Download Disrupted mRNA sorting in CNS neurons

3691
Journal of Cell Science 112, 3691-3702 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0835
The suppression of testis-brain RNA binding protein and kinesin heavy chain
disrupts mRNA sorting in dendrites
W. L. Severt1, T. U. L. Biber1, X.-Q. Wu2, N. B. Hecht2, R. J. DeLorenzo3 and E. R. Jakoi1,*
1Department of Physiology, Medical College of Virginia/Virginia Commonwealth University, Richmond, VA 23298, USA
2Center for Research on Reproduction and Women’s Health, and Department of Obstetrics and Gynecology, University
of
Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
3Department of Neurology, Medical College of Virginia/Virginia Commonwealth University, Richmond, VA 23298, USA
4Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA
*Author for correspondence (e-mail: [email protected])
Accepted 17 August; published on WWW 18 October 1999
SUMMARY
Ribonucleoprotein particles (RNPs) are thought to be key
players in somato-dendritic sorting of mRNAs in CNS
neurons and are implicated in activity-directed neuronal
remodeling. Here, we use reporter constructs and gel
mobility shift assays to show that the testis brain RNAbinding protein (TB-RBP) associates with mRNPs in a
sequence (Y element) dependent manner. Using antisense
oligonucleotides (anti-ODN), we demonstrate that blocking
the TB-RBP Y element binding site disrupts and mislocalizes mRNPs containing α-calmodulin dependent
kinase II (α-CAMKII) and ligatin mRNAs. In addition, we
show that suppression of kinesin heavy chain motor protein
alters only the localization of α-CAMKII mRNA. Thus,
differential sorting of mRNAs involves multiple mRNPs
and selective motor proteins permitting localized mRNAs
to utilize common mechanisms for shared steps.
INTRODUCTION
and trans-acting proteins that confer stability, govern
translation, and provide a means for movement. To date, most
studies have focused on the sequences and structural features
of mRNAs which are sufficient to direct localization when
introduced as reporter constructs (Mayford et al., 1996b;
MacDonald and Kerr, 1998; Muslimov et al., 1997; for review
see Bassell et al., 1999). While these can be limited to a
particular mRNA, others are present in a subclass of mRNAs
and thus may be candidates mediating common mechanisms
for shared steps. It is anticipated that many different transacting proteins will be involved including factors that direct
regional (dendrite versus soma) distribution and more specific
sorting to individual synapses, as well as those controlling
translation, stability and movement.
One of the structural features common to several localized
mRNAs in hippocampal neurons is the Y element which
permits formation of RNPs containing the TB-RBP (Translin)
(Han et al., 1995; Wu et al., 1999; Muramatsu et al., 1998;
Kobayashi et al., 1998). The TB-RBP is a microtubuleassociated protein (Han et al., 1995) that binds DNA and
mRNA in a sequence specific manner (Han et al., 1995; Kwon
and Hecht, 1991). In the mammalian testis, TB-RBP appears
to have the dual functions of translational suppression of stored
germ cell mRNAs (Kwon and Hecht, 1991, 1993) and of
mRNA transport through the intercellular bridges of male germ
cells, thereby, distributing mRNA between haploid cells
(Morales et al., 1998; Hecht, 1998). In neurons, the activity of
Cellular differentiation necessitates the regional distribution of
protein to establish polarity and functional microdomains. In
the polarized neuronal cell, definition of axonal-dendritic axes
and of individual synapses occurs early in development and is
subjected to structural reorganization throughout life in
response to synaptic activity. Although spatial heterogeneity of
protein can arise by the directed targeting of polypeptides,
regional localization of mRNA for localized translation has
recently emerged as an energetically and dynamically
advantageous alternative. Several mRNAs, RNPs, and
polyribosomes have been localized within distal dendrites and
synaptic areas of CNS neurons by biochemical fractionation
(Chicurel et al., 1993; Tiedge et al., 1991, 1993; Miyashiro et
al., 1994; Crino and Eberwine, 1996) and by in situ
hybridization (Garner et al., 1988; Kleinman et al., 1990;
Burgin et al., 1990; Benson et al., 1992; Bian et al., 1996; Gao
and Keene, 1996; Antic and Keene, 1998). These findings
provide support for the concept that structural organization
may be translationally regulated locally within synapses
(Steward, 1997; Comery et al., 1997; Weiler et al., 1997; Mohr,
1999). However, the proteins needed for regional distribution
of specific mRNAs and the functional significance of this
spatial heterogeneity are not well defined.
Localization of mRNA involves two classes of factors: cisacting regions of the mRNA that encode spatial information
Key words: mRNA sorting, Testis-brain RNA binding protein,
Kinesin, Hippocampal neuron
3692 W. L. Severt and others
the Y element may function in an analogous manner permitting
formation of TB-RBP associated RNPs required for mRNA
sorting.
How RNPs associate with the cytoskeleton and the identity
of the motor protein(s) involved are largely not known. In
neurons, directed movement of the RNP occurs as an
energy dependent process (Davis et al., 1987) directed by
microtubules. Because microtubules are bi-directionally
oriented within the dendritic compartment of CNS neurons
(Baas et al., 1988), members of the kinesin and/or dynein
motor protein families may be involved. Several studies report
microtubule-mediated movement of mRNAs into dendrites at
rates (10-21 µm/hour, Davis et al., 1987; 360 µm/hour,
Knowles et al., 1996; 250-400 µm/hour, Muslimov et al., 1997;
300 µm/hour, Wallace et al., 1998) consistent with calculated
velocities of members of the kinesin family of motor proteins
(Hirokawa, 1998). Whether these differences reflect the
properties of a single motor protein or of several members of
the superfamily of kinesin (or dynein) motor proteins is not
understood.
To fully understand the mechanisms that govern mRNA
localization and its functional significance in neurons,
identification and characterization of the trans-acting proteins
involved are necessary. Identification of trans-acting proteins
is typically done using gel mobility shift assays. To
characterize their function, transfection studies in cultured cells
using reporter constructs and chimeric/deletion mutants are
often performed. In such studies, over-expression and/or
introduction of a foreign mRNA encoding the trans-acting
protein may alter the localization process. An alternate
approach to understanding the role(s) of cis-acting elements
and trans-acting proteins is to perturb their function. To
achieve this, inactivation of mRNA by antisense
oligonucleotides (anti-ODN) is widely used (Phillips and
Gyurko, 1997). Typically, synthesis suppression of the cognate
protein by anti-ODN results from either ribonuclease H
degradation of the mRNA or inhibition of its translation.
