From the Division of Neuropathology, Department of Pathology and
Laboratory Medicine, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania 19104
Levels of neurofilament (NF) gene expression are
important determinants of basic neuronal properties, but overexpression
can lead to motoneuron degeneration in transgenic mice. In a companion study (Cañete-Soler, R., Schwartz, M. L., Hua, Y., and
Schlaepfer, W. W. (1998) J. Biol. Chem. 273, 12650-12654), we show that levels of NF expression are regulated by
altering mRNA stability and that stability determinants are present
in the 3'-coding region (3'-CR) and 3'-untranslated region (3'-UTR)
of the NF light subunit (NF-L) transcript. This study characterizes the
ribonucleoprotein complexes that bind to the NF-L mRNA when
cytoplasmic brain extracts are incubated with radioactive probes. Gel
retardation assays reveal ribonucleoprotein complexes that are
selectively competed with poly(C) or poly(U))/poly(A) homoribopolymers
and are referred to as C-binding and U/A-binding complexes,
respectively. The C-binding complex forms on the proximal 45 nucleotides of 3'-UTR, but its assembly is markedly enhanced by 23 nucleotides of flanking 3'-CR sequence. U/A-binding complexes form at
multiple binding sites in the 3'-CR and 3'-UTR. A pattern of reciprocal
binding suggests that the C-binding and U/A-binding complexes interact
and may compete for common components or binding sites. Cross-linking studies reveal unique polypeptides in the C-binding and U/A-binding complexes. The findings provide the basis for probing mechanisms regulating NF-L mRNA stability and the relationship between NF overexpression and motoneuron degeneration in transgenic mice.
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INTRODUCTION |
Neurofilaments (NFs)1
are the principle constituent of the axonal cytoskeleton, so that
levels of NF expression are believed to be a major determinant of
axonal size. This view is supported by the simultaneous up-regulation
of the light (NF-L), midsized (NF-M), and heavy (NF-H) NF subunit
mRNAs (2) that accompanies the enlargement and myelination of axons
during postnatal development of the nervous system (3). The postnatal
surge in NF expression is a posttranscriptional event (4) that is
mediated in part by stabilization of NF mRNAs (5). The extent of
axonal enlargement, and presumably the levels of NF up-regulation, are
determined by the nature of target cell innervations and can be
reconstituted in transected nerve upon successful reinnervation of the
target site (6). Moreover, the stability of NF mRNAs becomes
dependent upon continuity of axons with target sites in that the
transcripts are destabilized upon nerve transection or excision and
transfer of parent neurons to primary culture (7).
Interestingly, NF mRNAs are stabilized during the same postnatal
interval in which neurons acquire the ability to survive nerve
transection and mount a regenerative response (8). The overlapping
appearance of these phenomena raises the possibility that common
components of posttranscriptional pathways regulate neuronal
homeostasis and levels of NF expression. If so, then titration of
regulatory components could account for: (i) the motoneuron
degeneration in transgenic mice upon overexpression of a wild-type NF-L
(9) or NF-H (10) transgene or modest expression of a mutant NF-L
transgene (11), (ii) the preferential degeneration of NF-enriched
neurons in mice bearing a mutant SOD-1 transgene (12), and (iii) the
transient up-regulation of NF mRNA that precedes the spontaneous
motoneuron in Wobbler mice (13). Mechanisms regulating levels of NF
expression may therefore bear an important relationship to
neurodegenerative states.
We have begun to map stability determinants that regulate steady-state
levels of NF-L mRNA. Deletion of the 3'-untranslated region
(3'-UTR) from a mouse NF-L transgene stabilizes the NF-L transcript in
neuronal cell lines and alters the developmental up-regulation and
axotomy-induced down-regulation of the transgene in transgenic mice
(14). More recently, we have begun to apply an expression system with
tetracycline-regulated promoter to map stability determinants in NF
mRNAs. In a companion study (1), we demonstrate the presence of
stability determinants in the 3'-coding region (3'-CR) and 3'-UTR of
NF-L mRNA and have localized a stabilizing element at the junction
of 3'-CR and 3'-UTR that may account for the stabilizing properties of
the transcript.
