(Received for publication, May 10, 1995; and in revised form, August 24, 1995)
From the
High levels of neurofilament (NF) mRNA expression are attained
during early postnatal development and are a major determinant of
axonal size. High level NF expression is also dependent upon axonal
continuity since NF mRNA levels are down-regulated after nerve
transection. This study shows that both postnatal up-regulation and
axotomy-induced down-regulation are altered by deletion of 3`-UTR from
the mouse light NF subunit (NF-L). Transgenes with
(NF-L) or without (NF-L
) 3`-UTR
display similar patterns of neuron-specific expression but differ in
their respective levels of expression. Whereas changes in the level of
NF-L
mRNA parallel those of the endogenous mouse NF-L
mRNA, changes in the level of NF-L
mRNA differ from
the pattern of endogenous NF-L expression during postnatal
up-regulation and axotomy-induced down-regulation. Specifically, the
NF-L
transgene undergoes a 3-fold aberrant
up-regulation between embryonic days 15 (E15) and 18 (E18) and has lost
its susceptibility to axotomy-induced down-regulation. Studies of
transfected P19 cells show that 3`-UTR deletion leads to a severalfold
stabilization of NF-L mRNA and an increase in steady-state mRNA level.
The findings support the working hypothesis that the 3`-UTR contains
determinants that alter stability and that stabilization of NF-L mRNA
regulates the levels of NF-L mRNA in neuronal tissues and cells.
Large axons are composed predominantly of neurofilaments (NFs) ()so that their size and rate of impulse conduction are
dependent on the expression, assembly, and transport of the component
light (NF-L), midsized (NF-M) and heavy (NF-H) NF subunits. Expression,
assembly, and transport of the endogenous NF gene products are normally
coordinated so that NFs do not accumulate, and axonal size is
determined by the levels of NF subunit expression within the
cell(1, 2) . Expression of exogenous NF-L(3) ,
NF-H(4) , or mutant NF-H (5) transgenes can be
disruptive to NF assembly or transport and lead to proximal NF
accumulations and to reduced NF content and size of distal axons.
Reduced expression of endogenous NF-L also leads to NF accumulations
and reduction in axonal size as well as diminished expression of NF-M
and NF-H(6) . Axonal size may also be influenced by NF
phosphorylation or by interactions with Schwann
cells(7, 8) .
NF expression arises in post-mitotic neurons (9) but remains at low levels throughout embryonic development(10) . High level NF expression takes place during early postnatal development(11) , coincident with widespread enlargement and myelination of axons(12) . The postnatal up-regulation is manifested by coordinate increase in NF mRNAs and proteins during time frames that reflect regional differences in neuronal maturation(11) . In primary sensory neurons, the postnatal surge of NF expression correlates closely with increases of NF content and with enlargements of perikaryal size and axonal caliber(13) .
Studies in our laboratory have shown that the postnatal up-regulation of NF expression is accompanied by increases in stabilities of NF-L, NF-M, and NF-H mRNAs in primary cultures of rat sensory neurons (14) . The same NF transcripts in adult sensory neurons are destabilized in vitro in a transcription-dependent manner (15) by a process that resembles the coordinated down-regulation of NF mRNAs following axotomy(16) . These findings have led to the view that NF mRNAs are stabilized in high-expressing neurons and that changes in mRNA stabilities may account for the coordinate up-regulation and down-regulation of NF mRNA levels that occur during postnatal development and in axotomized neurons, respectively. Moreover, the conservation of 3`-UTR in NF mRNAs (14) and the involvement of 3`-UTR in mRNA stability (17, 18) suggest that the 3`-UTR in NF genes may harbor determinants of NF expression.
The present study has begun to
test the above hypothesis in vivo by comparing expression of
the endogenous mouse NF-L gene with that of a marked mouse NF-L
transgenes in which the 3`-UTR is intact (NF-L) or
deleted (NF-L
). Our findings identify the 3`-UTR of
the mouse NF-L gene as instrumental in mediating both the developmental
up-regulation and the axotomy-induced down-regulation of NF-L. The
findings indicate the importance of 3`-UTR in NF-L expression and
highlight the potential role of posttranscriptional mechanisms in
regulation of neuron-specific gene products.