In this report, we investigate the role(s) of cellular factors
important for sorting of mRNAs in hippocampal neurons. We
use gel mobility shift assays to identify the trans-acting RNAbinding protein and we use antisense oligonucleotides (antiODN) to disrupt formation of RNP complexes either by
blocking the cis-acting sequence or by suppressing the transacting motor protein. Subsequently, the location of the targeted
mRNA and its cognate protein are determined by fluorescence
in situ hybridization (FISH), confocal microscopy and
immuno-cytochemistry. We reason that if differential sorting
of dendritically targeted mRNA plays a key role in the
distribution of a given protein, then missorting of its encoding
mRNA will alter cognate protein expression. We study two
dendritically targeted mRNAs: one encoding the fatty-acylated
membrane-bound protein, ligatin; the other the cytosolic
protein, α-calmodulin dependent kinase II (α-CAMKII). Our
studies identify the microtubule-associated TB-RBP and
implicate kinesin heavy chain as trans-acting proteins
important for distribution of dendritically localized mRNAs
containing the Y element, but not for somata-restricted
mRNAs. Moreover, we show that differential sorting of
multiple mRNPs is involved, whereas mRNAs for cytosolic
protein(s) utilize different motor protein(s) from those used
sorting membrane-bound protein(s) mRNAs.
MATERIALS AND METHODS
Neuronal cell cultures
Rat hippocampi from neonate day 2 were trypsinized, triturated, and
plated (104 cells per coverslip) onto a confluent glia feeder bed as
described (Jakoi et al., 1992). Cells were maintained in defined
medium for 15-18 days before use. Pyramidal neurons were identified
morphologically. Neurons in culture were stained with specific
antiserum for α-CAMKII (Chemicon MAB1213) and for ligatin
(Jakoi et al., 1987); low levels of ligatin immuno-reactivity were
found in glia.
Recombinant β-galactosidase (βGAL) plasmid and
adenovirus
The protocols to obtain recombinant adenovirus have been described
in detail by Nevins et al. (1997). Briefly, the AdCMVβGAL is a
replication defective recombinant adenovirus (E1a/E1b minus)
obtained by in vivo recombination between the adenovirus mutant and
the pAdCMVβGAL. After isolation, the recombinant adenoviral
DNA was amplified in HEK293 cells, a trans-complementing cell line
for E1 function. Plaques were detected within 5 days post-infection
and viruses harvested 5 days later. Recombinant clones were amplified
in HEK293 cells to obtain a titer of ~1010 p.f.u./ml and triple plaque
purified to insure that viral suspensions used for the in vitro
experiments are free of wild-type virus. Two constructs were
used: pAdCMVβGAL and pAdCMVβGAL-3′UTR500. For
pAdCMVβGAL, the entire coding region of Escherichia coli lacZ was
excised from pGEMβGAL (Promega) and inserted into the HindIIIBamHI site of pAdCMV (gift of Drs Okatani and Nevins, HHMI Duke
University). For pAdCMVβGAL-3′UTR500, a 500-nt cDNA
fragment of ligatin 3′UTR was ligated onto the 3′ end of the lacZ
insert. This construct was then inserted into the HindIII-NotI region
of pAdCMV.
Rat hippocampal neuronal cells (2×104 per coverslip) in culture
were infected with 2×105 p.f.u./µl of viral suspension in 200 µl culture
medium and incubated for 30 minutes at 37°C. Culture medium
(400 µl) was then added to each well and the coverslips incubated for
60 minutes at 37°C. Subsequently, the media were replaced and the
cells were maintained for 4 hours at 37°C. Expression of βgalactosidase mRNA was detected by FISH and confocal microscopy
using a complementary oligonucleotide probe (βGAL, 5′CAGTGAATC CGTAATCAT-3′) (Operon) tailed with biotinylateddUTP (Clontech) as described (Panchision et al., 1995).
Constructs and RNA transcripts
Constructs derived from ligatin cDNA were cloned into pGEM-3Z (-)
(Promega) and screened by restriction analysis. The plasmids were
linearized with HinfI. Radiolabeled and unlabeled RNA transcripts
were synthesized in vitro with SP6 polymerase using the Riboprobe
In Vitro Transcription System (Promega). The single stranded
template of the UTR500 consisting of 500-nt and 59-nt of polylinker
was used to synthesize transcripts for gel shift assays. Sense αCAMKII transcripts were transcribed from 29-nt template consisting
of 17 nt of coding region (315-332) and 12-nt of polylinker. For
control transcripts, EcoRI linearized Riboprobe pGEM-11Z (-)
polylinker (58-nt) was used. For competition with the TB-RBP RNAbinding motif, sense RNA transcripts were synthesized from a 67-nt
protamine 3′UTR template (pGEMc) consisting of 42-nt of 3′UTR
and 25-nt of polylinker (Kwon and Hecht, 1991) using SP6
polymerase.
RNA gel mobility shift assays and UV cross-linking
Gel mobility shift assays were performed according to the method of
Gillis and Malter (1991). Hippocampal neuronal cell lysates were
prepared by repetitive (3×) freeze-thaw lysis in 25 mM Tris-HCl, pH
7.9, 0.5 mM ethylene glycol-bis (β-amino ethyl ether)-N,N,N′,N′-tetra
acetic acid (EDTA), and 0.1 mM phenylmethylsulfonyl fluoride
Disrupted mRNA sorting in CNS neurons 3693
(PMSF), followed by centrifugation at 15,000 g (4°C) for 15 minutes.
The supernatant was stored at −70°C. Cell lysate (12 µg) and
uniformly labeled [32P]RNA encoding a fragment of ligatin 3′UTR
(150,000-250,000 cpm) were incubated in the absence or presence of
non-radiolabeled competitors. Binding reaction mixes were incubated
at 30°C for 10 minutes in a buffer containing 10% glycerol, 20 mM
Tris-base, pH 7.9, 50 mM KCl, 0.3 mM EDTA, 1.1 mM dithiothreitol,
0.5 mM PMSF, 1% polyvinyl alcohol, 4mM ATP, 0.7 mM MgCl2, and
0.5 µg tRNA in a total volume of 25 µl. Heparin (40 µg) was added
and the reaction mixture was incubated in ice (10 minutes) prior to
digestion with RNase T1 (37°C, 30 minutes). Shift bands were
analyzed by electrophoresis on a 7% nondenaturing polyacrylamide
gel (80:1 acrylamide:bisacrylamide) in Tris-borate-EDTA buffer. The
gel was dried and RNA-protein complexes were detected by
autoradiography. For competition experiments, unlabeled competitor
RNAs were synthesized as above except no radiolabeled NTPs were
added to the reaction. Competitor RNAs were incubated with cell
lysate at 30°C for 10 minutes prior to addition of [32P]-3′UTR
probe. Competitor ODN [specific anti-ODN sequence 5′-AGCCCAGAGCTTG-3′; specific sense-ODN sequence 5′-CAAGCTCTGGGCT-3′; non-specific sequence 5′-TCTGTATAGACATGATGGCTG3′), was annealed to the radiolabeled probe (10 minutes at 65°C then
slow cooled to room temperature) before incubation with the
reaction mixture. Super shift assays were performed as described for
shift assays except that specific antibody (anti-α-CAMKII,
Chemicon MAB1213; anti-tubulin, Chemicon MAB065; anti-actin,
ICN C4; anti-68 kDa intermediate filament, gift of Dr Bigbee,
Medical College of Virginia; and anti-TB-RBP, Wu et al., 1997) was
added to the homogenate prior to the addition of radiolabeled probe.
