ABSTRACT
Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease that is
associated with a (CTG)
n
repeat expansion in the 3
'
-untranslated region of the myotonin protein kinase (Mt-PK) gene. This study reports the isolation and characterization of a
(CUG)
n
triplet repeat pre-mRNA/mRNA binding protein that may play an important role in DM
pathogenesis. Two HeLa cell proteins, CUG-BP1 and CUG-BP2, have been purified based upon their ability to bind
specifically to (CUG)
8
oligonucleotides
in vitro
. While CUG-BP1 is the major (CUG)
8
-binding activity in normal cells, nuclear CUG-BP2 binding activity increases in DM cells. Both CUG-BP1 and CUG-BP2 have been identified as isoforms of a novel
heterogeneous nuclear ribonucleoprotein (hnRNP), hNab50. The CUG-BP/hNab50 protein is localized predominantly in the nucleus and is
associated with polyadenylated RNAs
in vivo
.
In vitro
RNA-binding/photocrosslinking studies demonstrate that CUG-BP/hNab50 binds to RNAs containing the Mt-PK 3
'
-UTR. We propose that the (CUG)
n
repeat region in Mt-PK mRNA is a binding site for CUG-BP/hNab50
in vivo
, and triplet repeat expansion leads to sequestration of this hnRNP on mutant Mt-PK transcripts.
Myotonic dystrophy (dystrophia myotonica, DM) is the most common form of adult
onset muscular dystrophy (
1
). The clinical DM phenotype is highly variable both within and between
families, and is characterized by muscle weakness and myotonia in skeletal
muscle, dilated cardiomyopathy, and a number of extramuscular abnormalities
including cataracts and reduced cognitive function. Myotonic dystrophy is an
autosomal dominant disorder that shows genetic anticipation in which successive
generations show earlier onset and increasing disease severity. The gene
affected in DM has been mapped to chromosome 19q13.3, and encodes myotonin
protein kinase (Mt-PK) (
2
-
10
). Nearly all affected DM individuals possess a (CTG)
n
triplet repeat expansion in a region of the Mt-PK gene that corresponds to the 3'-untranslated region (3'-UTR) of the mRNA. The number of (CTG)
n
repeats is variable ranging from 5 to 37 triplet repeats in normal cells to
>700 repeats in the severe congenital form of the disease (CDM). This triplet
repeat expansion also shows somatic mosaicism with repeat size variable in
different tissues of affected individuals and a general increase in expansion
length with age (
11
-
13
).
The effect of the (CTG)
n
triplet repeat expansion on expression of the Mt-PK gene was unclear until recently. Early evidence indicated both
decreased expression of Mt-PK mRNA and protein in adult DM and increased expression in cells from a
single CDM patient (
9
,
14
-
17
). However, more recent reports have consistently demonstrated that the DM
expansion mutation leads to decreased expression in both adult onset DM and CDM
with some CDM individuals showing almost undetectable Mt-PK mRNA and protein levels (
18
-
20
) (Timchenko
et al
., manuscript in preparation). This decreased expression has been suggested to
be the result of a defect in the processing of Mt-PK pre-mRNA since the levels of mRNA are more severely affected than pre-mRNA (
16
,
19
,
20
). In support of the idea that loss of Mt-PK gene expression may lead to muscle disease in mammals, Mt-PK knock-out mice show a late onset myopathy in homozygotic (
Dmpk-
/-), but not heterozygotic, mutants (
21
). However, the degree of myopathy seen in
Dmpk-
/- mice is variable between studies, and loss of Mt-PK expression is not associated with any of the other phenotypes
commonly seen in human patients including myotonia and cataracts (
22
). These studies support the hypothesis that the expansion mutation is exerting
the dominant effect and disease is not simply the result of a loss of Mt-PK gene expression. Although the possible role of the (CTG)
n
expansion mutation in mediating loss of Mt-PK gene expression has remained a mystery, the corresponding (CUG)
n
RNA repeat might be a binding site for a pre-mRNA/mRNA-binding protein that is required for the biogenesis,
nucleocytoplasmic transport and/or translation of Mt-PK mRNA. Here we describe the isolation and characterization of a novel
heterogeneous nuclear ribonucleoprotein (hnRNP) that is identical to the
recently described (CUG)
8
-binding protein (CUG-BP) (
23
), and demonstrate that this hnRNP binds to RNAs containing the 3'-UTR of Mt-PK mRNA
in vitro
.
