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5474–5486 Nucleic Acids Research, 2007, Vol. 35, No. 16 Published online 15 August 2007
doi:10.1093/nar/gkm601
Muscleblind-like 1 interacts with RNA hairpins
in splicing target and pathogenic RNAs
Yuan Yuan
1
, Sarah A. Compton
2
, Krzysztof Sobczak
3
, Myrna G. Stenberg
1
,
Charles A. Thornton
3
, Jack D. Griffith
2
and Maurice S. Swanson
1,
*
1
Department of Molecular Genetics and Microbiology and the Genetics Institute, University of Florida, College
of Medicine, Gainesville, FL,
2
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel
Hill, Chapel Hill, NC and
3
Department of Neurology, University of Rochester Medical Center, Rochester, NY, USA
Received April 30, 2007; Revised July 18, 2007; Accepted July 24, 2007
ABSTRACT
The MBNL and CELF proteins act antagonistically to
control the alternative splicing of specific exons
during mammalian postnatal development. This
process is dysregulated in myotonic dystrophy
because MBNL proteins are sequestered by
(CUG)
n
and (CCUG)
n
RNAs expressed from mutant
DMPK and ZNF9 genes, respectively. While these
observations predict that MBNL proteins have a
higher affinity for these pathogenic RNAs versus
their normal splicing targets, we demonstrate that
MBNL1 possesses comparably high affinities for
(CUG)
n
and (CAG)
n
RNAs as well as a splicing target,
Tnnt3. Mapping of a MBNL1-binding site upstream
of the Tnnt3 fetal exon indicates that a preferred
binding site for this protein is a GC-rich RNA hairpin
containing a pyrimidine mismatch. To investigate
how pathogenic RNAs sequester MBNL1 in DM1
cells, we used a combination of chemical/enzymatic
structure probing and electron microscopy to
determine that MBNL1 forms a ring-like structure
which binds to the dsCUG helix. While the
MBNL1 N-terminal region is required for RNA bind-
ing, the C-terminal region mediates homotypic
interactions which may stabilize intra- and/or
inter-ring interactions. Our results provide a
mechanistic basis for dsCUG-induced MBNL1
sequestration and highlight a striking similarity in
the binding sites for MBNL proteins on splicing
precursor and pathogenic RNAs.
INTRODUCTION
To promote the assembly of functional ribonucleoprotein
(RNP) complexes, RNA-binding proteins possess sev-
eral different structural motifs which confer recognition
of RNA sequence and structural elements (1).
The RNA-recognition motif (RRM), which is the most
common RNA-binding domain, consists of a babbab-fold
in which specific amino acids in the two central b-strands
contact bases in a single-stranded (ss)RNA target site (2).
Additional ssRNA-binding motifs include the KH
domain, arginine-rich sequences and zinc-finger related
motifs. An important example of the latter motif is found
in the TTP/TIS11/ZFP36 proteins which promote
the deadenylation and turnover of target mRNAs which
contain a 30untranslated region (30UTR) class II AU-rich
element (ARE) (3). The NMR structure of a pair of
CX
8
CX
5
CX
3
H fingers in the TIS11d tandem zinc finger
(TZF) domain protein reveals that these zinc fingers,
which are separated by an 18-residue linker, bind
to adjacent 50-UAUU-30sequence elements (4). For
double-stranded (ds)RNAs, the most common motif is
the dsRNA-binding domain (dsRBD) which recognizes
the A-form helical conformation (5). The dsRBD struc-
ture, which consists of a abbba-fold with two highly
conserved basic loops, interacts with successive grooves
on the RNA helix (1,6). In addition, some zinc finger (ZF)
proteins recognize both sequence and structural
elements in their target RNAs, including TFIIIA which
simultaneously contacts ssRNA and dsRNA regions in
5S RNA (7).
Previous studies have suggested that the muscleblind-
like (MBNL) family of alternative splicing factors,
which possess multiple copies of a TTP/TIS11-related
CCCH motif (CX
7
CX
4-6
CX
3
H), also recognize both
ssRNA and dsRNA elements (8,9). Similar to TIS11d,
the CCCH motifs in MBNL proteins are organized
as tandem pairs separated by a 14–16-residue linker
(9–11). The MBNL proteins were first characterized as
factors involved in the pathogenesis of the neuromuscular
disease myotonic dystrophy (DM) (9). DM is caused
by the expansion of structurally similar microsatellites in
two functionally unrelated genes. Type 1 (DM1) disease is
associated with (CTG)
n
expansions in the 30-UTR of the
DMPK gene while (CCTG)
n
expansions in the first
*To whom correspondence should be addressed. Tel: +1 352 273 8076; Fax: +1 352 273 8284; Email: mswanson@ufl.edu
ß2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
intron of ZNF9 results in type 2 (DM2) disease (12).