For UV cross-linking, RNA-protein complexes were UV irradiated
(5 minutes) in ice under a germicidal lamp (Phillips G15T80)
located 5 cm above the reaction mixture. Irradiated shift complexes
were then heparinized and digested with RNase T1. For some
experiments, RNA-protein complexes were irradiated directly in the
gel and the excised band then subjected to SDS-PAGE. Prior to
electrophoresis, irradiated samples were suspended in SDS sample
buffer without β-mercaptoethanol. SDS-PAGE was performed
according to the method of Laemmli (1970) using 8%
polyacrylamide gels.
ODN incubation conditions
Disruption of RNP formation was performed as described by Jakoi
and Severt (1999). Sterile, non-derivatized, phosphodiester TB-RBP
antisense motif ODN [5′-AGCCCAGAGCTTG-3′](1 µM, OPERON)
was administered daily to cultured neurons for 3-4 days. As controls,
TB-RBP sense motif ODN [5′-CAAGCTCTGGGCT-3′] was given as
described. To suppress kinesin heavy chain function, sterile, nonderivatized phosphodiester ODN was administered to neurons in
culture (50 µM initially, then 25 µM every 12 hours for 3 days) as
described by Ferreira et al. (1992). For this purpose, ODN encoding
the following sequences were synthesized (Operon): antisense KHC
Table 1. The TB-RBP binding Y element on protamine,
ligatin and α-CAMKII mRNAs
mRNA
Protamine 2
Ligatin
α-CAMKII
Sequence
CTGAGCCCTGAGCT
CCAAGCTCTGGGCT
AGAAGCCCTATGCT
Location
Bases 442-455 (3′UTR)
Bases 1099-1112 (3′UTR)
Bases 316-329 (coding region)
The Y elements found in two dendritically targeted mRNAs share strong
homology with the first identified Y element from testicular protamine
2 mRNA (Kwon and Hecht, 1991). Bases printed in bold underline are
invariant and compose 71% of the total sequence.
(5′-CCGGGTCCGCCATCTTTCTGGCAG-3′)
(5′TGCCAGAAAGATGGCGGACCCGG-3′).
and
sense
KHC
FISH and confocal microscopy
FISH was performed as described (Panchision et al., 1995). Briefly,
cells were fixed in 5% paraformaldehyde in phosphate buffered
saline (PBS, 30 minutes), permeabilized with 70% ethanol, and
prehybridized in 50% formamide for 60 minutes (42°C).
Complementary oligonucleotide probe (ligatin, 5′-GTCTTCTGGGGCTTCTGAGAG-3′; α-CAMKII, 5′-GGTAGCATCCTGGCACT-3′; NSE, 5′-TCTGTATAGACATGATGGCTG-3′) (Operon) was
tailed with biotinylated-dUTP (Clontech) and added to the
hybridization mix. α-Tubulin cDNA probe was synthesized by SP6
polymerase using the RIBOPROBE kit and pBR322kα1(Gift of Dr
Cowan, New York Medical Center). Cells were hybridized for 60
minutes at 37°C, washed 3 times with 0.5× SSC, 0.1% SDS at 37°C
for 30 minutes and then blocked with 0.25% gelatin-0.1% saponin in
phosphate buffered saline (PBS) for 60 minutes. Bound probe was
detected with Ultra avidin-Texas red conjugate (Leinco Technologies)
(60 minutes).
Quantitation and image analysis
Neurons were selected for analysis if they exhibited: (1) a
characteristic pyramidal morphology (diamond- or triangular-shaped
soma of 20-30 µm diameter, bifurcated apical dendrite, 2-3 basal
dendrites) and (2) planar dendrites (continuous baseline fluorescence
within dendrites) at distances >200 µm from the soma. Cells were
scanned with a Zeiss LSM410 (Zeiss Inc., PA) using a C-Apo 40×
objective (1.2 NA). To permit an accurate comparison in a given
experimental set, the experimental and control images were obtained
under exactly the same scanning conditions (laser power,
photomultiplier sensitivity, filter settings, scanning duration,
averaging, pinhole settings, etc) that optimized the sense-ODN treated
specimens. Where indicated the soma signal of the experimental
image was normalized to that of the control image by changing the
photomulitplier sensitivity. Full frame images were analyzed with the
Carl Zeiss Laser Scanning Microscope System Software version 3.84.
The digitally recorded intensities of pixels (1 to 256) from selected
Fig. 1. Expression of β-galactosidase mRNA in
hippocampal neurons infected with 2×105 p.f.u.
AdCMVβGAL (A) and AdCMVβGAL-3′UTR500 (B).
Cultures (n=2 per group) were fixed 4 hours after
infection. Specific hybridization signal was detected by
FISH and confocal microscopy as described in Materials
and Methods. Micrographs are representative of 3
independent experiments. Insert depicts pseudocolor
intensity scale (1-256). Arrowhead denotes soma.
3694 W. L. Severt and others
regions were averaged and expressed as change in intensity per unit
area. Because the auto-fluorescence intensity and the relative change
in fluorescence from individual neurons within a given experiment
and among independent experiments did not vary significantly, no
correction for auto-fluorescence (background) was applied. Data are
given as the mean ± s.e.m. Statistical analysis was performed using
Student’s t-test. Values of <0.05 are considered significant.
The images presented are encoded with a pseudo-color table to
permit discrimination between different gray levels. Two color tables
were used: a standard glow color table and a customized color table
(Tbrain, see Fig. 5). For illustration, the experimental and control
images in a given set were printed in exactly the same manner.
Immunocytochemistry.
Immunocytochemistry was performed as described by Jakoi et al.
(1992). Briefly, cells were fixed in 5% paraformaldehyde in PBS for
30 minutes, permeabilized in 0.01% saponin-0.25% gelatin-PBS for
60 minutes and subsequently incubated with immune serum (rabbit
anti-ligatin, Jakoi et al., 1987; mouse anti-α-CAMKII, Chemicon
MAB1213; or mouse anti-SUK4, Developmental Studies Hybridoma
Bank, University of Iowa) for 60 minutes. Bound primary antibody
was detected indirectly with either biotinylated goat anti-rabbit serum
or biotinylated rat affinity purified goat anti-mouse serum (Vector
Labs) and Texas red-ultra avidin as described (Jakoi et al., 1992).
Cells were photographed with a Zeiss epi-fluorescence microscope
using a 25× objective (0.8 NA).
