wild-type and mutant
neurofilament (NF) light subunit (NF-L) mRNAs. Optimal conditions
for activation and inactivation of the target transgene were determined
using a luciferase reporter gene. Analyses of mRNA stability were
thereupon conducted on cells that were doubly transfected with
transactivator and inducible target genes and derived from pooled
clones of transfected cells. Rates of mRNA decay were compared upon
inactivation of the transgenes after high levels of mRNA had been
induced. Deletion of the 445-nucleotide (nt) 3'-untranslated region
(3'-UTR) (L/+++
) or 527 nt of the 3'-coding region (3'-CR) (L/++
+)
increased the stability of NF-L mRNA compared with the full-length
(L/++++) transcript in neuronal (N2a and P19 cells) and non-neuronal (L cells) lines. Deletion of both the 3'-UTR and 3'-CR (L/++
) led to a
further stabilization of the transcript. A major stability determinant
was then localized to a 68-nt sequence that forms the junction between
the 3'-CR and 3'-UTR of NF-L and is the binding site of a unique
ribonucleoprotein complex (Cañete-Soler, R., Schwartz, M. L., Hua, Y., and Schlaepfer, W. W. (1998) J. Biol. Chem. 273, 12655-12661). The studies establish a novel system for mapping determinants of mRNA stability and have applied the system to localize determinants that regulate the stability of the NF-L
mRNA.
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INTRODUCTION |
Altering mRNA stability is a universal post-transcriptional
mechanism for modulating gene expression during growth, development and
differentiation. Post-transcriptional regulation may be particularly important in cells, such as neurons, that undergo extensive
differentiation and change after cessation of cell division.
Alterations in mRNA stability are known to affect the levels of
several key proteins in post-mitotic neurons (2-5). Increases in
mRNA stability accompany the postnatal up-regulation in
neurofilament (NF)1
expression and are, therefore, instrumental in determining the size and
shape of neuronal processes (6, 7). Moreover, levels of NF expression
have been implicated in motoneuron disease by virtue of the selective
motoneuron degeneration that occurs when a light (NF-L) or heavy (NF-H)
subunit transgene is overexpressed in transgenic mice (8, 9). A
transient up-regulation of endogenous NF mRNA also precedes the
spontaneous motoneuron degeneration in the Wobbler mice (10).
Functional assays for mapping stability determinants use a variety of
methods to compare stabilities of mutant versus wild-type mRNAs (11). Most of these methods are limited by their disruptive or pleiotrophic effects on the host cells and are, therefore, especially problematic for assessing stabilities of long-lived mRNAs. An alternative method is provided by the development of the
tetracycline-responsive promoter system whereby target gene expression
is stringently controlled by a non-toxic antibiotic ligand (12-19).
Here, we have adapted this system to assess mRNA stability in
neuronal and non-neuronal cell lines and to map stability determinants
in the NF-L transcript. The method has enabled the identification of
stability determinants in the 3'-CR and 3'-UTR of the NF-L mRNA and
have localized a key determinant to a 68-nt sequence at the junction of
the 3'-CR and 3'-UTR.
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EXPERIMENTAL PROCEDURES |
Plasmids--
pUHD15.1, pUHD10.3, and pUHD13.3 were a generous
gift of Dr. H. Bujard. pUHD15.1M is a modified expression vector that
was rendered autoinducible (see Ref. 16) by placing an EcoRI
site and consensus translation start sequence immediately upstream of
the tTA open reading frame and subcloning the
EcoRI/BamHI PCR fragment from pUHD15.1 into the
polylinker of pUHD10.3.
Construction of Wild and Mutant NF-L cDNAs--
Full-length
and mutant mouse NF-L cDNAs (see Fig.
1) were constructed by splicing together
three PCR fragments. Fragment 1 extended from +1 to a BglII
site at +829 in exon 1, fragment 2 extended from +829 to a
KpnI site at +1208, and fragment 3 extended from +1208 to a
site (+2180) 25 bp upstream of the AATAAA termination signal. The PCR
template for fragment 1 was a genomic clone of NF-L, and templates for
fragments 2 and 3 were cDNAs generated from mouse spinal cord.
