Department of Biochemistry and Molecular Biology, The University of
Queensland, Qld 4072, Australia
1 Department of Neurosciences NC30, Cleveland Clinic Foundation, 9500 Euclid
Avenue, Cleveland OH 44195, USA
2 Wellcome/CR UK Institute, Tennis Court Rd, Cambridge CB2 1QR, UK
* Present address: Institute for Cellular and Molecular Biology, University of
Texas, 2500 Speedway, Austin, TX, USA
Author for correspondence (e-mail:
ross.s{at}uq.edu.au)
Accepted 3 September 2002
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Summary |
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Key words: RNA-protein interactions, Zipcode, RNA trafficking, Protein sequencing, Confocal microscopy
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Introduction |
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A general pathway for mRNA trafficking in somatic cells has been proposed,
in which mRNA molecules are packaged into granules in the nucleus, then
exported to the cytoplasm where they attach to microtubules or microfilaments
and are transported to their destination before being translated
(Barbarese et al., 1999). Two
of the best-described systems are those mediating localization of the mRNAs
encoding ß-actin (Bassell et al.,
1999
; Kislauskis et al.,
1994
; Oleynikov and Singer,
1998
) and myelin basic protein (MBP)
(Ainger et al., 1997
;
Ainger et al., 1993
;
Carson et al., 1997
). Both
mRNAs have small cis-acting segments: the 54-nucleotide zipcode in the chicken
ß-actin mRNA (Ross et al.,
1997
) and the 11-nucleotide hnRNP A2 response element (A2RE11) in
the MBP mRNA (Munro et al.,
1999
). These RNA segments appear to operate in more than one cell
type: zipcode-dependent transport has been demonstrated in fibroblasts
(Kislauskis et al., 1994
;
Ross et al., 1997
), where it
is actin-dependent, and in neurons (Bassell
et al., 1998
; Zhang et al.,
1999
), where it is microtubule dependent. Likewise, A2RE-dependent
transport occurs in oligodendrocytes (Hoek
et al., 1998
; Munro et al.,
1999
) and neurons (J. Shan et al., personal communication). An
important unanswered question is whether these mRNAs in different cell types
use the same, or different, trans-acting factors and trafficking pathways.
We show here that the ß-actin zipcode recognizes a set of at least ten rat brain polypeptides from rat brain. We have identified six of the most prominent proteins by Edman sequencing and western blotting as rat homologues of KH-type splicing regulatory protein (KSRP), far-upstream element binding protein (FBP), HuC and heterogeneous nuclear ribonucleoproteins (hnRNP) E1, E2 and L. All of these proteins possess established RNA-binding motifs. The RNA-sequence-specific binding of a group of brain proteins suggests that there are varied and complex interactions that govern the post-transcriptional actions and fate of mRNAs, which may involve segments such as the zipcode, perhaps in combination with other cis-acting sequences.
We further show that zipcode and the AU-rich response element (AURE), the
latter a cis-acting element that influences mRNA stability by recruiting
exosomes (Mukherjee et al.,
2002), bind a similar set of proteins. This set does not overlap
the A2RE11-binding proteins, Our results suggest that families of trans-acting
factors may recognize the same cis-acting element and participate in different
steps of mRNA processing and metabolism. These trans-acting factors may also
target different and multiple RNAs in the same cell. Finally, we show that
although KSRP and hnRNP A2 are both widely distributed in the brain they
differ in nuclear distribution and are segregated into different granules in
the cytoplasm of oligodendrocytes.
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Materials and Methods |
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Protein sequencing
Proteins isolated from eight individual magnetic particle experiments, each
with 50 µl of magnetic particles bearing 5' zipcode were combined,
partially concentrated by vacuum centrifugation, and run in a single well of a
12% SDS/polyacrylamide gel before staining with Coomassie brilliant blue.
Bands were excised from the gel and digested with trypsin as follows. The gel
slices were washed in 50% CH3CN for 5 minutes, 50%
CH3CN/50 mM NH4HCO3 for 30 minutes and 50%
CH3CN/10 mM NH4HCO3 for 30 minutes and then
vacuum dried. TPCK-treated trypsin (0.1 µg Promega, Sydney, Australia) in
15 µl of 10 mM NH4HCO3 was added to the dried gel,
which was then left at 37°C overnight. Peptides were extracted using two 1
hour incubations in 200 µl of 60% CH3CN in 0.1% TFA. Tryptic
fragments were dried by vacuum centrifugation and redissolved in 20 µl of
10% CH3CN/0.1% TFA before separation on a 2.1 mmx50 mm C18
reverse-phase HPLC column (Vydac, Hesperia, CA). Optimal separation was
achieved using a 10-40% acetonitrile gradient in 0.1% TFA over 60 minutes at a
flow rate of 30 µl/minute. Peptides from HPLC peaks that appeared well
resolved were selected for Edman microsequencing on an Applied Biosystems
(Foster City, CA) Procise cLC sequencer. The levels of the tryptic peptides
sequenced were generally below 1 pmol, and several were sequenced at the
200-250 femtomol level.