RESULTS
Reporter construct containing the Y element sorts
into dendrites
Our initial interest was to identify proteins that act as transacting factors to localize mRNAs in hippocampal neurons. The
discovery that the TB-RBP binds localized mRNAs encoding
tau and myelin basic protein (Han et al., 1995), led us to ask
whether dendritically localized mRNAs contain the TB-RBP
consensus sequence and whether these mRNAs associate with
the TB-RBP. Using the GenBank database, we found ligatin
and α-CAMKII mRNAs contain sequences with high
homology (>70%) to a TB-RBP binding sequence (Y element)
(Table 1). This Y element resides within the 3′UTR of ligatin
mRNA, in α-CAMKII mRNA, it is within the coding region.
A chimeric βGAL construct was tested to determine whether
a 500-nt region of ligatin mRNA containing the Y element was
active in localizing mRNA within dendrites. For these
experiments, we used a gene transfer vector from adenovirus
(Nevins et al., 1997) that efficiently delivers genes into the
post-mitotic neurons with no adverse effects on cell
morphology, viability, or function (Moriyoshi et al., 1996;
Caillaud et al., 1993). The molecular chimera was constructed
by inserting the ligatin mRNA 3′UTR containing the Y element
between the vector lacZ coding region and the SV40 3′UTR
polyadenylation signal. The adenovirus constructs were
infected at 2×105 p.f.u. for 90 minutes at 37°C. Subsequently,
the virus was removed, and four hours later the encoding
mRNAs were located by FISH and confocal microscopy. Both
glia and neurons were infected under these conditions; the
efficiency of infection was ~40%. The construct containing the
500-nt region of ligatin mRNA 3′UTR was effective in
localizing β-galactosidase mRNA within dendrites of cultured
neurons (Fig. 1B). In the absence of this 3′UTR region (control
construct), the β-galactosidase mRNA remained confined to
the somata (Fig. 1A). These data establish that a 500 nt region
of ligatin 3′UTR is sufficient for sorting.
Formation of RNA-protein complexes in lysates of
hippocampal neurons
To identify the RNA binding protein(s) involved, we next
studied the in vitro formation of RNA-protein complexes using
this 500-nt region in RNA gel mobility shift and UV crosslinking assays. Hippocampal cell lysates were incubated with
32P-labeled transcripts containing the same 3′UTR region of
ligatin mRNA used in Fig. 1. To reduce formation of nonspecific RNA protein complexes, reaction mixtures were
incubated in the presence of heparin and then digested with
RNase T1. Radiolabeled complexes were detected by
polyacrylamide gel electrophoresis (PAGE) under nondenaturing conditions. Transcripts generated from the distal
500-nt sequence of this message formed a single shift band
(Fig. 2A, lanes 2-5). This RNA-protein complex could be
competed (>50%) by unlabeled 3′UTR-500 transcript at 10fold excess (Fig. 2A, lanes 3 and 5). Non-specific competitor
RNAs encoding pGEM11z(-) polylinker (58-nt) or tRNA did
not compete (see below, Fig. 4A, lane 5 and Fig. 4B, lane 1,
respectively).
The apparent molecular mass of the trans-acting protein(s)
involved in formation of this RNA-protein shift complex was
determined by UV cross-linking and SDS-PAGE. RNA-protein
complexes were generated, UV irradiated, excised from the gel
and then analyzed by SDS-PAGE and autoradiography. A
Fig. 2. Formation of a specific RNA-protein complex between 32Plabeled ligatin 3′UTR region and hippocampal neuronal cell lysate.
(A) RNA mobility shift assays of cell extracts from untreated
neurons were performed as described in Materials and Methods. The
binding mixture was analyzed by electrophoresis on a 7% nondenaturing polyacrylamide gel. Lane 1 contains the 32P-labeled
ligatin 3′UTR probe alone. Lanes 2-5 contain the ligatin 3′UTR
region probe and cellular extracts from two different preparations
alone (lanes 2 and 4) and in the presence of unlabeled 10-fold excess
specific competitor RNA (lanes 3 and 5). Arrowhead denotes the
position of the band shift complex. (B) Identification of polypeptide
interacting with the ligatin 3′UTR transcript by UV cross-linking and
SDS PAGE. RNA-protein binding reactions using 32P-labeled ligatin
3′UTR region as substrate were carried out as in A. 32P-labeled
RNA-protein shift complexes were UV irradiated for 5 minutes and
the shift bands excised. Cross-linked products were resolved directly
by SDS PAGE on an 8% polyacrylamide gel. Lane 1 contains
radiolabeled probe incubated with cell lysate. Lane 2 contains
radiolabeled probe alone. Arrow denotes single polypeptide resolved.
Prestained molecular mass standards (Pharmacia) were used as size
markers.
Disrupted mRNA sorting in CNS neurons 3695
Fig. 3. Effect of specific antibody on
RNA-protein shift complex. Shift
assays of ligatin 3′UTR region with
cell lysate of hippocampal neurons
were performed as described in the
presence or absence of specific
antibody. Lanes 1-5 contain
radiolabeled probe and cellular
extract. Lanes 1-4 contain
radiolabeled probe and specific
antibody to TB-RBP (1), tubulin (2),
actin (3), 68 kDa intermediate
filament (4), respectively. Lane 5
contains RNA-protein complex alone
(control). Migration of the single
shift band is retarded (super shift) in
lane 1. Lower arrow, specific RNAprotein complex. Upper arrow, supershifted complex.
single 32 kDa polypeptide was resolved (Fig. 2B, lane 1). In
the absence of cell lysate, no radiolabeled polypeptides were
found (Fig. 2B, lane 2). In other experiments, RNA-protein
complexes were irradiated in solution prior to RNase T1
digestion and, again, a single 32 kDa polypeptide was resolved
(data not shown). Because the efficiency of UV cross-linking
is low, these results provide a minimal estimate of the transacting proteins that bind to this region of the 3′UTR of ligatin
mRNA.
Identification of TB-RBP as trans-acting protein
To investigate the possibility that the 32 kDa protein in the
ligatin RNA-protein complexes was TB-RBP, we used affinity
purified TBP antibody in super shift mobility assays (Wu et al.,
1997). In these assays, the RNA-protein complexes were
incubated with the specific antibody, digested with RNase T1
and then analyzed by PAGE. In the absence of added antibody,
one band was detected (Fig. 3, lane 5, lower band). In the
presence of TB-RBP antibody, a slower migrating shift band
(super shift band) was detected (Fig. 3, lane 1). This super shift
was specific, since antibodies to cytoskeletal proteins, tubulin
(Fig. 3, lane 2), α-actin (Fig. 3, lane 3), and a 68 kDa
intermediate filament (Fig. 3, lane 4) did not alter the migration
of the RNA-protein complex. Collectively, our findings
strongly suggest that the TB-RBP of hippocampal neuronal
lysates interacts with a 500 nt region of ligatin mRNA (Fig. 3,
lane 5).