HindIII and XbaI sites were placed in the
flanking primers to facilitate cloning of cDNAs into the
HindIII/XbaI polylinker sites of pRC/RSV
(Invitrogen, San Diego, CA) and pRC/tet (modified pRC expression vector
in which the Rous sarcoma virus promoter was replaced with the
heptamerized Tn-10 tet operator sequence). The pRC/tet vector
containing the full-length NF-L cDNA was designated L/++++. A
deletion mutant lacking 445 bp of 3'-UTR (L/+++
) was created by
splicing PCR fragment 3, which extended 3' to a site (+1740) just
beyond the TGA stop codon. A deletion mutant lacking 445 bp of 3'-UTR
and 527 bp of the coding region (L/++
) was generated by splicing
PCR fragments 1 and 2 and placing a TGA stop codon at +1212. A deletion
mutant lacking only the 3'-coding region (L/++
+) was made by splicing
with PCR fragment 3 containing a KpnI site, TGA stop codon,
and 3'-UTR. A 68-bp deletion at the junction of 3'-CR and 3'-UTR
(+1735) was generated by splicing upstream and downstream PCR fragments
that lacked sequence between +1712 (BspMI site) and +1979
(HincII site) and placing a stop codon at +1712. Wild-type
sequence in L/++++ was then replaced with the PCR fragment and
designated L/++/del/++. Constructs were sequenced to confirm the
integrity of junctional and Tn-10 tet operator sequences.
Cell Lines/Transfections--
HeLa, P19, N2a, and 3T3 (L) cell
lines were obtained from the American Type Culture Collection
(Rockville, MD) and transiently or stably transfected using calcium
phosphate (20) or LipofectAMINE (7). Cells were cotransfected with
transactivator (pUHD15.1 or pUHD15.1M) and pSVZeo (Invitrogen),
selected with Zeocin, and pooled (>100 clones). Cells were then
transfected with target genes bearing tetracycline-responsive
promoters, selected with G418 and Zeocin, and pooled (>100 clones).
Growth and selection were conducted in the presence of tetracycline
(0.5 µg/ml). The presence of the transgenes was monitored by PCR.
Assay of Tetracycline-responsive Reporter Gene--
The
luciferase reporter gene driven by the tetracycline-responsive promoter
(pUHD13.3) was used to test tetracycline-mediated regulation of
transcription. Pooled clones of N2a, P19, and L cells bearing pUHD15.1
or pUHD15.1M were transiently transfected with pUHD13.3 and assayed for
luciferase activity after 24 h in the absence of tetracycline.
pUHD13.3 was also stably transfected by cotransfecting with pcDNA3
(Invitrogen). Luciferase activity was measured by luminometer (Lumat LB
9501; Berthold, Wildbad, Germany) using a luciferase kit (Promega,
Madison, WI) according to manufacturer's instructions.
NF-L expression was compared in pooled clones of N2a, P19, and L cells
that had been doubly transfected with pUHD15.1M and pRC/tet vectors
bearing wild-type or mutant NF-L cDNAs. Cells were grown in the
absence of tetracycline, split into replicate plates, and harvested in
triplicate for NF expression at varying time points after readdition of
tetracycline. Ribonuclease protection assays were conducted as
described previously (3).
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RESULTS |
Characterization of Transactivator (tTA) Function on P19, N2a, and
L Cells--
A luciferase reporter gene with tetracycline-responsive
promoter (pUHD 13.3) was used to test the ability of the tTA
transactivator expression vectors (pUHD15.1 or pUHD15.1M) to confer
tetracycline-sensitive activation in neuronal (P19 and N2a)
versus non-neuronal (L cells) cell lines. Preliminary
studies with transient transfections revealed increases in luciferase
activity of 2-4 orders of magnitude in neuronal and non-neuronal cell
lines by 24 h after withdrawal of tetracycline (data not shown),
as reported previously in other cell lines (12-14). A similar range of
induction was observed in cell lines that were transiently
cotransfected or that had been stably transfected with the tTA
transactivator vector. Expression vectors with constitutive (pUHD15.1)
and autoinducible (pUHD15.1M) promoters were both effective in
conferring tetracycline-responsive activation.