Whole cell extracts were prepared as described previously (
23
). To prepare the cytoplasmic fraction, cells were washed twice with phosphate-buffered saline (PBS), pelleted and resuspended in buffer A (10 mM Tris-HCl, pH 7.6, 1.5 mM MgCl
2
, 10 mM KCl, 0.5 mM DTT). After a 15 min incubation on ice, cells were
homogenized by passage through a 23 g needle (eight strokes), and the sample
was centrifuged for 5 min at 10 000 r.p.m. (Sorvall Microspin 12S) to pellet
nuclei which were used for the preparation of nuclear extracts (NE) as
described previously (
23
). The supernatant (cytoplasmic fraction) was collected and stored at -80oC. For CUG-BP purification, HeLa cells (200 plates, 15 cm, 50%
confluency) were grown in MM medium (
23
), and cytoplasmic proteins were fractionated by the denaturation/elution
technique (
24
). Briefly, proteins were resolved by SDS-PAGE (12% gels) and transferred to a nitrocellulose filter. The fraction
containing proteins in the 40-50 kDa range (p46) was isolated by cutting a region of the membrane near
the position of the 46 kDa ovalbumin marker, and proteins were eluted in 100 [mu]l of renaturation buffer (20 mM HEPES, pH 7.9, 50 mM KCl, 5 mM MgCl
2
, 10% glycerol, 1% Triton X-100, 0.1 mg/ml Fraction V BSA) at 4oC. The p46 fraction, which contained 15-20 proteins as determined by SDS-PAGE and silver staining, was subjected to DEAE
chromatography, and the proteins were eluted with a NaCl step gradient (0.1-1.0 M NaCl). The polypeptide composition of the DEAE-flowthrough fraction containing CUG-BP2 was analyzed by SDS-PAGE and found to contain a single 51 kDa polypeptide.
The CUG-BP1 protein was eluted by 0.2-0.3 M NaCl and further purified by FPLC MonoQ chromatography using
a linear salt gradient (0.02-1.0 M NaCl). The CUG-BP1 binding activity was present in fractions 18-26 with maximal activity in fraction 22 which contained a
major 49 kDa polypeptide as determined by SDS-PAGE and silver staining. Each protein fraction was analyzed by bandshift
analysis with the (CUG)
8
probe and polyacrylamide gel electrophoresis.
Binding reactions for the bandshift assay were performed at room temperature for
30 min in a 10 [mu]l reaction mixture containing 0.1-0.5 ng of
32
P-labeled DNA or RNA probe, 5-10 [mu]g NE or 20-30 [mu]g cytoplasmic extract, 2 [mu]g of poly(dI-dC), 20 mM Tris-HCl, pH 7.6, 100 mM KCl, 5 mM MgCl
2
, 5 mM DTT and 10% glycerol. When the (CUG)
8
and (CGG)
8
RNA probes were used in the binding reactions, 2 [mu]g of total HeLa cell RNA was also added as a non-specific competitor. For single-stranded DNA probes, poly(dI-dC) was heated to 95oC for 10 min followed by incubation on ice prior to
addition to the binding reaction. To determine binding specificity, a 100-fold molar excess of unlabeled (CUG)
8
or (CGG)
8
was added to the binding reaction prior to addition of the labeled probe. The
ss(CTG)
8
DNA, ss(CUG)
8
RNA
and ss(CGG)
8
RNA oligonucleotides, and the ds(CTG) DNA fragment, were synthesized and end-labeled as described (
23
). For supershift experiments, 0.5-3 [mu]g of affinity-purified mAb 3B1, specific for CUG-BP/hNab50, was added to the binding reaction and
incubated at room temperature for 15 min prior to addition of the
32
P-labeled probe. Protein-DNA and protein-RNA complexes were separated from free probe by
polyacrylamide gel electrophoresis as described (
23
). For bandshift analysis of DM lymphoblast cytoplasmic and nuclear extracts
(Fig.