The present study examines the ribonucleoprotein (RNP) complexes that
assemble on NF-L mRNA when nascent radioactive fragments of the
transcript are incubated with cytosolic brain extracts. The RNP
complexes have been characterized by their abilities to be competed
with homoribopolymers, thereby taking advantage of the high affinity
and selective binding of many RNA-binding proteins for specific
homoribopolymers (15). A similar approach has been very helpful in
characterizing RNP complexes that are believed to stabilize
-globin
mRNA (16, 17). The goal of the present study is to correlate
biochemical with functional studies (1) and thereby develop a working
model for elucidating the mechanisms that regulate the stability of the
mouse NF-L transcript.
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EXPERIMENTAL PROCEDURES |
Preparation of Cytosolic Extracts--
Rat brain and liver
(10-15 g) were minced, washed in phosphate-buffered saline, and
homogenized in a Dounce homogenizer with two volumes of 50 mM potassium acetate, 3 mM magnesium acetate, and 2 mM dithiothreitol in 20 mM HEPES buffer,
pH 7.4 (Buffer A), and a mixture of protease inhibitors (18). Crude
supernatant from a microcentrifuge spin (10,000 × g
for 10 min) was centrifuged at 100,000 × g for 60 min,
yielding cytosolic extracts with 7-9 or 15-20 mg/ml protein from
brain and liver, respectively. Glycerol (5%) was added, and the
extracts were aliquoted, snap-frozen, and stored at
80 °C. All
operations were conducted on ice or at 4 °C.
Multiple dishes (>108 cells) of confluent P19, N2a, or L
cells, originally obtained from the American Type Culture Collection, were washed twice in phosphate-buffered saline, detached with a
scraper, pelleted in a clinical centrifuge (1,000 × g
for 5 min), and homogenized in a Dounce homogenized with two volumes of
Buffer A containing 0.5% Nonidet P-40. Crude supernatants from a
microcentrifuge spin (10,000 × g for 10 min) were
centrifuged at 100,000 × g for 60 min, yielding
cytosolic extracts (protein of 5-8 mg/ml). Extracts were admixed with
glycerol, aliquoted, and stored at
80 °C. Some extracts were also
concentrated 3-fold in a Centricon-10 (Amicon Corp.).
Preparation of RNA Probes--
Templates for RNA probes (Fig. 1)
were prepared by PCR with restriction sites or a T7 promoter sequence
in the primers to facilitate cloning or to generate RNA products
directly from PCR templates. The parent A680 template (+1482/+2161) was
cloned into HindIII/BamHI sites of the
pBluescript II SK+ vector (Stratagene) and was cut with
BamHI (at +2161), HincII (at +1779), or
BspMI (at +1712) to yield the full-length A680 template
(+1482/+2161), the A680/H template (+1482/+1779), or the A680/B
template (+1482/+1712). The SalI (and HincII)
site in the polylinker was mutated to preserve the continuity of T7
promoter sequence with the A680/H template. The A680/del template was
generated by a second PCR reactions that fused upstream and downstream
PCR products with sequence between +1712 and +1779 deleted. Probes 1-5
were cloned into the SK+ vector, and probes A, B, and X were generated
off PCR templates. Templates were sequenced to confirm junctional and
mutational sites.
RNA probes were uniformly labeled with [32P]UTP using T7
polymerase (7). Full-length probes were excised from denaturing acrylamide gels, eluted overnight into 0.5 M
NH4 acetate, 0.1% SDS, and 1 mM EDTA, and
ethanol-precipitated. Probes were diluted to 2.5 × 104 cpm/µl in Buffer A immediately prior to use.