Western blot analysis
Proteins were separated on 12% SDS/polyacrylamide gels before
electrophoretic transfer onto PVDF (Millipore, Sydney, Australia) and
incubation with primary antibody. The secondary antibodies were 1 in 1000
dilutions of either goat anti-rabbit or rabbit anti-chicken antibodies
conjugated to alkaline phosphatase (Sigma). Phosphatase activity was
visualized using a 4-nitro blue
tetrazolium/5-bromo-4-chloro-3-indoyl-phosphate substrate (NBT). The method of
Stenoien (Stenoien and Brady,
1997) was used to stain for the kinesin heavy chain, but the
kinesin antibody was detected with a 1 in 12,000 dilution of rabbit
anti-chicken antibody conjugated to horseradish peroxidase (Sigma) and
visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech,
UK).
Cell culture
Brains were excised from newborn Wistar rats and homogenized in DMEM
containing 10% foetal bovine serum by triturating repeatedly with a 10 ml
syringe. The homogenate was allowed to settle, and the supernatant was removed
and placed in 75 cm2 tissue culture flasks. After 10 days the
cultures were vigorously shaken and the medium was removed to generate
secondary cultures on poly-L-lysine coated coverslips. Secondary cultures were
fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) before
processing for immunostaining.
Immunostaining
15-day-old Wistar rats were perfused intracardially with PBS, then 4%
paraformaldehyde in PBS. Brains were fixed overnight, cryoprotected in 0.5 M
sucrose in PBS, frozen, then sectioned in a cryotome to generate 30 µm
thick coronal sections. Sections were rinsed three times for 5 minutes in PBS
containing 0.0001% Triton X100, then microwaved twice until boiling in 0.01 M
citrate buffer, pH 6.0. They were then incubated in 10% Triton X-100 PBS for
30 minutes, then in 3% normal goat serum containing 1% hydrogen peroxide for
30 minutes. After rinsing with PBS, sections were incubated in primary
antibodies overnight at 4°C (Table 3.1), in biotinylated secondary
antibodies for 1 hour at room temperature, and then in a 1:1000 dilution of
ABC solution (Avidin Biotin Complex Elite Kit, Vector Laboratories,
Burlingame, CA) for 1 hour at room temperature. Peroxidase activity was
detected with DAB reaction mix (Sigma), following the manufacturer's
instructions, before mounting sections in glycerol.
Fluorescence immunostaining
The procedure for fluorescence immunostaining of tissue is as described
above, until the incubation with the secondary antibody. Sections were
incubated either with FITC- or TRITC-conjugated secondary antibodies or with
biotinylated secondary antibodies for 2 hours at room temperature. Sections
incubated with biotinylated secondaries were then incubated with FITC- or
Texas Red (TR)- conjugated avidin for 1 hour at room temperature. Cultured
cells were incubated in 0.1% Triton X-100 for 10 minutes, 0.2% fish skin
gelatin for 15 minutes, primary antibody for 30 minutes and secondary antibody
for 30 minutes. Tissue sections and cultured cells were mounted in Vectashield
containing 4'-6-diamidino-2-phenylindole (DAPI, Molecular Probes Inc,
Portland, OR). Tissues and cells were imaged with a Zeiss Axiophot 2
fluorescence microscope equipped with a Variocam camera (Carl Zeiss,
Oberkochen, Germany), and x40 (0.75 NA), x63 (1.4 NA) and
x100 (1.3 NA) lenses. Confocal images were produced with Leica TCS-NT
(Leica, Inc., Deerfield, IL) or BioRad MRC 600 (BioRad, CA) confocal
microscopes equipped with x63 (1.4 NA) lenses. Multiple Z-plane images
were merged, where required, using NIH Image v1.62 (National Institutes of
Health, Washington, DC).