To confirm the necessity of having an accessible Y element
on the mRNA for formation of the mRNA-protein complexes,
we performed competition assays as described in Fig. 2 in
which neuronal cell lysates were preincubated with unlabeled
RNA and ODN competitors (10 minutes, 30°C). Compared to
the control (Fig. 4A, lanes 2 and 8), RNA-protein complex
formation was disrupted by unlabeled transcripts encoding the
testis protamine 2 Y element (Fig. 4A at 10- and 50-fold
excess, lane 3 and 4) and by unlabeled antisense ODN
encoding the ligatin Y element (Fig. 4A at 1:5000, lane 6).
Neither tRNA (1:50) nor the TB-RBP Y element sense-ODN
(1:5000) competed (Fig. 4A non-specific competitors, lanes 5
and 7). When the unlabeled competitor RNA encoding the Y
element of testis protamine 2 mRNA was added to the reaction
mixture without preincubation with the cell lysate, no
inhibition of the RNA-protein complex was seen (data not
shown). The RNA-protein complexes were also diminished by
hybridizing the 32P-labeled transcript with unlabeled anti-ODN
(complementary to the TB-RBP Y element of ligatin) at a
molar ratio of 1:5 (specific competitor) (Fig. 4B, lane 3).
However, hybridization with unlabeled RNA encoding
pGEM11z (-) polylinker (Fig. 4B, lane 1) or ODN
complementary to the N-methyl-D-aspartate receptor
(NMDAR1, non-specific competitor) (Fig. 4B at 1:5, lane 4)
did not interfere with RNA-protein interactions. These results
show that RNA encoding a sequence >70% homologous to the
ligatin Y element and ODN complementary to the ligatin Y
element can disrupt RNA-protein complex formation.
Fig. 4. Formation of RNA-protein complex involves TBRBP Y element. (A) Gel mobility shift assays using
radiolabeled ligatin 3′UTR region were performed as
described in Fig. 2 and formed complexes resolved by
electrophoresis in non-denaturing polyacrylamide gels
(80:1, acrylamide: bisacrylamide). To test for
competition of RNP formation, hippocampal neuronal
lysates were preincubated with unlabeled competitor for
10 minutes at 30°C. Lane 1 contains radiolabeled
transcript alone. Lanes 2 and 8 contain lysate and
radiolabeled probe alone (control). Lanes 3-7 show
competition of RNA-protein complex by RNA and ODN.
Unlabeled RNA transcript encoding TB-RBP Y element
sense sequence (lanes 3 and 4, specific competitor of 67
nt) and tRNA (lane 5, unrelated competitor) were added at 1:10, 1: 50, and 1:50, respectively. Unlabeled ODN encoding anti-sense TB-RBP Y
element ODN (lane 6, specific sequence competitor) was added at 1:5000. Sense TB-RBP motif ODN (lane 7, non-specific DNA competitor)
was added at 5000-fold excess. (B) The gel mobility shift assay using 32P-labeled ligatin 3′UTR region and hippocampal cell lysates was
performed as described in Fig. 2. The control RNA-protein complex is seen in lane 2. In lane 1, pGEM11(-) polylinker of 58 nt (non-specific
competitor) was added with radio-labeled transcript. In lanes 3 and 4, unlabeled ODN encoding 13 nt of Y element anti-sense (specific
competitor of 13 nt) at 1:5 (lane 3) or 21 nt of N-methyl-D-aspartate receptor subunit 1 (NMDAR1, non-specific competitor) at 1:5 (lane 4).
(C) RNA-protein shift complex with TB-RBP Y element of α-CAMKII coding region. The gel mobility shift assay using 32P-labeled mRNA
encoding the TBP Y element (17-nt coding and 12-nt polylinker) and hippocampal cell lysates was performed as described in Fig. 2. Lane 1
contains radiolabeled probe and cellular lysate. Unlabeled TB-RBP anti-ODN (lane 2, 13-nt sequence competitor) and TB-RBP sense ODN
(lane 3, 13-nt competitor) were added at 1:5000. Arrowhead denotes specific RNA-protein complexes.
3696 W. L. Severt and others
To address the question of whether the Y element per se can
participate in forming a complex with TB-RBP, we used a
radiolabeled transcript of 29-nt corresponding to the αCAMKII Y element (17-nt coding and 12-nt polylinker) in gel
mobility shift assays. A single shift band (Fig. 4C, lane 1) was
detected. Formation of this shift complex could be competed
(>60%) by unlabeled anti-ODN corresponding to the antisense
and sense Y elements (Fig. 4C, lanes 2 and 3, respectively).
Collectively, these findings identify the TB-RBP as a common
factor bound to ligatin and α-CAMKII mRNAs in hippocampal
neurons.
Antisense ODN to the TB-RBP motif alters mRNA
distribution
To evaluate the functional role of TB-RBP in mRNA
localization, we used anti-ODN to inactivate the Y element of
endogenous mRNA and thereby perturb in situ RNP formation.
If the somato-dendritic transport of RNPs was disrupted, then
the affected mRNAs would be expected to either (1) increase
within the soma relative to the dendrites or (2) decrease within
the soma due to selective degradation of the misrouted mRNA.
In the first instance, the soma to dendrite ratio would therefore
increase; in the second case; this ratio may decrease or remain
the same depending upon the turnover rate of the mRNA within
the dendrites.
Non-derivatized, phosphodiester ODN complementary to
the TB-RBP Y element (5′-AGCCCAGAGCTTG-3′) (1 µM)
was administered daily to cultured hippocampal neurons;
control cultures were given a sense-ODN of 5′-CAAGCTCTGGGCT-3′. After 3-4 days of treatment, the cellular
distribution of ligatin mRNA was examined using FISH and
confocal microscopy. The scanning parameters were optimized
for the sense-ODN treated cells (controls) (Fig. 5A).
Measurements from several neurons (n=7 total, sense-ODN,
n=4; antisense-ODN, n=3) were pooled and the mean values
calculated. A significant (P=0.015) mean increase of 32% in
signal intensity was found in the somata of the anti-ODN
treated neurons (Fig. 5B).
To evaluate further the ODN-treated cells, we normalized
the somata signals of anti-ODN versus sense-ODN neurons
(Fig. 5C) by altering the scanning parameters. Under these
conditions, the dendrite signal decreased 66% (P=0.001) at a
distance of 40-50 µm (sense-ODN, n=11, anti-ODN, n=8) (Fig.
6); at a distance of 100-110 µm (sense-ODN, n=14, anti-ODN,
n=8), the relative signal intensity decreased by 72% (P=0.001).
Thus the ratio of soma to dendrite signal increased ~3.0-fold
in the anti-ODN versus sense-ODN treated neurons. These data
indicate that the increased signal within the soma was not due
to a general increase in transcription but involved changes in
mRNA distribution. These findings suggest that the Y element
Fig. 5. Pseudocolor micrographs depicting subcellular distribution of
ligatin mRNA in hippocampal neurons treated with anti-ODN and
sense-ODNs to the Y element. (A-C) Distribution of ligatin mRNA.