Transection of sciatic nerves and
processing of axotomized and control L4/L5 dorsal root ganglia (DRG)
were conducted, as described in rats (15) . Tissues from
transgenic mice as well as cultured cells were solubilized in 4 M guanidinium thiocyanate and processed for RNA by phenol
extraction(23) . RNA (10-20 µg) was hybridized with
NF-L and -actin cRNAs (4
10
cpm/probe) in 20
µl containing 80% formamide, 400 mM NaCl, 1 mM EDTA, and 40 mM MOPS buffer, pH 7.0, at 65 °C for
14-18 h. Samples were digested with RNase T1 (15) or with
S1 (300 units/200 µl) for 30 min at 37 °C, extracted with
phenol/chloroform, and precipitated with ethanol. Protected fragments
were resolved on acrylamide/urea gels, and radioactivities were
quantitated by phosphor-imaging analysis.
Figure 1:
Schematic diagram of mouse NF-L
extending from -325 (relative to the transcription start site) to
+5.5 kilobases (NF-L-325/+5.5), the same construct that
is marked with a 66-bp insert at the BglII site
(NF-L*-325/+5.5), the full-length NF-L transgene
(NF-L) with marked insert and intact 3`-UTR to
+4100, and the deletion mutant transgene (NF-L
)
with marked insert and deleted 3`-UTR sequence between +3615 and
+3984.
The NF-L transgenes
also contained an insert at the BglII site (+829) that
enables transcripts from the transgenes and endogenous mouse NF-L gene
to be distinguished by RNase protection assay (see Fig. 1). With
an antisense probe that spans the insertion site, fragments of 190 and
110 nucleotides are generated from the transgene and endogenous mouse
NF-L genes, respectively. Protection assays show that NF-L and NF-L
mRNAs are readily expressed in stably
transfected L cells and are coexpressed with endogenous mouse NF-L mRNA
in P19 cells (Fig. 2). The NF-L
and
NF-L
transgenes generate similar steady-state mRNA
levels in L cells but not in P19 cell lines. Levels of NF-L
mRNA are higher than NF-L
mRNA in stably
transformed P19 cells even though the lines were derived from pooled
transformants and contained similar average copy numbers of the
respective transgenes (5-10 copies/pool). The presence of
NF-L
or NF-L
increased levels of
endogenous NF-L (Fig. 2), whereas the presence of
NF-L
also increased levels of NF-M and NF-H mRNA
(data not shown).
Figure 2:
Protection assay showing mRNA levels of
NF-L transgene (NF-L*), endogenous NF-L (NF-L), and -actin
(
-actin) in untransfected L cells (lane 2), L cells transfected
with NF-L
(lane 3) or NF-L
(lane 4) in untransfected P19/clone 48 cells (lane
5), P19/clone 48 cells transfected with NF-L
(lane 6) or NF-L
(lane 7) in
untransfected P19/clone 43 cells (lane 8), P19/clone 43 cells
transfected with NF-L
(lane 9) or
NF-L
(lane 10). Lane 1 shows
undigested probes for NF-L*/NF-L and
-actin. These probes generate
protected fragments of 190 bp for the marked transgenes (NF-L*), 97 bp
for endogenous NF-L (NF-L), and 83 bp for
-actin (
-actin).
P19/clone 48 and P19/clone 43 express low levels of NF-L. P19/clone 43
also expresses low levels of NF-M and NF-H.
Expression of the transgene versus endogenous
NF-L genes was analyzed in neural and non-neural tissues of F1 adult
mice from each cell line by RNase protection assay. Transgene
expression was detected in every line, most readily in brain and spinal
cord, but levels of expression did not correlate with transgene copy
number. Steady state levels of NF-L mRNA were, on
average, at least 2-fold higher than that of NF-L
mRNA. Ectopic expression occurred in non-neural tissue but at
levels that were at least 10-fold lower than that of brain and spinal
cord. Minute amounts of transgene mRNA were detected in thymus, kidney,
lung, testis, spleen, and liver.
Lines A and D expressed the highest
levels of NF-L and NF-L
transgenes
and were used to assess the effects of 3`-UTR deletion on the regional
expression of the transgenes in the nervous system. Both
NF-L
and NF-L
transgenes showed
similar rank order patterns of expression relative to the expression of
the endogenous NF-L gene (Fig. 3). Increased
transgene/endogenous mRNA expression was also noted in brain versus spinal cord tissues of other transgenic lines (data not shown).
The findings indicate that the NF-L
and
NF-L
transgenes are expressed in a similar
distribution, albeit at differing levels, in the nervous system.
Although the expression of the transgenes differed from that of the
endogenous NF-L gene, the differences were not due to deletion of the
3`-UTR. Interestingly, the pattern of NF-L transgene expression is
consistent with that of other neuronal transgenes that have been
examined at the cellular level (see (24) ), namely partial (or
mosaic) expression in neurons that express the endogenous gene and
ectopic expression in neurons that do not express the endogenous gene.