Gel Retardation and Cross-linking
Studies--
Homoribonucleotides (Sigma) and antisense
oligonucleotides were solubilized in Buffer A. Radioactive probes
(5 × 104 cpm), cytosolic extract (120 µg), and
varying amounts of homoribonucleotides or antisense oligonucleotides
were incubated for 30 min at 20 °C in 20 µl, digested for 10 min
with 2 units of RNase T1 (Sigma), dissociated with heparin (5 mg/ml),
and electrophoresed on a 5% acrylamide gel in 0.5× Tris-borate-EDTA
buffer. Dried gels were subjected to autoradiography. For cross-linking
analyses, the complexes were cross-linked on ice for 30 min at 3 cm
distance underneath a UV light (4 × 106
J/cm2) and further digested for 15 min at 20 °C with
RNase T1 (10 units) or RNase A (10 µg). Radioactive cross-linked
polypeptides were solubilized by boiling for 5 min in SDS sample
buffer, separated by SDS-polyacrylamide gel electrophoresis, and
identified by autoradiography of dried gels.
Complex formations were compared on probes that were denatured at
95 °C for 2 min and rapidly or slowly cooled to 20 °C prior to
the addition of extract. Probes were also heat-denatured with antisense
oligonucleotide in excess of EDTA (5 mM) and incubated with
RNase H (1 unit) for 20 min at 37 °C in excess magnesium (3 mM) prior to addition of extract.
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RESULTS |
RNP Complexes Bind to the NF-L mRNA and Are Selectively
Competed with Specific Homoribopolymers--
An NF-L mRNA probe
(A680) that spans the entire 3'-UTR (427 nt) and distal 253 nt of 3'-CR
(see Fig. 1) was used to identify binding
factors and binding sites that could account for stability determinants
in these regions of the transcript (1). When brain extracts were
incubated with the radioactive A680 probe and electrophoresed on
non-denaturing gels, multiple gel-shifted bands were detected (Fig.
2, lane 3). These bands were
characterized by their abilities to be competed by addition of
increasing amounts of poly(C) (lanes 4-6), poly(U)
(lanes 7-9), poly(A) (lanes 10-12), or
poly(G) (lanes 13 and 14)
homoribopolymers. A set of faster migrating bands (solid arrowheads) were competed by 20 ng of poly(C), but not by 100-fold higher concentrations of poly(G), poly(A), or poly(U). Similarly, a set
of slower migrating bands (open arrowheads) were competed by
200 ng of poly(A) or poly(U), but not by 10-fold higher concentrations of poly(C) or poly(G). The faster migrating bands are referred to as
the C-binding complex, whereas the slower migrating bands are referred
to as the U/A-binding complex. Gel-shifted bands were not seen when the
probes were incubated without extract (lane 1) or when the
extract was treated with protease K (lane 2) or heated to
65 °C (data not shown).

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Fig. 1.
Schematic diagram of RNA probes in
relationship to the mouse NF-L cDNA. Nucleotides are
numbered according to the transcription start site.
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Fig. 2.
Gel shift assay of RNP complexes that bind to
probe A680 (+1482/+2161) when incubated with cytosolic brain extract
(120 µg) in the presence of 0 ( ), 20 (+), 200 (++), or 2000 (+++)
ng of poly(U), poly(A), poly(C), or poly(G) homoribopolymers. A
set of slower migrating bands (open arrowheads) are competed
with poly(U) or poly(A) and are referred to as the U/A-binding complex.
A set of faster migrating bands (closed arrowheads) are
competed with poly(C) and are referred to as the C-binding complex.
Gel-shifted bands do not occur in the absence of extract (lane
1) or when the extract is treated with protease K (lane
2).