Neurons (15 days old) were treated for 4 days with 1 µM sense-ODN
(A) or 1 µM anti-ODN (B) complementary to the TB-RBP Y
element. Levels of ligatin mRNA were measured by FISH and
confocal microscopy. In anti-ODN treated neurons, increased mRNA
levels are found in the somata (**) and decreased levels within
dendrites (*). (C) The signal intensity of the soma in B normalized to
that in A. When the soma intensities are equalized, the dendritic
signals in the anti-ODN treated neurons are significantly reduced (P=
0.001) (I). (D) No probe. (E and F) Distribution of α-CAMKII
mRNA in sense-ODN (E) and anti-ODN (F) treated neurons.
Confocal microscopic analysis of representative cells of 2
experiments is shown. Specific labeling of α-CAMKII mRNA is
altered by anti-ODN within the somata (**) and dendrites (*). (G and
H) Distribution of α-tubulin mRNA in sense-ODN (G) and antiODN (H) treated cells.
Disrupted mRNA sorting in CNS neurons 3697
Fig. 6. Quantitation of ligatin and αCAMKII mRNA levels of ODN treated
neurons described in Fig. 5. Left panel,
representative images showing relative
area (box) measured along dendrite.
Right panel, mean amounts of specific
signal within soma or dendrite. n=7 cells
for each mRNA. Insert, pseudocolor
intensity scale (1-256). Bar, 25 µm.
is involved in delivery of ligatin
mRNA within dendrites.
To investigate the role of the Y
element in a second mRNA known to
be transported (Benson et al., 1992;
Chicurel et al., 1993), we analyzed
the effect of the Y element anti-ODN
on the cellular distribution of αCAMKII mRNA. We found
decreased
hybridization
signal
throughout the neurons, especially
within the dendritic arbors (compare
Fig. 5E to F). The measurements
from several neurons (n=7 total,
sense-ODN, n=3; antisense-ODN,
n=4) were pooled and the mean values calculated (Fig. 6).
Significant (P=0.001) decreases in α-CAMKII mRNA signal
intensities were found with a 58% decrease in the somata, 67%
within dendrites at 30-40 µm distant from the somata, and 70%
at 40-50 µm. The ratio of soma to dendrite signal for αCAMKII mRNA was increased 1.6-fold.
To assess whether the anti-ODN depleted and/or altered the
distribution of control mRNAs that do not contain the Y
element, we examined α-tubulin mRNA which lacks Y and H
elements and is restricted to the somata of hippocampal
neurons. Administering oligonucleotides as described above,
no effect on the subcellular distribution of α-tubulin mRNAs
was found (Fig. 5G and H). This establishes that the anti-ODN
was specific for localized mRNAs containing the Y element
and suggests that inactivation of the Y element alters the
subcellular distribution of the ligatin and α-CAMKII mRNAs.
Antisense ODN to the TB-RBP binding motif
suppresses protein expression
The consequences of blocking mRNA transport by anti-ODN
were examined using immunofluorescence to quantitate ligatin
and α-CAMKII proteins in ODN treated cells. Following
treatment with anti-ODNs, marked reductions in ligatin and αCAMKII protein levels were found (2 separate experiments,
n=11 and 19, respectively) (Fig. 7B and D). In sense-ODN
treated neurons (controls) (ligatin, n=15; CAMKII, n=25),
expected amounts of these proteins were detected throughout
the somato-dendritic compartment (Fig. 7A and C). Although
the reduced levels of α-CAMKII protein reflected the
decreased levels of its mRNA and ligatin protein levels were
reduced, ligatin mRNA was increased (32%) under these
conditions within the soma. These data indicate that anti-sense
ODN binding to the Y element and/or mis-localization of
ligatin mRNA may affect translation of the mRNA.
Kinesin heavy chain motor is required for
localization of select mRNAs
To determine whether specific motor proteins could be
involved with TB-RBP in mRNA movement, we used the
procedure of Ferreira et al. (1992) to suppress kinesin heavy
chain (KHC). Initially, an anti-ODN of 24-nt targeted to the
initiator AUG region of KHC was used and the cellular
distribution of ligatin mRNA and of α-CAMKII mRNAs were
examined in neurons. Although no changes were seen in ligatin
mRNA distribution (compare Fig. 8A and B) with anti-sense
ODN treatment, there was a marked decrease in the cytosolic
level of α-CAMKII mRNA throughout the somatic-dendritic
compartment of these cells (compare Fig. 8C and D, E and F).
Quantitation of the decline in α-CAMKII mRNA revealed
significant decreases (36.6%, P=0.002) in somata.
Measurements from four separate experiments were pooled
and mean values obtained for somata (sense-ODN, n=9, antiODN, n=8) and within dendrites at 30-40 µm (sense-ODN,
n=24, anti-ODN, n=20) and at 40-50 µm (sense-ODN, n=24,
anti-ODN, n=20) from the soma (43.3% and 42.5%, P<0.001
and P<0.001, respectively). No significant decrease was found
when neurons were treated with a KHC sense-ODN (Fig. 8C
and E).
To ascertain whether the dendritic localization of αCAMKII mRNAs was perturbed, we normalized the intensities
of the somata of KHC sense-ODN and KHC anti-ODN treated
cells from 2 separate experiments. Under these scanning
conditions, significant decreases (27%, P=0.01 and 49%,
P<0.001, respectively) in signal intensity were found at
80-90 µm (sense-ODN, n=11; anti-ODN, n=13) and at 120130 µm (sense-ODN, n=12, anti-ODN, n=11) from the somata.
To control for specificity of the anti-KHC ODN effects, we
targeted a second non-overlapping site located immediately
downstream from our first sequence with an anti-ODN of
3698 W. L. Severt and others
25 nt. Again, α-CAMKII mRNA levels were reduced
throughout the somato-dendritic compartment (data not
shown), suggesting that the effect was due to suppression of
KHC. Moreover, the levels and distribution of the somatarestricted mRNA encoding neuron specific enolase (NSE) were
unaffected by the ODNs used to target KHC (Fig. 8G and H).
To evaluate whether the KHC anti-ODN altered the
accumulation of α-CAMKII mRNA directly, we performed
FISH at 24 hours, a time before KHC protein levels decrease
from the ODN treatment (50% suppression is seen at 36 hours).
No significant decrease in the level of α-CAMKII mRNA was
found (data not shown). Whereas several sense ODNs
(encoding KHC and Y element) had no effect on α-CAMKII
mRNA levels and specific anti-ODNs complementary to the Y
element and to KHC led to decreased α-CAMKII mRNA
levels, we propose that mis-localization of α-CAMKII mRNA,
rather than transcriptional inhibition, leads to decreased levels
of this transcript.