To monitor the tetracycline-responsive promoter system for both
activation and inactivation of the test gene, the luciferase reporter
gene was stably transfected into cell lines containing the
autoinducible tTA transactivator expression vector (pUHD15.1M). To
reduce position effects and disparity in copy numbers, multiple clones
were pooled for each transfection. The luciferase gene in doubly
transfected cell lines was highly sensitive to tetracycline (Fig.
2A) and was rapidly activated
and inactivated upon withdrawal and readdition of tetracycline (Fig. 2,
B and C). Induction factors ranged from 100- to
300-fold after 24 h and reached maximal levels by 72 h after
withdrawal of tetracycline in the neuronal and non-neuronal cell lines.
There was also rapid decreases of luciferase activity upon readdition
of tetracycline, with less than 5% and 0.5% of residual activities
after 24 and 48 h, respectively. Levels of tetracycline-inducible
luciferase expression did not differ appreciably in cell lines
transfected with varying admixtures of tTA expression vectors driven by
constitutive (pUHD15.1) and autoinducible (pUHD15.1M) promoters.

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Fig. 2.
Luciferase activity from a luciferase
reporter transgene with tetracycline-responsive promoter (pUHD10.3)
transfected into N2a, P19 and L cells showing effects of tetracycline
concentration (A), time course of transgene activation upon
withdrawal of tetracycline (B), and time course of
transgene inactivation upon readdition of tetracycline (0.5 µg/ml) to
the cultures (C). The cells were initially transfected
with an expression vector bearing the tTA transactivator gene
(pUHD15.1M); see "Experimental Procedures."
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Tetracycline-induced Transactivation of NF-L Expression--
NF-L
cDNAs driven by the tetracycline-responsive promoter were readily
inducible upon withdrawal of tetracycline in P19 and N2a cells (Fig.
3) as well as in L cells (data not
shown). Increases of 50-100-fold in NF-L mRNA levels occurred in
P19 and N2a cell lines in which the tTA expression (pUHD15.1M) and NF-L
target (L/++++) vectors had been transfected. NF-L expression was not induced in the parental N2a cells lacking a NF-L transgene (Fig. 3,
parental); however, there was low level leakage from the
L/++++ transgene in the presence of tetracycline (Fig. 3, 0 h). Background in P19 cells was also derived from endogenous NF-L
gene expression. Interestingly, marked increases of NF-L transgene
expression in P19 cells was accompanied by up-regulation of endogenous
NF-M gene expression. Occasionally, there also appeared to be slight increases in NF-H expression.

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Fig. 3.
Protection assay of NF-H (H),
NF-M (M), NF-L (L), and -actin
( -A) mRNA levels in P19 (above) and N2a
(below, right) cells doubly transfected with the tTA
transactivator expression vector (pUHD15.1M) and the full-length NF-L
cDNA target gene (L/++++). The target transgene was activated
by withdrawal of tetracycline at the 0 time point. Parental N2a cells
(below, left) contained the tTA expression vector
but not the NF-L target transgene. Note that increases of NF-L
(L) mRNA in P19 cells is accompanied by increases of
NF-M (M) mRNA.
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Tetracycline-induced Inactivation of Wild-type Versus Mutant NF-L
Transgenes Demonstrates Stability Determinants in the 3'-CR and 3'-UTR
of the NF-L mRNA--
The stabilities of wild-type
versus mutant NF-L mRNAs were compared by inducing NF-L
transgene expression for 72 h and then measuring mRNA levels
at varying time points after the readdition of tetracycline. Fig.
4 depicts a representative experiment of mRNA decay in P19 cells containing wild-type (L/++++) or mutant (L/++
+, L/+++
, and L/++
) NF-L target transgene. Fig.