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C-binding Complexes Require the Proximal 45 nt of 3'-UTR and Distal
23 nt of 3'-CR for Binding--
To localize binding sites, gel shift
assays were conducted with probes that extended to varying distances
into the 3'-UTR (Fig. 3). The C-binding
complex (solid arrowheads) formed on the full-length A680
probe extending to +2161 (lanes 1-3), to a much lesser
extent on probe A680/H extending to +1779 (lanes 4-8), but
not on probe A680/B extending to +1712 (lanes 9-12) or on probe A680(del) in which sequence between +1712 and +1779 is deleted (lanes 13-15). Variations in relative intensities of
gel-shifted bands of the U/A- and C-binding complexes occurred when
examined with different probes. In addition, the C-binding complex was markedly enhanced on probe A680/H when U/A-binding complex was competed
off with poly(U) or poly(A) (compare lanes 4 with
lanes 6-8). The findings indicate that formation of the
C-binding complex requires sequence between +1712 and +1779. This
sequence comprises the distal 23 nt of 3'-CR (+1712/+1734) and the
proximal 45 nt of 3'-UTR (+1735/1779).

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Fig. 3.
Gel shift assay of RNP complexes that bind to
probe A680 (+1482/+2161), probe A680/H (+1482/+1779), probe A680/B
(+1482/+1712), and probe A680/del (sequence deleted between
+1712/+1779) after incubation with brain extract in the presence of 0 ( ) or 2000 ng (+++) of poly(A), poly(U), or poly(C)
homoribopolymers. Gel-shifted bands of the C-binding complex
(closed arrowheads) form on probes A680 and A680H, but not
on probes A680/B or A680/del. Gel-shifted bands of the U/A-binding
complex (open arrowheads) form on all probes.
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To confirm the location of the binding site for the C-binding complex,
gel shift studies were conducted with probe A (+1712/+1779), which
contained the 3'-CR and 3'-UTR components of the putative binding site,
and with probe X (+1735/+1779), which contained only the 3'-UTR
component of the binding site. Fig. 4
shows that the C-binding complex (solid arrowhead) readily
formed on probe A (lane 1), but not very well on probe X
(lane 6). Upon prolonged exposures of autoradiograms,
similar but markedly reduced amounts of C-binding complex can be
identified on probe X (data not shown). Low (20 ng), medium (200 ng),
and high (2000 ng) amounts of poly(C) competed the C-binding complex
from probe A (lanes 2-4) and probe X (data not shown). Note
that the addition of poly(C) not only abolished the C-binding complex
but also enhanced the formation of the slow-migrating U/A-binding
complex (open arrowhead) on probe A (lanes 2-4)
and probe X (data not shown). Additional gel-shifted bands formed on
probe A and, especially, on probe X, but were not competed with poly(C)
or poly(U), and their relationship to the C- and U/A-binding complexes
is unclear. Their presence does not obscure the principle finding,
i.e. that the C-binding complex binds weakly to the proximal
45 nt of 3'-UTR and that this binding is markedly enhanced by the
addition of upstream flanking sequence.

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Fig. 4.
Gel shift assay of the C-binding complex
(closed arrowhead) and U/A-binding complex (open
arrowhead) that form on probe A (+1712/+1779) and on probe X
(+1735/+1779) after incubation with brain extract in the presence of 0 ( ), 20, (+), 200 (++), or 2000 (+++) ng of poly(U) or poly(C).
Other RNP complexes that form on the probes are not competed with
homoribonucleotides.
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Further evidence that 3'-CR is instrumental in the formation of the
C-binding complex is seen in gel shift assays using a series of
5'-deletion probes extending from +2064 (probe 1), +1870 (probe 2),
+1768 (probe 3), +1735 (probe 4), and +1519 (probe 5) to +2161 (see
Fig. 1). Whereas probes 1-4 contained increasing amounts of 3'-UTR,
only probe 5 extended beyond the 3'-UTR and into the 3'-CR. Fig.