Depletion of KHC from neurons after the specific antiODN treatments was assayed by using immunocytochemistry
with a specific KHC antibody (SUK4) (sense-ODN, n=27;
anti-ODN, n=44; 4 independent experiments) (Fig. 9A and
B). Under identical conditions of staining and exposure, the
specific immuno-labeling of neurons incubated with
antisense ODN was reduced. The cytoarchitecture and
viability of the ODN treated cells were not affected as
previously reported by Ferreira et al. (1992). The partial
suppression of KHC protein by KHC anti-ODN is consistent
with previous reports of a 50-80% reduction in KHC (Ferreira
et al., 1992) and is likely the reason we detect a residual
localization of α-CAMKII mRNA.
DISCUSSION
Spatial heterogeneity of mRNAs in hippocampal neurons has
been previously reported using isolated nerve terminal
(synaptoneurosomes) preparations (Chicurel et al., 1993;
Tiedge et al., 1991, 1993; Miyashiro et al., 1994) and in situ
hybridization (Garner et al., 1988; Kleinman et al., 1990;
Burgin et al., 1990; Steward and Wallace, 1995; Steward et
al., 1998). These earlier studies lend support to the hypothesis
that sorting of RNA plays a vital role in the establishment of
axonal-dendritic polarity (Garner et al., 1988; Bruckenstein
et al., 1990; Litman et al., 1993; Behar et al., 1995; Kanai
and Hirokawa, 1995; Marsden et al., 1996) and of synapses
(Steward, 1997; Steward and Banker, 1992; Miyashiro et al.,
1994; Chircurel et al., 1993; Tiedge et al., 1993; Weiler et al.,
1997; Steward et al., 1998). Transfection studies performed
in primary cultured somatic cells, including neurons, have
used reporter constructs and chimeric/deletion mutants to
show that cis-acting element(s) residing within the 3′UTRs
of mRNAs encode spatial information. Moreover, in vivo
expression of reporter constructs in transgenic mice
demonstrate that cis-acting elements within the 3′UTR of αCAMKII mRNA are sufficient for dendritic localization
(Mayford et al., 1996b). In this study, we utilized neuronal
cell cultures for analysis of the differential distribution of
dendritically localized mRNAs to identify elements important
for this process. We were able to alter the distribution of two
localized mRNAs by inactivating a common cis-acting
Fig. 7. Cellular distribution of ligatin and α-CAMKII proteins in
neurons treated with ODNs complementary to the TB-RBP motif.
Neurons are treated with ODN as described in Fig. 5. Following 4
days of ODN treatment, specific anti-ligatin staining is seen
throughout the somato-dendritic compartment of sense-ODN (A) and
anti-ODN (B) treated neurons. Specific staining of antiserum to the α
subunit of CAMKII in Y element sense-ODN (C) treated neurons
and anti-ODN (D) treated cells. n=2 independent experiments. Bar,
25 µm.
element. Administration of anti-ODN complementary to the
TB-RBP Y element disrupted RNA-protein interactions (Fig.
4) and sorting of α-CAMKII and ligatin mRNAs within
dendrites (Fig. 5).
Although the anti-ODN treatment is highly selective for its
complementary mRNA (Phillips and Gyruko, 1997),
sequence-independent toxic effects have been reported to
occur with this experimental approach. In most instances
where toxic effects are seen, phosphothioate derivatized ODN
and/or high dosages of ODN have been used. Because the
hippocampal neurons are grown in defined media in the
absence of serum, we use unmodified ODN given daily at
concentrations below toxic levels. During treatment, neurons
maintained well-differentiated morphology and did not
decrease in cell number. Moreover, the ODN-induced effect
is reversed with removal of the ODN. Because the use of
ODNs to perturb cell function necessitates numerous
controls, we provide the following to show sequence
specificity of the anti-ODN we have used: (1) the
corresponding sense ODN has no effect (Figs 4, 5 and 7), (2)
anti-ODN does not affect either the distribution or the amount
of mRNAs lacking the Y element, indicating that cellular
toxicity is not involved (Fig. 5), (3) several different ODN
Disrupted mRNA sorting in CNS neurons 3699
give the same inhibitory effect for KHC, and (4) anti-ODN
directed against the Y element disrupt RNA-protein complex
formation in vitro (Fig.4). Moreover, we show that a reporter
construct containing this Y element encodes spatial
information sufficient to sort mRNA to dendrites in cultured
cells (Fig. 1). Our findings support a general role for the TBRBP in sorting mRNAs within the somato-dendritic
compartment. Additionally, our results suggest that TB-RBP
does not mediate stability of these mRNAs.
The specific RNA binding protein, TB-RBP, is a
microtubule-associated protein that binds mRNA in a
sequence specific manner (Han et al., 1995; Kwon and Hecht,
1993). In the mammalian testis, TB-RBP appears to have the
dual functions of translational suppression and of
intracellular and intercellular mRNA transport (Morales et
al., 1998). In brain, the TB-RBP is primarily expressed in
neurons (Wu et al., 1999) and forms 11.5S RNPs with
untranslated BC1 and BC200 RNAs (Muramatsu et al., 1998;
Kobayashi et al., 1998) which are localized within dendrites.
Here we show that the TB-RBP binds localized mRNAs
encoding ligatin and α-CAMKII and that disruption of these
interactions alters somato-dendritic sorting. We suspect that
the TB-RBP is involved in tethering specific mRNAs to
microtubules and hence permitting their movement.
Moreover, we propose the TB-RBP in association with other
proteins suppress translation of bound mRNAs in an
analogous manner to its role in testis. In vitro and in vivo
assays have demonstrated that these proteins, the transitional
endoplasmic reticulum ATPase, Trax and a cytoskeletal
gamma actin, interact with TB-RBP in diverse tissues
including brain (Wu et al., 1999). It is likely that posttranslational regulation of TB-RBP by synaptic activity may
provide a mechanistic link important in regulating both
spatial and temporal expression of specific proteins several
hundreds of microns from the nucleus.
Many studies have focused on the localization of mRNA
at synaptic areas because of its implied role in local synthesis
of protein. In particular, preparations of isolated nerve
terminals (synaptoneurosomes) have been used to study
neurotransmitter effects on local translation (Weiler et al.,
1997). In synaptoneurosomal preparations, functional
polyribosomes form in vitro in response to K+ depolarization
and to glutamate (Weiler and Greenough, 1993). Activation
of the metabotropic subtype of glutamatergic receptors
initiates protein synthesis, while activation of the ionotropic
NMDA receptor subtype attenuates it. These findings indicate
that synaptic activity regulates translation of localized
mRNAs locally and that distinct signaling cascades may be
involved. In our studies, we directly tested whether local
translation is a prerequisite for gene expression by disrupting
mRNA delivery. We studied two localized mRNAs, αCAMKII and ligatin, that are post-transcriptionally regulated
in models of glutamate-induced injury (Liang and Jones,
1997; Murray et al., 1995; Panchision et al., 1995; Jakoi et
al., 1992). Using immunocytochemistry, we show that
preventing TB-RBP binding to its Y element by anti-ODN
decreases the amounts of α-CAMKII and ligatin proteins we
can detect within hippocampal neurons.