2
) the following cell lines were obtained from the NIGMS Human Genetic Mutant
Cell Repository: DM1 (GM03986A), DM2 (GM03756), DM3 (GM03990A). Normal
lymphoblasts (HH) were obtained from the Baylor College of Medicine Tissue
Culture Core Facility,
The hNab50 protein was isolated using a yeast two hybrid interaction system
(Clontech). The yeast strain HF7c was transformed with pNAB2.GBT9 and fusion
protein expression confirmed by immunoblot analysis using the anti-Nab2p mAb 3F2 (
25
). Cells expressing the Gal4pBD-Nab2p fusion protein were subsequently transformed with a HeLa cell cDNA
library cloned into the pGADGH plasmid. Cells were selected on SD-Leu-Trp-His plates, and clones initially tested for [beta]-galactosidase activity using a plate assay
following a protocol provided by the manufacturer. Cells that were dark blue by
the plate assay were subsequently tested for [beta]-galactosidase activity using a quantitative liquid assay. Three cDNA
clones encoding the full-length CUG-BP/hNab50 protein were isolated from both human osteosarcoma and
HeLa cell libraries by hybridization with the pGADGH-hNab50 clone. DNA sequences for all cDNA clones were determined and
analyzed as described (
25
,
26
).
For the preparation of anti-hNab50 polyclonal antisera, BALB/c mice were injected with an hNab50-maltose-binding protein (hNab50-MBP) fusion protein which was prepared by expression of
the pMAL50.1 plasmid in
Escherichia coli
TB1 cells followed by amylose resin affinity chromatography (New England Biolabs). The pMAL50.1 plasmid was constructed by cloning a partial hNab50 cDNA clone
(encoding amino acids 44-482) behind the
malE
gene
.
Antisera were tested by immunoblot analysis using both purified hNab50-MBP protein as well as HeLa whole cell lysates. The mAb 3B1 was prepared
by fusing spleen cells from the best responding mouse with SP2/O cells,
hybridoma supernatants were screened by immunoblotting and cell
immunofluorescence, and positive hybridomas cloned as described (
25
). Affinity purified mAb 3B1 was prepared by growing hybridoma cells in Dulbecco's modified Eagle's medium (DMEM) containing 10% ultralow IgG FBS (Gibco-BRL) followed by affinity purification on Protein G-Sepharose. For immunoblot analysis of CUG-BP1 and CUG-BP2, proteins were fractionated on 12% SDS-PAGE gels and electroblotted onto
nitrocellulose. The membrane was blocked with 10% milk in PBS for 1 h at room
temperature, and subsequently incubated with the following monoclonal
antibodies and dilutions: 3B1 (1:500); 4B10 (1:2000) (specific for the hnRNP A1
protein); 4F4 (1:2000) (specific for the hnRNP C proteins). After incubation
for 1 h at room temperature, membranes were incubated for 1 h with a sheep anti-mouse secondary antibody conjugated with horseradish peroxidase and
washed. All other immunoblot analyses were performed as described (
25
,
26
), and proteins were detected by ECL (Amersham). Indirect cellular
immunofluorescence was performed essentially as described previously using a
1:500 dilution for mAbs 3B1 and 1D8 (specific for the hnRNP M proteins) (
26
,
27
) and either HeLa, Hep2, A549 or normal patient myoblast cell lines.
For RNA-protein photocrosslinking
in vivo
, HeLa S3 cells were grown in DMEM supplemented with 10% calf serum and 1%
penicillin-streptomycin to subconfluent densities. Cells were washed with ice-cold PBS and irradiated with UV light (Stratalinker, Stratagene)
for 5 min in 5 ml of PBS at 4oC. Polyadenylated RNPs and immunopurified hnRNP complexes were isolated as
described previously (
27
,
28
). For
in vitro
RNA binding studies, plasmids containing the 3'-UTR regions of the Mt-PK (MTPK.2, linearized with
Hin
dIII) and actin [pSP6[gamma]-actin (
29
), linearized with
Bam
HI] genes were transcribed
in vitro
in the presence of [
32
P]UTP, and purified by denaturing gel electrophoresis. The MTPK.2 plasmid was
constructed by subcloning a
Bam
HI-
Hin
dIII fragment (nt 2212-2849, DDBJ/EMBL/GenBank accession no. M87312) into pSP72 (Promega).