5 shows that addition of poly(C)
abolished the set of fast-migrating gel-shifted bands (solid
arrowheads) that formed on probe 5 (compare lanes 1 and
3) but did not abolish the set of fast-migrating gel-shifted
bands that formed on probe 4 (compare lanes 4 and
6), probe 3 (compare lanes 7 and 9),
probe 2 (compare lanes 10 and 12) or probe 1 (compare lanes 13 and 15). The ability to be
competed away with poly(C) identifies the fast-migrating set of bands
on probe 5 as the C-binding complex. This complex was also enhanced in
the presence of poly(U) (compare lanes 1 and 2).
Gel-shifted bands of similar migration also formed on probes 1-4, but
were not competed with poly(C), so that their relationship to the
C-binding complex is unclear. The findings provide additional evidence
that formation of the C-binding complex is dependent upon sequences in
the proximal 3'-UTR and distal 3'-CR.

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Fig. 5.
Gel shift assay of C-binding (closed
arrowheads) and U/A-binding (open arrowheads)
complexes that form on probes 1 (+2064/+2161), probe 2 (+1870/+2161),
probe 3 (+1768/+2161), probe 4 (+1735/+2161), and probe 5 (+1519/+2161)
after incubation with brain extract in the presence of 0 ( ) or 2000 (+++) ng of poly(U) or poly(C). Other RNP complexes that form on
the probes are not competed with homoribonucleotides.
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U/A-binding Complexes Bind at Multiple Sites in 3'-UTR of
NF-L mRNA--
U/A-binding complexes are defined as sets of
slow-migrating gel-shifted bands that are competed with poly(U)
or poly(A). Fig. 5 shows that similar U/A-binding complexes (open
arrowheads) were competed with poly(U) on probe 5 (lanes
1 and 2), probe 4 (lanes 4 and
5), probe 3 (lanes 7 and 8), probe 2 (lanes 10 and 11), and probe 1 (lanes
13 and 14). Gel-shifted bands of U/A-binding complexes
on probes 1-5 differed in their relative abundance. Likewise, similar
gel-shifted bands in different proportions comprised the U/A-binding
complex that formed on probes containing only 3'-CR sequence (A680/B),
with the addition of proximal 3'-UTR (A680/H) or with the addition of
the full 3'-UTR (A680) (see Fig. 3). U/A-binding complexes were also
present on short probes (see Fig. 4) and were generally composed of
fewer and fainter bands. Moreover, the U/A complexes were sometimes
competed more effectively with poly(U) or poly(A), depending on the
size and position of the probe. Finally, in many instances, competing
off the U/A-binding complex with poly(U) or poly(A) enhanced the
formation of the C-binding complex. We conclude that slow-migrating
U/A-binding complexes of varying composition form at multiple sites in
the 3'-CR and 3'-UTR of NF-L mRNA and that formation of U/A-binding complexes may adversely affect the formation of C-binding
complexes.
Formation of C-binding Complexes Requires Multiple C-rich Sequences
in the Proximal 3'-UTR--
The sequence necessary for the formation
of the C-binding complexes extends from +1712 to +1779 of the NF-L
cDNA and spans the distal 23 nt of 3'-CR and proximal 45 nt of
3'-UTR, as follows.
To assess specific sequence requirements for the formation of the
C-binding complex, gel shift assays were conducted with brain extracts
and probe A (+1712/+1779) that had been preincubated with 15-mer
antisense oligonucleotides to the proximal (oligo P), middle (oligo M),
or distal (oligo D) 3'-UTR sequence in probe A (as schematically shown
above). Fig. 6 shows that formation of
the C-binding complex was abolished by preincubation with antisense probes P or M and markedly reduced by antisense probe D. Preincubation with the same amounts (100 ng) of 15-mer oligos to sequence in the SK+
vector (oligo SK) or 5'-flanking region of NF-L (oligo 5') did not
affect the formation of C-binding complexes. The findings indicate that
assembly of the C-binding complexes can be disrupted at multiple sites,
thereby requiring an extended stretch of intact sequence in the
proximal 3'-UTR that is enriched in cytosine and pyrimidine
residues.