Antibodies
Rabbit anti-ZBP-1 antibody (Ross et
al., 1997), used at a 1:100 dilution, was generously supplied by
R. Singer (Albert Einstein College of Medicine, NY), chicken anti-CRD-BP
antibody (Leeds et al., 1997
)
(1:3000) by J. Ross (McArdle Laboratory for Cancer Research, University of
Wisconsin-Madison, WI), 4606 rabbit anti-FBP antibody (1:1000) by David Levens
(Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland),
chicken anti-kinesin heavy chain antibody (KHC 555-772) (1:5000) by R.
Diefenbach (Westmead Institute of Health Research, Westmead Hospital, Sydney,
Australia) and both rabbit KSRP antibodies, one (which stains nucleoli; DBKS)
raised against a C-terminal peptide and the other (C2742) against residues
172-711 (Markovtsov et al.,
2000
; Min et al.,
1997
) (1:1000) by D. Black (UCLA, CA). The specificities of our
antibodies to peptides from hnRNPs A1, A2/B1, B1 and A3 have been verified
previously (Ma et al., 2002
)
(T. P Munro, A2RE-mediated RNA transport, PhD thesis, University of
Queensland, 2002).
Other primary antibodies included mouse anti-adenomatous polyposis coli protein (CC1 antibody; Oncogene Research Products, San Diego, CA) and mouse anti-CNP antibody (Sigma). Biotin-labeled goat anti-rabbit antibody (Jackson Immunoresearch Laboratories, West Grove, PA), goat anti-rabbit TRITC (Sigma), goat anti-rabbit FITC (Sigma), goat anti-mouse FITC (Jackson), goat anti-mouse TRITC (Sigma), mouse anti-chicken FITC (Sigma) and Texas red avidin (Vector Laboratories) were used as secondary antibodies.
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Results |
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Identification of zipcode-binding brain proteins
Microsequencing of tryptic peptides generated by in-gel digestion after
separation on SDS/polyacrylamide gels was used in an attempt to identify the
most prominent of the 5'-zipcode-binding proteins. Four bands (a-d in
Fig. 1, arrowheads on 5'
ZIP lane) yielded levels of peptide sufficient for Edman microsequencing, all
in the range of 200-500 femtomol. Three peptides from an 83 kDa band
(Fig. 1, band a) matched the
amino-acid sequence of human KSRP (Table
2), an RNA-binding protein that contains several KH domains; a 72
kDa band (band b) matched FBP exactly over one 11 residue peptide; three
peptides from the 61 kDa band (band c) had 100% sequence identity with human
hnRNP L; and a 38 kDa band (band d) yielded three peptides of which two
matched the sequence of mouse HuC and one matched hnRNP E. The only difference
in this region between the isoforms hnRNPs E1 and E2 is at residue 107, which
is Thr in hnRNP E1 and Ser in E2: both PTH derivatives were unequivocally
present in the corresponding Edman cycle, suggesting that both isoforms, which
differ in mass by only 1054 Da, were present in the gel band and, therefore,
that both bind the zipcode. hnRNP L appeared not to bind AURE although it
bound the zipcode (band c in Fig.
1). Lower molecular weight bands corresponding in position to
hnRNPs E and HuC were also evident in AURE-binding proteins. No other
sequences in the translated DNA databases matched as well as those identified
above, even allowing for minor ambiguities or gaps in some of the peptide
sequences. Several other bands were subjected to digestion with trypsin, HPLC
and Edman degradation but yielded no reliable sequences.
|
Western blots highlight the specificity of RNA-protein
interactions
As antibodies were available for two of the identified zipcode-binding
proteins, KSRP and FBP, these proteins were selected for further study. Their
specific association with zipcode was verified by western blotting. Antibodies
to KSRP and FBP recognized 83 kDa (calculated mass 72 kDa) and 72 kDa
(calculated mass 68 kDa) proteins, respectively, eluted from immobilized
5' zipcode but not from A2RE11 or NS1
(Fig. 2A), in accordance with
the results shown in Fig. 1.
Conversely, antibodies to hnRNPs A1, A2/B1, B1 and A3 recognized proteins
eluted from immobilized A2RE11 but none from 5' zipcode. AURE did not
bind to hnRNP A2 and is thus unlikely to bind to the other hnRNP A/B proteins
(data not shown). These results demonstrate the highly sequence-specific
nature of these RNA-protein interactions.
|
Fig. 2B shows NS1-, AURE- and 5'-zipcode-binding proteins detected with anti-KSRP (C2742) and verifies that the co-migrating AURE- and 5'-zipcode-binding proteins marked `a' in Fig. 1 are both KSRP. In the original gel, a closely spaced doublet was observed, which may represent isoforms of KSRP.