Following incubation of the ~600 nt labeled actin and Mt-PK RNAs (2.0 * 10
5
c.p.m.) in a 25 [mu]l reaction mix (11 [mu]l HeLa cell nuclear extract, 20 mM HEPES, pH 7.6, 1.3 mM MgCl
2
, 1.5 mM ATP, 20 mM creatine phosphate) at 30oC for 10 min, 5 [mu]g tRNA were added, samples were exposed to UV light (Stratalinker) for
5 min and RNAs were digested with 2.5 [mu]g RNase A (30 min at 37oC). Both total and immunopurified proteins photocrosslinked to RNAs
were detected by label transfer/autoradiography following SDS-PAGE. Total protein samples fractionated by SDS-PAGE corresponded to 7.5 [mu]l of the initial 25 [mu]l reaction. Since the hnRNP C proteins crosslink more
efficiently than other hnRNPs, the amount of the crosslinked reaction volume
used for immunopurification varied from 25 [mu]l (for mAb 4F4) to 190 [mu]l (mAb 3B1). Immunopurifications were performed at 4oC for 20 min essentially as described previously (
28
)
except that Protein G-Sepharose was used and crosslinked samples were treated at 100oC in 1% SDS prior to dilution in PBS containing 1 mM EDTA, 1% Triton
X-100, 0.5% deoxycholic acid, 0.1% SDS, 0.5% aprotinin.
Numerous reports have documented a correlation between the size of the (CTG)
n
repeat expansion and expression of the Mt-PK gene at both the mRNA and protein levels (
9
,
14
-
20
). The isolation of (CUG)
8
RNA-binding proteins permitted a direct test of the hypothesis that these
proteins might be involved in the regulation of Mt-PK gene expression and DM pathogenesis (
23
). We first analyzed the RNA-binding activities of CUG-BP1 and CUG-BP2 by bandshift analysis using nuclear and cytoplasmic
extracts derived from lymphoblast cell lines obtained from both normal and DM
patients. A previous study identified a single-stranded (ss)CTG-repeat recognizing protein (ssCRRP) that was localized in the
cytoplasmic fraction (
23
). We therefore also assayed binding actitivites for ssCTG and double-stranded (ds)CTG repeats. The dsCTG binding activity was similar in both
normal and three different DM lymphoblast lines (Fig.
2
A). In agreement with previous results using HeLa cell extracts, ssCRRP was
localized in the cytoplasmic fraction and showed only a slight increase in
binding to ss(CTG)
8
in DM cells (Fig.
2
B). In contrast, CUG-BP1 and CUG-BP2 were distributed in both the cytoplasmic and nuclear fractions
(Fig.
2
C). In normal lymphoblasts, the majority of the (CUG)
8
binding activity of CUG-BP1 was in the cytoplasmic fraction, but significant activity was also
present in the nucleus while CUG-BP2 was predominantly nuclear. Although the (CUG)
8
binding activity of CUG-BP1 declined in both the cytoplasmic and nuclear fractions in DM
lymphoblasts, there was a consistent ~2-fold increase of binding activity associated with CUG-BP2 in nuclear extracts from DM cells. This increase in nuclear
CUG-BP2 activity was confirmed using four additional lymphoblast, as well as
two myoblast, cell lines (data not shown). These results demonstrated that the
CUG-BP proteins were distributed in both nuclear and cytoplasmic fractions,
and that nuclear CUG-BP1 and CUG-BP2 activities were altered in DM cells.
Although CUG-BP1 and CUG-BP2 specifically bound CUG repeats
in vitro
, no evidence existed that these proteins were associated with mRNAs
in vivo
. Proteins that directly bind to pre-mRNAs and mRNAs in the nucleus are heterogeneous nuclear
ribonucleoproteins (hnRNPs) while cytoplasmic mRNA binding proteins are mRNPs (
30
,
31
). However, some hnRNPs shuttle between the nucleus and cytoplasm, and
subcellular fractionation invariably results in the presence of hnRNPs in the
cytoplasmic fraction (
32
). Since the CUG-BP proteins were present in both nuclear and cytoplasmic fractions, we
determined whether CUG-BP1 or CUG-BP2 were previously identified nuclear RNA-binding proteins by immunoblot analysis using monoclonal
antibodies against several human hnRNPs. Remarkably, a monoclonal antibody
(mAb) to the hNab50 protein, mAb 3B1, reacted against both CUG-BP1 and CUG-BP2 in cytoplasmic and partially purified p46 fractions (Fig.
3
A). Fractionation of the two different CUG-BP activities by DEAE-Sepharose chromatography and subsequent immunoblot analysis
indicated that both CUG-BP1 and CUG-BP2 were specifically recognized by mAb 3B1. In contrast, monoclonal
antibodies against two other abundant hnRNPs, the hnRNP A1 and C proteins,
detected the corresponding proteins only in the cytoplasmic fraction (Fig.
3
A).