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Fig. 6.
Gel shift assay of the C-binding complex
(closed arrowheads) that forms when probe A is preincubated
with 15-mer antisense oligonucleotides (100 ng) to the proximal
(P), middle (M), or distal (D)
sequence of the putative C-binding site and then incubated with brain
extract in the presence of 0 ( ) or 2000 (+++) ng of poly(C) or
poly(U). Control 15-mer antisense oligonucleotides were from the
SK+ vector (SK) and from the 5'-flanking sequence of NF-L
(5').
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Components for Assembly of C-binding Complexes Are Present in
Cytosolic Extracts from Neuronal and Non-neuronal Cell Lines but Are
Enriched in Neuronal Tissues--
Cytosolic extracts from neuronal
(P19 and N2a) and non-neuronal (L cells) cell lines were tested in gel
shift assays and were found to generate C-binding complexes (data not
shown). Studies were then conducted to compare the C-binding complexes
from neuronal (brain) and non-neuronal (liver) tissues. Fig.
7 shows that very similar C-binding
complexes formed when brain (lanes 3-8) or liver (lanes 9-14) extracts were gel-shifted with probe A. However, at least a 10-fold larger amount of C-binding complex formed
with brain extracts, based upon radioactivity per microgram of protein in the cytosolic extract.

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Fig. 7.
Gel shift assay of the C-binding complex
(closed arrowhead) that forms when probe A is incubated
with cytosolic extracts from rat brain (80 µg) or rat liver (160 µg) in the presence of 0 ( ) or 2000 (+) ng of poly(C) or poly(U),
poly(A), or poly(G). Control includes the undigested probe
(lane 1), an incubation without extract (lane 2),
and competition with 100-fold excess of cold probe A (lanes
8 and 14).
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C-binding Complexes Contain 18- and 36-kDa Polypeptides, whereas
the U/A-binding Complexes Contain 72- and 80-kDa
Polypeptides--
Components of the C-binding and U/A-binding
complexes were identified by UV cross-linking of the complexes that
form on radioactive probes, then digested away the radioactive residues
that are not cross-linked to protein and using the cross-linked
radioactivity to detect polypeptides in autoradiograms after their
separation by SDS-polyacrylamide gel electrophoresis. When polypeptides
in rat brain extracts were cross-linked to probe B (+1670/+1779) and
separated on a 15% acrylamide gel, polypeptides with migrational rates
of 39- and 18-kDa were identified as components of the C-binding complexes (Fig. 8A). Their
association with the C-binding complexes was attributed to their
ability to be competed with poly(C). The 39-kDa polypeptide was
competed with low levels (20 ng), whereas loss of the 18-kDa
polypeptide required higher levels (2000 ng) of poly(C). Cross-linking
of the 18-kDa and, especially, the 39-kDa polypeptides was enhanced by
the addition of poly(U). Digestion of the cross-linked complexes using
RNase A (in addition to T1 nuclease) increased the migrational rate of
the 39-kDa polypeptide to that of a 36-kDa polypeptide. Addition of
poly(U) also competed away the cross-linking of an 80-kDa
polypeptide.

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Fig. 8.
A, polypeptide in mouse brain extracts
cross-linked to probe B (+1640/+1779) in the presence of 0 ( ), 20 (+), or 2000 (+++) ng of poly(U) or poly(C), with (+) and without ( )
further digestion with RNase A or T1, and separated on a 15%
SDS-polyacrylamide gel. Sizes of radioactive polypeptides
(left-hand margin) are estimated by their rates of
comigration relative to those of known standards. B,
polypeptides in mouse brain extracts cross-linked to probe A
(+1712/+1779) in the presence of 0 ( ) or 2000 (+++) ng of poly(U) or
poly(C), digested with RNase T1, and separated on a 10%
SDS-polyacrylamide gel. Polypeptide sizes (left-hand margin)
are estimated from the comigration of known standards. Control samples
without extracts are shown in lanes 1.