Figure 3:
Expression of NF-L (panel A) and NF-L
(panel B)
transgenes in different regions of the nervous system. Protection
assay shows mRNA levels of NF-L
or NF-L
transgenes relative to endogenous NF-L (NF-L) mRNA in cerebrum (lane 1), cerebellum (lane 2), brainstem (lane
3), spinal cord (lane 4), and dorsal root ganglia (lane 5) of adult transgenic mice from line D
(NF-L
) and line A (NF-L
). Levels of
transgene/endogenous NF-L mRNA expression in the different tissues,
relative to the level of expression in cerebrum, are shown in panel
C.
Figure 4:
Developmental expression of transgene versus endogenous NF-L mRNAs in brain of F2 mice bearing
NF-L (panel A) and NF-L
(panel B) transgenes at embryonic day 15 (lanes
1-4), embryonic day 18 (lanes 5-8), and
postnatal day 2 (lanes 9-12). Protection assay generates
fragments of 190 nucleotides from NF-L
and
NF-L
mRNA, 97 nucleotides from endogenous NF-L mRNA,
and 83 nucleotides from
-actin mRNA. Panel C shows the
up-regulation in NF-L
, NF-L
, and
endogenous NF-L mRNA at embryonic day 18 and at postnatal day 2
relative to levels of expression at embryonic day 15. All NF-L mRNA
levels were normalized to
-actin mRNA levels. Endogenous NF-L mRNA
values were averaged from NF-L
and NF-L
transgenic mice. Each lane represents analysis of tissue
from a littermate.
Figure 5:
Developmental expression of transgene versus endogenous NF-L mRNAs in spinal cord of F2 mice bearing
NF-L (panel A) and NF-L
(panel B) transgenes at embryonic day 15 (lanes
1-4), embryonic day 18 (lanes 5-8), and
postnatal day 2 (lanes 9-12). Panel C shows the
up-regulation in NF-L
, NF-L
, and
endogenous NF-L mRNA at embryonic day 18 and at postnatal day 2
relative to levels of expression at embryonic day 15. Analyses were
identical to those described in Fig. 4.
Analyses of NF-L
expression in transgenic mice (line A) show that NF-L mRNA and endogenous NF-L mRNA are down-regulated in axotomized
sensory neurons at 4 days (data not shown) and at 7 days (Fig. 6, lanes 1 and 2) after sciatic nerve
transection. In mice bearing the NF-L
transgene (line
D), mRNA from the endogenous NF-L gene but not from the transgene is
down-regulated at 4 days (data not shown) and at 7 days (Fig. 6, lanes 3 and 4) after axotomy. Down-regulation of
endogenous NF-L mRNA but not NF-L
mRNA also occurs in
sensory neurons of line E (Fig. 6, lanes 5 and 6) and line F (Fig. 6, lanes 7 and 8)
at 7 days following sciatic nerve transection.
Figure 6:
Panel A, the expression of NF-L
transgene (NF-L*), endogenous NF-L (NF-L), and -actin
(
-actin) mRNAs in axotomized (lanes A) versus control (lanes C) L4/L5 DRG at 7 days after unilateral
sciatic nerve transection. Axotomy-induced changes were examined in a
transgenic mouse line (panel A) bearing the NF-L
transgene (lanes 1 and 2) and in three lines
(lines D, E, and F) bearing the NF-L
transgene (lanes 3 and 4, 5 and 6, and 7 and 8, respectively). Protection assay generates
fragments of 190 nucleotides (NF-L*) from NF-L
and
NF-L
mRNAs, 97 nucleotides (NF-L) from endogenous
NF-L mRNA, and 83 nucleotides (
-actin) from
-actin mRNA. Panel B, the extent of axotomy-induced down-regulation of
NF-L, NF-L
, and NF-L
mRNA in
axotomized DRG (lanes A in panel A) compared with the
mRNA levels in unaxotomized contralateral control DRG (lanes C in panel A). NF-L
,
NF-L
, and NF-L mRNA levels are normalized to
-actin mRNA levels.
Figure 7:
Protection assay showing mRNA levels of
NF-Land NF-L
transgenes (NF-L*),
endogenous NF-L (NF-L), and
-actin (
-actin) in P19/clone 48
cells transfected with NF-L
or NF-L
following the addition of 4 µg/ml actinomycin. Panel A shows a representative experiment, while panel B shows
average (n = 6) levels of NF-L
,
NF-L
, and NF-L mRNAs relative to
-actin mRNA at
0, 4, 8, and 16 h in actinomycin-treated
cells.