Although reduced levels of α-CAMKII and ligatin proteins
in dendrites reflect directly the ODN-induced reduction in the
encoding mRNAs, suppression of translation within the somata
may result from either RNA mis-sorting or DNA binding to the
cis-acting TB-RBP Y element on the α-CAMKII and ligatin
mRNAs. Our data can not discriminate between these
possibilities. Collectively, our findings implicate translational
control as a critical regulatory site governing local expression
of proteins important for synaptic activity.
The movement of numerous mRNAs within cells including
neurons has been documented. Both microfilament and
microtubule mediated movement have been implicated.
However, only the microfilament associated motor protein,
Myo04, has been identified. This protein localizes ASH1
mRNA in budding Saccharomyces cerevisiae (Long et al.,
1997). In neurons, microtubule associated movement of
mRNA within dendrites may utilize either kinesin and/or
dynein motors. Several studies have reported microtubule
mediated movement of mRNAs into dendrites at rates
consistent with kinesin mediated movement (Davis et al.,
1987; Knowles et al., 1996; Muslimov et al., 1997; Wallace
et al., 1998). Because KHC-null (knock out) animals are not
viable due to neuronal death (Saxton et al., 1991), and no
pharmacological agent is available to selectively inhibit
movement by individual motor proteins, we have suppressed
the function of a specific motor protein with an anti-ODN.
Earlier studies by Ferreira et al. (1992) showed that after 36
hours of anti-ODN treatment, KHC protein levels are reduced
50% and the movement of organelles within the axon of
newly plated hippocampal neurons is perturbed in a reversible
manner. To perturb movement of mRNAs with an anti-ODN
to KHC, we have used well-differentiated neonatal
hippocampal neurons in culture. Using FISH and confocal
microscopy, we have demonstrated that KHC anti-ODN has
no effect on levels and distribution of α-CAMKII mRNA
levels at 24 hours. However, at 72 hours, α-CAMKII mRNA
levels were decreased throughout the somato-dendritic
compartments, when the level of kinesin heavy chain protein
is reduced. We conclude that this general decline in αCAMKII mRNA by KHC anti-ODN was a specific effect
because (1) the corresponding sense-ODN had no effect, (2)
two different ODNs complementary to KHC had the same
effect, making suppression of a protein different from KHC
unlikely, (3) no reduction in α-CAMKII mRNA was seen at
24 hours a time when KHC protein was not yet affected by
ODN treatment, (4) ODN against the Y element (a nonrelated sequence to the KHC anti-ODN) disrupted αCAMKII mRNA sorting and also reduced its cellular levels,
and (5) the stability of other mRNAs such as ligatin and
neuron specific enolase was not affected, indicating a lack of
cellular toxicity. Collectively, these findings indicate that
ODN treatment did not alter the net accumulation of αCAMKII mRNAs. Our data indicate that the motor protein
KHC plays a necessary role in the localization of at least one
dendritically targeted mRNA although the roles of other
motor proteins cannot be excluded.
In situ hybridization and biochemical fractionation were
used previously in hippocampal neurons to localize mRNAs
(Chicurel et al., 1993; Tiedge et al., 1991, 1993; Miyashiro et
al., 1994; Crino and Eberwine, 1996; Garner et al., 1988;
Kleinman et al., 1990; Burgin et al., 1990; Steward and
Wallace, 1995; Comery et al., 1997; Weiler et al., 1997;
Steward et al., 1998). Here we alter the distribution of two
transported mRNAs by inhibiting the formation of a RNA-
3700 W. L. Severt and others
Fig. 8. Effect of KHC anti-ODN on the relative distributions of
somato-dendritic mRNAs in hippocampal neurons. Neurons are
given ODNs (50 µM initially then 25 µM every 12 hours for 3 days)
as described. For FISH, cells are hybridized with biotinylated probe
complementary to the mRNA of ligatin (A, B), α-CAMKII (C-F),
and NSE (G, H). Bound probe is visualized with Texas red-ultra
avidin conjugate. (A,C,E,G) FISH/confocal micrographs of neurons
treated with KHC sense-ODN show somato-dendritic distribution of
mRNAs. (B,D,F,H) Neurons treated with KHC anti-ODN show a
marked decrease in labeling for α-CAMKII mRNAs (D and F). The
regional distribution of ligatin (B) and NSE (H) mRNAs are not
affected. * Dendrite; ** soma. Relative intensity scale as in Fig. 5.
Bars: 50 µm (A-D,G,H); 20 µm (E and F).
protein complex and by depleting a specific motor protein. We
conclude that TB-RBP and KHC are important contributors in
the localization of mRNA within the dendritic compartment of
hippocampal neurons and implicate microtubules as the transit
route. Our data and the interactions of TB-RBP with the
transitional endoplasmic reticulum ATPase, the ATPase
involved in intracellular transport and vesicle fusion (Wu et al.,
1999), suggest structural heterogeneity of RNA-protein
complexes involved in the regional localization of mRNA in
hippocampal neurons. Additionally, we show that local
translation of targeted mRNA is critical for expression of the
cognate protein within post-synaptic areas. Our working
hypothesis is that the formation of RNA-protein complexes
important for mRNA sorting may be governed by synaptic
activity and thus provides a mechanistic link by which neural
differentiation and plasticity can be regulated locally within
dendrites and post-synaptic areas of CNS neurons.
The authors thank Drs J. Nevins, J. DeGregori, E. Ellis, J. Feher,
E. Raff, L. Jakoi and M. Sheetz for editorial comments in the
preparation of this manuscript. We thank Drs G. Leone, J. DeGregori,
and J. Nevins (HHMI Duke University Medical Center, NC) for
preparation of the recombinant adenoviruses. Appreciation is
expressed to Dr Okatani (HHMI Duke University) for the pAdCMV,
Dr Cowan (New York Medical Center, New York) for the pBR322kα1 encoding α-tubulin and Dr Bigbee (Virginia Commonwealth
University, VA) for anti-intermediate filament antibody. We thank C.
Gerwin for technical assistance. This work was supported by A. D.
Williams grants 6-46917 (T.B.) and 6-46576 (E.R.J.) and AHAWilliam Randolph Hearst award 92009590 (E.R.J.), by NIH Center
grant and Jacob Javits award (R.J.D.) and by the Sophie and Nathan
Gumenick Neuroscience Research Endowment, and by NICHD grant
HD28832 (N.B.H.).
Fig. 9. Regional distribution of KHC protein in hippocampal
neurons treated with KHC sense-ODN (A) and KHC antiODN (B). ODNs were administered as described in Fig. 8.
After 72 hours, neurons were stained with specific antibody
to KHC (1:10). Results are representative of 4 experiments.
* dendrite; ** soma. Bar, 25 µm.
Disrupted mRNA sorting in CNS neurons 3701
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