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When polypeptides in brain extracts were cross-linked to probe A
(+1712/+1779) and separated on a 10% gel (Fig. 8B), 80- and 72-kDa polypeptides were competed away by the addition of poly(U) (compare lanes 2 and 4) and were slightly
increased by the addition of poly(C) (compare lanes 2 and
3). A 39-kDa polypeptide was competed away by the addition
of poly(C) (compare lanes 2 and 3) and was slightly increased by the addition of poly(U) (compare lanes
2 and 4). The presence of an 18-kDa polypeptide
(lane 2) and its ability to be competed by poly(C)
(lane 3) and enhanced by poly(U) (lane 4) were
obscured by the cross-linking of additional polypeptides that migrated
in the 20-30-kDa range (compare lanes 1 and 2). The findings identify 18- and 36-kDa core polypeptide components of the
C-binding complex and 80- and 72-kDa components of the U/A-binding
complex.
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DISCUSSION |
With this study, we have begun to identify the RNP binding factors
and binding sites that regulate the stability of NF-L mRNA. Competition with homoribopolymers (15-17) has been very helpful in
characterizing specific RNP components and in localizing their binding
sites. In particular, the ability to be selectively competed with
poly(C) has identified a prominent C-binding complex that binds to the
proximal 3'-UTR of NF-L mRNA. In a companion study (1), we have
mapped the stability determinants in the NF-L transcript and have shown
that the major determinant of NF-L mRNA stability is localized to
the binding site of the C-binding complex. The identification and
characterization of RNP complexes on the NF-L transcript are therefore
directly relevant to the mechanisms that regulate the stability of the
NF-L transcript.
The C-binding complex is unusual, in that its binding site is located
in the proximal 45 nt of 3'-UTR but the binding reaction is markedly
enhanced by the addition of the 23-nt flanking upstream sequence in the
3'-CR. The ability of distal 3'-CR to enhance formation of C-binding
complexes could be due to its participation in secondary (or tertiary)
structure. Computer modeling based on the free energy minimization
algorithm of Zuker et al. (19) indicates that the distal
3'-CR forms a stable stem structure with the proximal 3'-UTR.
Alternatively, it is also possible that other factors bind to the 3'-CR
and facilitate formation of the C-binding complex. Immediate upstream
flanking sequences are necessary for binding to a 29-nt destabilizing
element in the 3'-UTR of human amyloid precursor protein mRNA (20).
Remote upstream sequences may also be required to form an essential
stem structure (21).
The binding site for the C-binding complexes in the proximal 3'-UTR of
NF-L mRNA is phylogenetically conserved (Table
I). Several pyrimidine-rich stretches are
present in proximal 45 nt of the 3'-UTR and may be instrumental for
binding of the complex. Disruption of RNA structure by incubating
probes with antisense oligonucleotides shows that the C-binding complex
requires the integrity of sequences spanning the proximal, middle and
distal pyrimidine-rich sites. Flanking and intervening sequences are also conserved (Table I) and may also be important for the formation of
the C-binding complex.
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Table I
Comparison of the sequences in the proximal 3'-UTR (lowercase) and
adjacent 3'-CR (uppercase) of the mouse (38), human (39), rabbit (40)
and cow (W. D. Hill, L. Zhang, B. J. Balin, and T. J. Sprinkle,
GenBankTM accession no. U83919P NF-L mRNAs indicating conserved
residues (vertical bars)
Sequences have been aligned to optimize the extent of homologous
sequence.