5 shows the average decline of
NF-L/
-actin mRNA levels in N2a and P19 cells from three
experiments. Similar pattern of NF-L mRNA decay was observed in L
cells (data not shown). In all instances, wild-type (L/++++) NF-L
mRNA was relatively unstable, comparable to measurements using
actinomycin-induced decay (3). Deletion of 3'-CR (L/++
+) or 3'-UTR
(L/+++
) diminished the loss of NF-L mRNA in each of the cell
lines tested. Moreover, deletion of both 3'-CR and 3'-UTR (L/++
)
was additive in that it further diminished the loss of the NF-L
transcript. Short term (6 h) losses of NF-L mRNAs from wild-type
and mutant transgenes in P19 cells showed intermediate levels of
decline (data not shown), supporting the view that decreases in the
rate of loss of NF-L/
-actin mRNA over time may be due to
increasing admixtures of mRNA from the endogenous NF-L gene. The
loss of NF-L mRNA upon inactivation of the transgene in P19 cells
was accompanied by a parallel loss of NF-M mRNA expression that had
been up-regulated by induction of the transgene. Interestingly, the
up-regulation (and subsequent down-regulation) of the endogenous NF-M
gene did not occur with high level expression of the L/++
mutant
NF-L mRNA.

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Fig. 4.
Protection assay of NF-H (H),
NF-M (M), NF-L (L), and -actin
( A) mRNA levels in P19 cells in which transcription
of wild-type (L/++++) and mutant (L/++ +, L/+++ , and L/++ ) NF-L
transgenes was activated for 72 h in the absence of tetracycline
and then inactivated by addition of tetracycline (0 time
point).
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Fig. 5.
NF-L mRNA decay in P19 and in N2a cells
following activation and inactivation of transcription of wild-type
(L/++++) and mutant (L/++ +, L/+++ , and L/++ ) NF-L cDNAs, as
described in Fig. 4. NF-L mRNA levels were quantitated by
phosphorimager and normalized to levels of -actin mRNA. Data
were averaged from three experiments.
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Localization of Stability Determinant to the Distal 23 nt of
3'-Coding Region and Proximal 45 nt of 3'-UTR of the NF-L
Transcript--
To localize stability determinants in NF-L transcript,
a NF-L transgene (L/++/del/++) was constructed with a 68-nt segment deleted from the junction between the coding region and 3'-UTR. The
deleted sequence corresponded to the binding site of the C-binding RNP
complex (1) and comprised the 23 nt of distal 3'-CR and 45 nt of
proximal 3'-UTR. Loss of NF-L mRNA from the deleted transgene (L/++/del/++) was much less than that from the wild-type transgene (L/++++) upon tetracycline-induced inactivation of the transgenes in
N2a cells (Fig. 6). In fact, Table
I shows that the NF-L transcript was
stabilized to a similar extent either by deletion of the 68-nt segment
from the junction of 3'-CR and 3'-UTR (L/++/del/++) or by deletion of
527 nt from the 3'-CR and 445 nt of 3'-UTR (L/++++). Deletion of either
the 3'-CR (L/++
+) or 3'-UTR (L/+++
) led to intermediate levels of
stabilization. A diminished rate of mRNA decay from the deleted
transgene (L/++/del/++) was also observed in L cells (data not shown).
The findings indicate that a major stability determinant is localized
to the junction between the 3'-CR and 3'-UTR.

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Fig. 6.
A, protection assay of NF-L
(L) and -actin ( A) mRNAs in N2a cells
transfected with mutant (L++/del/++) or wild-type (L/++++) transgene
after tetracycline-induced inactivation of the transgenes.
B, loss of NF-L/ -actin mRNAs in N2a cells with mutant
(L++/del/++) or wild-type (L/++++) transgene. Data were quantitated by
phosphorimager and averaged from three experiments.