Zipcode-binding proteins from chicken and rat were also detected on western blots with an anti-CRD-BP antibody, which recognizes ZBP-1 and the homologous KSRP protein (16% amino acid identity and 42% amino acid similarity), to determine whether ZBP-1 is a rat brain zipcode-binding protein. Both 83 kDa (data not shown) and, at a much lower level, 68 kDa ZBP-1-like proteins were present in foetal brain (E15, Fig. 2C), but in 21-day-old rat brain (P21) the 83 kDa protein was much less abundant and the 68 kDa band was not detected even after prolonged exposure. The 68 kDa CRD-BP-immunoreactive protein was not detected in the P21 chicken or rat brain proteins eluted from magnetic particles bearing either A2RE or 5' zipcode. ZBP-1 was, therefore, not one of the brain zipcode-binding proteins shown in Fig. 1. Only the 83 kDa protein was detected with anti-CRD-BP antibody in the chicken and rat brain proteins bound to magnetic particles bearing the zipcode, most probably reflecting the very low level of ZBP-1 in mature rat brain.
Conventional kinesin, which has been implicated in the cytoplasmic trafficking of RNAs, was evident on western blots of whole rat brain extracts but not in 5'-zipcode- or A2RE11-binding proteins (Fig. 2D). This suggests that if kinesin does bind, directly or indirectly, these RNA-binding proteins the interaction does not persist in the pull-down experiments.
The 3' zipcode binds a subset of the 5'-zipcode-binding
proteins
Pull-down experiments were performed with rat brain proteins and the two
segments of the zipcode, the 5' and 3' zipcodes, and the bound
proteins analyzed on SDS/polycrylamide gels. In this experiment, as in some
others, the level of hnRNP L isolated was higher than in the experiment shown
in Fig. 1.
Fig. 3 shows that some, but not
all, of the proteins that bound the 5' zipcode also bound the 3'
zipcode. KSRP and FBP, but not hnRNP L, HuC or hnRNPs E1 and E2, bound the
3' zipcode, suggesting that within the full 51 nucleotide rat zipcode
there are two sequence-specific binding sites or a site that overlaps the
junction between the 5' and 3' zipcodes for the first two
proteins. There is some sequence similarity between short sections of the two
zipcode fragments: one possible common motif is (G/C)UUUNNNA, which is also
found in AURE.
|
A2RE11- and zipcode-binding proteins are co-expressed but have
different distributions in brain
KSRP and FBP are widely distributed in rat brain. They were detected by DAB
staining in different cell types in the white (wm) and grey (gm) matter of
15-day-old animals. From their position and morphology, most cells, including
oligodendrocytes, astrocytes and cortical and hippocampal neurons, appeared to
be positive for KSRP, FBP and hnRNP A2.
The distribution of KSRP and FBP in the cortex is shown in
Fig. 4A,C. Intense KSRP and FBP
fluorescence was found in the nuclei of cells stained for the oligodendrocyte
cell body marker CC1 (Fig.
4B,D). These cells also expressed hnRNP A2
(Fig. 4E,F), suggesting that
A2RE-binding proteins (hnRNP A2) and zipcode-binding proteins
(KSRP-immunoreactive proteins) are co-expressed. Although KSRP and FBP were
detected primarily in the nuclei by DAB staining the hnRNP A2 appeared to be
present in both the nuclei and cytoplasm
(Fig. 4E). However, confocal
fluorescence microscopy showed that most hnRNP A2 is nuclear in
oligodendrocytes (Fig. 4F).
hnRNP A3, which, like hnRNP A2, may play a role in cytoplasmic trafficking of
RNA (Ma et al., 2002) was also
apparent in oligodendrocyte nuclei. hnRNP A1, which appears not to associate
with A2RE, or to do so more weakly, was not evident, and hnRNP B1, a far less
abundant isoform of hnRNP A2, was detected in only a small subset of
oligodendrocytes (Fig.