The results described above suggested that different isoforms of hNab50 might be
responsible for both the CUG-BP1 and CUG-BP2 (CUG)
8
RNA-binding activities. To test this possibility, we investigated the effect of affinity-purified mAb 3B1 on (CUG)
8
binding
in vitro
by bandshift/supershift analysis. The p46 fraction containing both CUG-BP1 and CUG-BP2 was incubated with the (CUG)
8
probe in the presence of increasing amounts of mAb 3B1. Addition of low amounts
of mAb 3B1 (1 [mu]g) to the binding reaction resulted in the complete disappearance of CUG-BP2 and the appearance of a new supershifted band (Fig.
3
B). Higher antibody concentrations (>3 [mu]g) were required to neutralize formation of the CUG-BP1 complex. Bandshift/supershift experiments with purified CUG-BP1 and CUG-BP2 proteins yielded identical results (Fig.
3
C). To determine if recombinant hNab50 could bind specifically to the (CUG)
8
probe, a hNab50 cDNA clone (see Materials and Methods) was fused in-frame with the
E.coli
maltose-binding protein (MBP)
malE
gene to form pMAL50.1. The pMAL50.1 fusion protein was synthesized in
E.coli
, purified to homogeneity by affinity chromatography, and assayed for RNA
binding by bandshift analysis. The MBP-hNab50 protein bound to the (CUG)
8
probe and formed a shifted complex (Fig.
3
D). This interaction was specific since addition of a 100-fold excess of unlabeled (CUG)
8
, but not (CGG)
8
, abolished binding (data not shown). No detectable binding of the MBP alone was
observed (Fig.
3
D). We conclude that CUG-BP1 and CUG-BP2 are isoforms of hNab50.
The hNab50 protein was identified during studies on the function of a yeast
hnRNP, Nab2p (
25
). To elucidate conserved pathways in which Nab2p might play an important role,
we sought to identify human proteins that interact with yeast Nab2p
in vivo
using the two hybrid interaction system. Several human cDNAs were isolated from
a HeLa cell cDNA-activation domain library. A full-length cDNA clone encoding the hNab50 protein was isolated from a
human osteosarcoma cDNA library, and the deduced amino acid sequence indicated
that hNab50 is a basic (pI = 8.75) ~52 kDa protein (Fig.
4
A). The hNab50 protein is related to a family of RNA-binding proteins which possess three RNA-binding domains (RBDs) and are differentially expressed in the
vertebrate nervous system (
33
,
34
). Two proteins were detectable in HeLa cells by immunoblot analysis using an
anti-hNab50 monoclonal antibody. The sizes of these two proteins, 49 and 51
kDa, were identical to CUG-BP1 and CUG-BP2, respectively. Proteins immunologically related to hNab50 were
also present in a variety of vertebrate cells from human to frog, but were not
present in
Saccharomyces cerevisiae
(Fig.
4
B).
The results described above demonstrated that CUG-BP/hNab50 bound to (CUG)
n
repeats
in vitro
and was a pre-mRNA/mRNA-binding protein
in vivo
. To test if this hnRNP was able to bind to the 3'-UTR of Mt-PK mRNA, we employed an
in vitro
photocrosslinking assay. Labeled RNAs containing the 3'-UTRs of the Mt-PK and actin genes were prepared by
in vitro
transcription, incubated in HeLa cell nuclear extracts, and proteins were
crosslinked to the RNAs by exposure to UV light. Following RNase digestion,
proteins crosslinked to RNAs were fractionated by SDS-PAGE and detected by autoradiography. The majority of proteins in HeLa
cell nuclear extracts, including the hnRNP C proteins, crosslinked more
efficiently to the actin 3'-UTR than to the Mt-PK RNA (Fig.
7
). In contrast, the CUG-BP/hNab50 protein crosslinked preferentially to the Mt-PK RNA. These results demonstrate that CUG-BP/hNab50 binds to RNAs containing the 3'-UTR of Mt-PK, and suggest the possibility that this
hnRNP may possess transcript-specific binding properties.