Transgenic mice have provided a model for identifying
regulatory components in the 3`-UTR of mouse NF-L that function in
neuronal tissues of the mouse. NF-L transgenes with
(NF-L) or without (NF-L
) 3`-UTR are
expressed in a neuron- specific manner, like other human and mouse NF-L
transgenes with similar or more extensive 5`- and 3`-flanking
sequence(26, 27, 28, 29, 30, 31) .
Deletion of 3`-UTR in NF-L
does not change the
pattern of tissue-specific expression but alters the level of gene
expression during developmental up-regulation and axotomy-induced
down-regulation of NF-L. In addition, deletion of 3`-UTR is associated
with increased steady-state mRNA levels in transgenic mice and in
stably transfected P19 cells.
Alterations in the developmental
expression of the 3`-UTR-deleted transgene (NF-L)
occur during the transition from low level to high level NF gene
expression. This transition normally occurs during early postnatal
development and is part of a coordinated postnatal up-regulation of all
three NF genes(11) . Deletion of the 3`-UTR leads to an
aberrant up-regulation of the NF-L transgene at E18 during late
embryonic development. The aberrant up-regulation of the
NF-L
transgene at E18 does not alter the expression
of the endogenous NF-L gene or prevent the subsequent postnatal
up-regulation of the mutant transgene. Hence, a direct relationship
between the aberrant embryonic and postnatal up-regulations is not
readily apparent.
Deletion of NF-L 3`-UTR also prevents the down-regulation that occurs in DRG neurons following sciatic nerve transection. This axotomy-induced loss of NF mRNAs in high expressing adult DRG is related to postnatal NF up-regulation in the sense that it does not occur in axotomized DRG neurons if the transections are made prior to the period of postnatal up-regulation(32) . Hence, both postnatal up-regulation and axotomy-induced down-regulation are associated with high level expression.
The phenotype of the mutant
transgene (NF-L) is complex in that it results in a
gain of function (premature up-regulation) during development and a
loss of function (sensitivity to axotomy) in mature neurons. A gain of
function in a deletion mutant implies that the 3`-UTR contains
repressive elements during development, while the loss of function
suggests that factors that lower NF-L expression can no longer function
in mature neurons. Such diverse effects in a deletion mutant suggest
that positive and negative elements are deleted or that the cognate
binding factors are inoperative in the respective target neurons. The
changes could reflect alterations at transcriptional or
posttranscriptional levels of function. The latter is favored by the
complexity of the phenotype and its limitation to quantitative, not
distributional, features of expression.
Moreover, the ability of the
3`-UTR deletions to increase the half-life of NF-L transcripts in P19
cells provides direct evidence that increased NF-L expression can
result from stabilization of NF-L transcripts and that differential
stabilization is manifest by mRNAs derived from
NF-L/NF-L
transgenes. Additional
studies in our laboratory indicate that NF-L
mRNA is
also more stable than NF-L
mRNA in brain polysome
preparations from transgenic mice. Although limited by variable
nonspecific degradation, this in vitro model indicates that
factors regulating mRNA stabilities of the
NF-L
/NF-L
transgenes are present in
primary neurons as well as in P19 cells.
While our findings support
the view that the NF-L 3`-UTR is instrumental in regulating mRNA
stability, the regulatory elements and underlying mechanisms are
unknown. For example, it is unclear whether the aberrant up-regulation
(E18) reflects a differential maturation of stabilizing or
destabilizing factors that can selectively affect the NF-L or NF-L
transgenes. It is also possible that the
aberrant embryonic (E18) and postnatal up-regulations may involve
common regulatory components that destabilize (or stabilize) NF-L mRNA
and become functional at different times during development. Deletion
of 3`-UTR may also alter the balance of stabilizing versus destabilizing components and lead to increased steady-state levels
of NF-L mRNA in cell lines and transgenic tissues that contain active
cognate binding factors.
Involvement in mRNA stability may partly account for the conservation of 3`-UTR sequence in the NF (14) and other genes(33) . However, transcript stabilization/destabilization often involves sequences within the coding region or multiple cis-acting elements and may be closely associated with translation (for reviews, see Refs. 17, 18, and 35-37). Hence, 3`-UTR deletions may alter only a part of the stabilizing or destabilizing pathway. It is also likely that stabilization of NF-L mRNA serves to coordinate levels of NF-L mRNA with those of NF-M and NF-H mRNAs and with the levels of NF metabolism in the cells. The present study identifies the 3`-UTR as a key regulatory region in NF-L expression and provides a framework for identifying additional components that regulate NF mRNA levels within the cell.