4G,H).
|
KSRP and hnRNP A2 have different nuclear locations in
oligodendrocytes
The co-expression of KSRP-immunoreactive proteins and hnRNP A2 was also
evident in cultured glial cells, where both are present predominantly in the
nuclei (Fig. 5A,B). A single
confocal optical section shows that KSRP-immunoreactive proteins and hnRNP A2
had a reticular distribution in the nucleoplasm and appeared to be excluded
from many small circular nuclear regions. There was some overlap in the
reticular staining patterns, but both proteins were concentrated in different
regions. The difference in the distributions of these proteins was most
striking in mitotic cells in which KSRP was widely distributed in the mitotic
cell (Fig. 5D), whereas hnRNP
A2 (Fig. 5E) was primarily
associated with the segregated chromosomes
(Fig. 5F,G).
|
KSRP and hnRNP A2 are in different cytoplasmic granules
In oligodendrocyte processes, antibodies to KSRP and hnRNP A2 bound
cytoplasmic granules, which resemble the mRNA transport particles observed in
many different cell types (Fig.
6A,B). An analysis of the KSRP (arrowheads in B, red) and hnRNP A2
(arrows, green) fluorescence in each granule demonstrated that
KSRP-immunoreactive proteins and hnRNP A2 were concentrated in separate
granule populations. The relative levels of KSRP and hnRNP A2 fluorescence in
each granule were calculated, and a histogram of these values showed that the
majority of granules contained just KSRP [red, r, r/(r+g)=1] or hnRNP A2
[green, g, r/(r+g)=0] (Fig.
6C). Few granules appeared to contain both proteins. This suggests
that there are transport complexes that transport zipcode-containing mRNAs and
separate granules that transport A2RE-containing mRNAs.
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Discussion |
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The post-natal rat brain zipcode-binding proteins identified here include
KSRP, FBP, hnRNP L, hnRNPs E1 and E2 and HuC. In recent experiments Gu and
colleagues (Gu et al., 2001)
identified ZBP-2, FBP, ssDBF (a single-stranded DNA binding protein) and ABBP
(an hnRNP A/B type protein) as zipcode-binding proteins in embryonic chicken
brain. There is overlap, with FBP and KSRP/ZBP-2 being common to both groups.
ZBP-1 is the predominant zipcode-binding protein of embryonic fibroblasts
(Ross et al., 1997
), but
expression of this protein declines after birth, and it was not detected in
the zipcode-binding proteins from postnatal brain in our experiments. Little
CRD-BP (a ZBP-1 homologue) was recently found in adult human brain using
RT-PCR, although it was detected in a few adult tissues, in many foetal
tissues including the brain and in mesenchymal tumors
(Ioannidis et al., 2001
).
We saw only one closely spaced protein doublet in the molecular weight
range expected for KSRP and ZBP-2: these proteins are most probably isoforms
of KSRP, a rat homologue of the chicken ZBP-2
(Gu et al., 2001) and rat
MARTA1 (Rehbein et al., 2000
).
The chicken ZBP-2 and human KSRP have over 86% identity at the amino-acid
level but ZBP-2 has an additional segment near the N-terminus
(Gu et al., 2001
): searches of
the human and mouse databases for this ZBP-2 47-residue segment did not reveal
any cognate sequence.
The identification of several zipcode-binding proteins raises the question:
do they interact with the zipcode-containing RNA in the nucleus, cytoplasm or
in both locations? KSRP (Min et al.,
1997) was reported to be located primarily in the nucleus, where
it acts as a splicing factor and forms part of the perinucleolar structure
(Huang, 2000
), but our
experiments show that it is also present in cytoplasmic granules that have
been implicated in RNA trafficking (Hoek
et al., 1998
; Kiebler et al.,
1999
; Kohrmann et al.,
1999
) and binds zipcode, a known cis-acting sequence for
cytoplasmic RNA trafficking.
Each of the other proteins has been implicated in the cytoplasmic
metabolism of RNAs. FBP targets a far upstream cis-acting element of
c-myc, regulating its transcription
(Bazar et al., 1995), but it
also binds a 26 nucleotide pyrimidine-rich sequence in the 3' UTR of
GAP-43 mRNA, modulating its stability
(Irwin et al., 1997
;
Wang et al., 1998
). hnRNP E
(Hahm et al., 1993
) regulates
cap- and IRES-dependent translation
(Ostareck et al., 1997
). It
also binds and possibly stabilizes the pyrimidine-rich 3' UTR regions of
erythropoietin (Czyzyk-Krzesk,
1999
) and
-globin
(Kiledjian et al., 1995
)
mRNAs. hnRNP L (Piñol-Roma et al.,
1989
) was originally described as being localised to the nucleus
(Huang, 2000
;
Kamma et al., 1995
) but
extranuclear functions are suggested by its association with gliomas that
manifest translational repression and mRNA instability
(Hamilton et al., 1999
). It
also binds the 126 nucleotide hypoxia stability region of human vascular
endothelial growth factor mRNA (Shih and
Claffey, 1999
) and the AU-rich cis-acting destabilization sequence
of glucose transporter Glut1 mRNA
(Hamilton et al., 1999
).