Myotonic dystrophy is one of several diseases in humans which are associated
with the expansion of a trinucleotide repeat (
37
,
38
). These trinucleotide repeat expansions occur in various regions of the
affected gene, and generally result in either loss of the correct gene product
or a dominant gain-of-function phenotype in which the structure of the gene product is
altered. Three models have been proposed to explain the puzzling observation
that the (CTG)
n
triplet repeat expansion in the 3'-UTR of the Mt-PK gene results in an autosomal dominant and variable
phenotype. First, the precise level of the Mt-PK protein may be critical to normal cellular function and repeat
expansion in one of the Mt-PK alleles may lead to haploinsufficiency and disease (
9
). Second, the mutant DM allele may alter chromatin structure by changing
nucleosome positioning and affect the expression of both Mt-PK and other linked genes (
39
-
41
). Third, the (CTG)
n
repeat expansion may be a dominant gain-of-function mutation either exerted in trans at the RNA level or this
repeat may be a binding site for a specific nuclear RNA-binding protein (
19
,
23
). Recent studies have provided evidence which support the proposal that Mt-PK gene expression may be affected at the post-transcriptional level in DM cells. Hoffman and co-workers have shown that (CTG)
n
repeat expansion results in the reduction of poly(A)
+
mRNAs from both the normal and DM mutant alleles even though there is only a
minor effect on the transcription and accumulation of Mt-PK pre-mRNAs (
19
). Another study using Mt-PK mRNA-specific
in situ
hybridization analysis has shown that transcripts from the DM mutant allele
accumulate within intranuclear foci although both wild-type and mutant Mt-PK mRNAs are detectable in the cytoplasm (
42
).
In this study, we identify a novel human hnRNP as a candidate for the first
triplet repeat eukaryotic RNA-binding protein to be characterized. We present three lines of evidence
that hNab50 is responsible for (CUG)
n
triplet repeat RNA-binding activity: (i) polyclonal and monoclonal antibodies against hNab50
specifically recognize purified CUG-BP1 and CUG-BP2; (ii) anti-hNab50 antibodies supershift/neutralize CUG-BP activity; (iii) recombinant hNab50 and CUG-BP isolated from human cells have identical and
specific (CUG)
8
RNA-binding activities. In addition, the CUG-BP/hNab50 protein binds to RNAs containing the 3'-UTR of Mt-PK mRNA
in vitro
, and nuclear extracts from DM cells show alterations in CUG-BP activities compared with normal cells. We conclude that CUG-BP/hNab50 is a (CUG)
n
triplet repeat RNA-binding protein in human cells, and propose that the Mt-PK (CUG)
n
triplet repeat is a binding site for CUG-BP/hNab50
in vivo
.
How might a (CUG)
n
RNA-binding protein be involved in the regulation of gene expression and DM
pathogenesis? In prokaryotes, a triplet repeat RNA-binding protein has been previously characterized that regulates both
transcription and translation. The
trp
RNA-binding attenuation protein (TRAP) of
Bacillus subtilis
binds specifically to an RNA secondary structure, the antiterminator region, in
the nascent
trp
operon leader transcript. Binding of TRAP, which is dependent on the presence
of 11 G/UAG triplet repeats within the leader transcript, results in the
disruption of the antiterminator and transcriptional termination upstream of
the
trp
structural genes (
43
-
45
). TRAP also appears to play a role in translation by binding to a G/UAG-rich region that overlaps the ribosome binding site in
trpG
transcripts. In eukaryotes, hnRNPs bind to RNA polymerase II transcripts
following transcripitional initiation (
30
,
31
). The association of these abundant nuclear pre-mRNA/mRNA-binding proteins to nascent transcripts is believed to play an
important role in facilitating the formation of pre-mRNA structures amenable to subsequent pre-mRNA processing events. Recent work has also demonstrated that
hnRNPs are not exclusively nuclear proteins, and therefore they could
potentially function in the nucleocytoplasmic export, translation and turnover
of mRNAs (
30
-
32
). Expansion of the Mt-PK 3'-UTR triplet repeat in DM cells would lead to a large increase
in the number of potential binding sites for a (CUG)
n
triplet repeat mRNA-binding protein. If CUG-BP/hNab50 is important for the biogenesis and/or turnover of both Mt-PK as well as other mRNAs, then a reduction in the
availability of this hnRNP might affect the processing and/or turnover of these
mRNAs. This RNA processing/turnover defect could manifest itself by affecting
different RNAs in different tissues, thus accounting for the highly variable DM
phenotype seen in humans.
We thank L. Green and L. Matthews for assistance with antibody preparation, M.
Sardana for help with the purification of CUG-BP, G. Dreyfuss for providing antibodies to hnRNP A1 and C. S. Willingham
for photography, and members of our laboratories for comments on the
manuscript. This work was supported by grants from the NIH (GM46272), the NHLBI
Specialized Centers of Research (HL54313-01), the NIH Training Center in Molecular Cardiology (T32-HL07706), the AHA Burgher Foundation Center for Molecular Biology
(86-2216) and an AHA Medical Student Research Fellowship to J.W.M.
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