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DISCUSSION |
The tetracycline-responsive promoter system provides for
stringent, non-toxic, and high level control of specific gene
expression using either the addition or withdrawal of the antibiotic
ligand (or derivatives) to activate or repress transcription (12-19). The present study has adapted this system to study mRNA stability using the ligand to turn off transcription after high levels of the
test gene mRNA are attained. For this purpose, the use of tetracycline as a repressor is advantageous in that the kinetics of the
off reaction is mediated by the addition rather than by the
disappearance (i.e. half-life) of the ligand. Our methods have also incorporated some safeguards such as the pooling of stably
transfected clones to equalize test gene copy number, reduce position
effects, and limit alterations from genetic perturbations during
passage of the transgenes. Pooling of transfectants may reduce levels
of induction but generates sufficient and comparable levels of
transgene mRNAs for ready assessment of transcript decay.
The relative persistence in steady-state levels of mutant
versus wild-type NF-L mRNA after inactivation of the
transgene indicates that deletion of either the 3'-coding region
(L/++
+) or the 3'-UTR (L/+++
) removes destabilizing components from
the NF-L transcript. The increased stability of the double mutant
(L/++
) mRNA suggests that sequences in the 3'-CR and in the
3'-UTR may also act in an additive or cooperative manner. Multiple
determinants in the 3'-coding region and 3'-UTR have also been detected
in other neuronal transcripts (21, 22) and could function in separate
pathways (see Refs. 23 and 24) or as separate components of a common pathway. The latter possibility is supported by the localization of a
major stability determinant to a 68-nt sequence at the junction of
3'-CR and 3'-UTR of NF-L. Both the 3'-CR and 3'-UTR sequence in the
68-nt fragment are necessary for assembly of a unique C-binding RNP
complex at this site (1). Interestingly, the stability of the
-globin mRNA is also regulated by a determinant on the proximal
edge of the 3'-UTR that is dependent upon sequence in the adjacent
3'-CR (25, 26).
The increased stability of the mutant NF-L transcripts in both neuronal
and non-neuronal cells suggests that destabilization of NF-L mRNA
involves common components that are widely present in different cell
lines. Moreover, the relative instability of wild-type NF-L mRNA in
both neuronal and non-neuronal cell lines, compared with the
extraordinary stability of the transcripts in primary neurons (3),
raises the possibility that NF mRNA levels are regulated by
altering a default destabilization pathway. Such putative stabilizing
conditions (or factors) might help to explain the discrepancy between
the high levels of NF expression that are attained in vivo
and the low levels of NF expression that occur in vitro. It
also indicates that destabilization of NF mRNA may provide key
insights into the mechanisms regulating levels of NF expression and
their role in motoneuron degeneration.
The increases in steady-state levels of endogenous NF-M mRNA that
accompany high level expression of the wild-type (L/++++) or single
mutant (L/++
+ or L/+++
) transgenes in P19 cells could be due to the
titering of a component(s) that serves to destabilize the NF-L and NF-M
transcripts. The inability of the double mutant (L/++
) transgene to
up-regulate NF-M expression may indicate that the function of the
putative component(s) in destabilizing NF-L and NF-M mRNAs
requires both the 3'-CR and 3'-UTR. Similar changes were not observed
in N2a or L cells but would have been precluded by methylation and
nonexpression of the NF-L and NF-M genes in these cell lines (27).
Up-regulation of NF-L upon overexpression of an NF-M transgene has been
reported in transgenic mice (28), although the relationship between
transgene and endogenous gene expressions may not be comparable to that
in P19 cells by virtue of the greater stability of NF mRNA in
vivo as well as the much higher levels of trangene expression
achieved in vitro.
Our biochemical studies have not only identified the binding site of a
unique C-binding RNP complex at the juction of 3'-CR and 3'-UTR but
have also detected multiple binding sites of a U/A-binding RNP complex
in the 3'-CR and 3'-UTR of NF-L mRNA (1). Moreover, binding of the
C-binding and U/A-binding complexes show a pattern of reciprocal
interactions, as if competing for common factors or binding sites. To
what extent the different complexes enhance or alter the stability of
NF-L transcript is presently unknown. The tetracycline-responsive
promoter system, herein described, provides a functional assay that
will enable a more precise mapping of regulatory elements and, thereby,
facilitate the identification and cloning of cognate binding factors
that regulate neurofilament gene expression.