Finally, HuC belongs to the ELAV family of RRM-containing proteins which,
although found in the nucleus, binds AURE
(Abe et al., 1996
) and is
colocalized with ribosomes in microtubule-associated granules in neuronal
dendrites (Antic and Keene,
1998
; Gao and Keene,
1996
). FLAG-tagged HuC is localized to the cytoplasm of
transfected PC-12 cells (Akamatsu et al.,
1999
).
Thus, there is evidence to suggest that each of these zipcode-and AURE-binding proteins can bind to, and modulate the function of, cytoplasmic RNA, primarily by controlling mRNA stability or translation. This group includes proteins that contain KH domains, and some with RRMs. Hence, the observed RNA binding cannot be attributed to a single type of RNA-binding domain: modules with divergent tertiary structures bind the ß-actin zipcode sequence selectively.
The binding of these proteins to the zipcode and AURE can be partly
rationalised in terms of their RNA-binding preferences. None of the RNA
sequences that have been reported to bind to these proteins overlaps with
A2RE11, but several have segments that match parts of the zipcode or AURE
(Table 1): (1) FBP binds a 26
nucleotide sequence (Irwin et al.,
1997) that shares the sequence UAUUU with AURE; (2) hnRNP E binds
repeated CCUCCC sequences (Holcik and
Liebhaber, 1997
) overlapping with the zipcode CCCU; (3) hnRNP L
binds the 33 nucleotide Glut1 stability element
(Hamilton et al., 1999
), which
contains both zipcode (GUUUUA and UUACUG) and AURE (UUUA and UUUUUA) segments.
It also binds the 21 nucleotide bovine vascular endothelial growth factor
3' UTR sequence (Shih and Claffey,
1999
), which has limited overlap with the zipcode (UACA, ACCC);
(4) HuC binds AUUUA and related AU-rich sequences
(Abe et al., 1996
;
Inoue et al., 2000
) and hence
overlaps with the UUUA segment of the zipcode and the AURE sequence AUUUA.
Thus, the identified zipcode- and AURE-binding proteins appear to favour
U-rich tracts, which have more common in AURE than with zipcode.
Are the RNA segments identified above, which are often only four to six
nucleotides, sufficient to mediate specific protein binding? In the few
RNA-protein complexes of known 3D structure, linear stretches of eight to nine
nucleotides are sufficient to fill the binding sites, and the essential
nucleotides may be smaller in number. An 11 nucleotide segment of the A2RE is
sufficient for recognition and transport by the hnRNP A2 targeting pathway
(Munro et al., 1999), and of
these 11 nucleotides, substitution in only five non-contiguous positions
results in a marked lowering of binding to hnRNP A2
(Munro et al., 1999
)
(Moran-Jones et al., personal communication). It is possible that the
zipcode-binding proteins recognise different segments of the 51 nucleotide
zipcode oligonucleotide or the 22 nucleotide AURE. The KH3 domain of Nova has
been shown to have an absolute requirement for the tetranucleotide UCAY
presented in the loop region of a hairpin structure, although the two base
pairs proximal to the loop also contribute to binding
(Jensen et al., 2000
).
In summary, we have shown that two cis-acting elements, zipcode and AURE, sequence selectively bind a similar group of proteins, six of which have recognized RNA-binding motifs. The A2RE11 also binds several proteins but they do not overlap with the zipcode/AURE-binding proteins. Although the zipcode-binding protein KSRP and the A2RE-binding hnRNP A2 are abundant in the nuclei of many CNS cells, they appear not to be colocalized. Similarly, both are present at lower levels in the processes of oligodendrocytes, but they are concentrated in mutually exclusive sets of putative transport granules. These experiments reinforce the view that binding of trans-acting factors to small cis-acting elements is sequence selective but also demonstrate the possibility of competition between proteins for the same RNA element and, conversely, between different cis-acting elements for a single protein. The regulation of RNA metabolism is thus likely to involve a complex interplay between multiple RNA elements and trans-acting factors.
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Acknowledgments |
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![]() |
Footnotes |
---|
Rehbein et al. have recently shown that rat MARTA1 is the orthologue of human KSRP, with 98% amino acid identity.
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