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Pyrimidine-rich stretches in the proximal 3'-UTR are also assembly
sites for RNP complexes that regulate stability of other mRNAs (16,
17, 22-24). The pyrimidine composition of some of these sites is
C-rich and complex formation is also inhibited by poly(C)
homoribopolymers (16, 22). Moreover, pyrimidine-rich sites often
contain tandem repeats of sequence-specific motifs that are not
functionally redundant in that complex formation is markedly impaired
if a single motif repeat is disrupted (16, 22, 24). Whereas point
mutations in some residues can be very disruptive, complex formation
may tolerate or even be enhanced by exchanging the C and U pyrimidine
residues in the binding site (22, 25). Interestingly, the sequence as
well as the recognition site in the cognate binding factors have
diverged during evolution so that the complex on the
-globin
mRNA binds to a C-rich motif in the human but C/U-rich motif in the
mouse (17). Splicing factors that bind to polypyrimidine tracts of
intervening sequences have distinctive but overlapping sequence
specificities (26-28). Indeed, competition between multiple
trans-acting factors for binding to polypyrimidine tracts is believed
to be instrumental in splicesome assembly and in the selection of the
3'-splice sites.
An unusual feature of the C-binding complex is that its assembly is
effected by the presence of other RNP complexes on the transcript. Gel
shift studies suggest that the C- and U/A-binding complexes have
reciprocal binding interactions, as if the two sets of complexes
compete for the same components or use the same or nearby sites. These
binding features were most readily observed with short probes that
spanned the C-binding and nearby U/A-binding sites. Moreover,
reciprocal binding phenomena were readily apparent in cross-linking
experiments, suggesting that interactions between the complexes alter
core (i.e. RNA contacting) binding components of the
respective complexes.
The assembly of similar U/A-binding complexes on probes to different
regions (e.g. probes A, 1, and A680/B) of the NF-L
transcript indicates that the complexes bind to multiple sites in the
3'-UTR and 3'-CR. Such a multiplicity of binding sites in the 3'-UTR and 3'-CR is reminiscent of some adenylate/uridylate-rich elements (ARE) that were originally described as destabilizing determinants in
short-lived mRNAs of proto-oncogene, cytokines, and transcription factors (32). For example, destabilization of
-interferon mRNA is mediated by multiple AREs that compete with each other and with
poly(U) or poly(A) in the binding of a 65-kDa polypeptide (33).
Agonist- or hypoxia-induced stabilization of mRNAs may be mediated
by masking destabilizing AREs (34) or by a different set of AREs (35).
Binding of the same set of factors to multiple sites in the 3'-UTR is
also believed to regulate the stability of the neuronal GAP-43 mRNA
(31). Moreover, the GAP-43 binding factors are neuron-specific,
including a factor that was recently identified as an Elav-like protein
(36). Interestingly, Elav-like proteins are essential for the
differentiation and maintenance of neurons and have been identified as
the target immunogens that mediate autoimmune neurodegenerative disease
(37).
The extent to which different components of the C- and U/A-binding
complexes are neuron-specific is presently unknown. It is quite
possible, for example, for neuron specificity to be mediated by
altering (e.g. phosphorylation) a common component, by
changing the concentration of a critical component(s), or by adding a
novel component to the RNP complex in a neuronal setting. The 18- and 36-kDa polypeptide core binding components of the C-binding complexes are novel RNP components, which are enriched in neuronal tissues. They
could represent subunits with similar properties or monomer/dimer components of a common subunit. In the latter instance, the ability of
poly(C) to compete preferentially with the binding of the 36-kDa polypeptide would indicate a greater effect on the assembly of the
dimer in the complex. In either case, the subunits are smaller than
those of other RNP components that bind to C-rich sites and are
competed with poly(C) (16, 22, 29-31).
The role of the C-binding and U/A-binding complexes in regulating the
stability of the NF-L mRNA is presently unknown. The identification
of the complexes and their binding sites will now enable further
characterization of the RNP components and their role in stabilizing
NF-L transcript. It is quite conceivable that factors regulating NF-L
mRNA stability could also play a major role in the
post-transcriptional regulation of neuronal metabolism. In particular,
the findings could provide important insights into the relationship
between overexpression of an NF-L transgene and the selective
degeneration of motor neurons in transgenic mice.