The RNA-mediated pathogenesis model for DM suggests
that these expansions are toxic at the RNA level because
DM1 and DM2 mutant RNAs fold into stable RNA
hairpins which bind MBNL proteins with high affinity.
MBNL proteins are pre-mRNA alternative splicing
factors and loss of MBNL1 activity in DM tissues leads
to persistence of fetal splicing patterns in the adult.
In support of this model, MBNL1 binds to (CUG)
n
and
(CCUG)
n
expansion RNAs (CUG
exp
, CCUG
exp
) and
co-localizes with nuclear RNA (ribonuclear) foci in
DM1 and DM2 skeletal muscle and brain, with a
concomitant reduction in the nucleoplasmic level (13,14).
Moreover, DM1 ribonuclear foci are lost following
siRNA-induced downregulation of MBNL1 expression
(15). Finally, Mbnl1 knockout mice recapitulate several
clinical defects characteristic of DM, including myotonia
and subcapsular particulate cataracts (16).
These observations indicate that a critical initiating
event in the DM pathogenesis pathway is loss of MBNL
function leading to defective pre-mRNA splicing.
Apparently inconsistent with this view is a recent finding
that several of the cardinal features of DM, including
skeletal muscle myotonia and cardiac conduction defects,
are recapitulated in a transgenic mouse model which
overexpresses an inducible GFP-DMPK 30-UTR trans-
gene that contains only five CTG repeats (14).
Surprisingly, these mice do not develop ribonuclear foci
and the subcellular localization pattern of Mbnl1 is not
altered. This study raises the question of whether foci
formation is required for loss of MBNL1 function and
highlights the need to further investigate the interactions
of muscleblind-like proteins with both normal target and
pathogenic RNAs.
Here, we report that the MBNL1 protein preferentially
recognizes GC-rich RNA helices containing a pyrimidine
mismatch on both normal splicing substrates and patho-
genic RNAs. Furthermore, MBNL1 binds selectively to
the stem region of CUG
exp
RNA and can be visualized as
a ring-like structure in the electron microscope. This study
introduces the possibility that the tandem arrangement of
MBNL high affinity binding sites present on CUG
exp
RNA results in a stacked ring complex which effectively
traps MBNL1 and inhibits its role as an alternative
splicing factor during postnatal development.
MATERIALS AND METHODS
Plasmids
To construct pGEX-6P-His, a XhoI–NotI fragment
encoding the His
6
tag from pGEX-MBNL1 was inserted
into XhoI–NotI digested pGEX-6P-1. pGEX-6P-
MBNL1-His was constructed by inserting a BamHI–
XhoI fragment from pGEX-MBNL1 into BamHI–XhoI
digested pGEX-6P-His. The MBNL1 N-terminal region
(residues 1–253) was amplified using MSS2759 and
MSS2760, digested with BamHI and XhoI and inserted
into pGEX-6P-His to create pGEX-6P-MBNL1-N-His.
For the yeast two-hybrid system, either full length,
amino terminal (residues 1–264) or carboxyl terminal
(residues 239–382) MBNL1 cDNAs were PCR amplified
using the following primers and inserted into pGBKT7
or pGADT7 (Clontech, Mountain View, CA, USA) at
SmaI and BamHI sites: (i) full length, MSS1163 (forward
primer) and MSS1166 (reverse primer); (ii) N-terminal
region, MSS1164 (forward) and MSS1166 (reverse);
(iii) C-terminal region, MSS1163 (forward) and
MSS1165 (reverse). To create pcDNA3-V5, MSS3045
and MSS3046 were subjected to a 10-cycle PCR reaction:
948C30s,508C20s,728C 20 s. The resulting DNA
fragment was gel purified followed by digestion with NheI
and BamHI, and inserted into NheI–BamHI digested
pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA).
The BamHI–XhoI fragment from pGEX-6P-MBNL1-
His was inserted into pcDNA3-V5 at BamHI and XhoI
sites to create pcDNA-V5-MBNL1. The BamHI–XhoI
fragments from pGEX-6P-MBNL1-His and pGEX-
6P-MBNL1-N-His were inserted into pcDNA3.1(+)/
myc-His A (Invitrogen) to create pcDNA3-MBNL1-
mycHis and pcDNA3-MBNL1-N-mycHis, respectively.
The CUGBP1-coding sequence was PCR amplified using
MSS2699 and MSS2700 and inserted into pcDNA3.1(+)/
myc-His A at BamHI and XhoI sites. The Tnnt3 minigene
was prepared by amplifying the mouse Tnnt3 genomic
region between exons 8 and 9 using MSS1949 and
MSS1950 and inserting the PCR product into
pSG5(Stratagene, La Jolla, CA, USA) at the EcoRI site.
The mutant Tnnt3 minigenes, pSG5-Tnnt310 and pSG-
Tnnt3/gg and pSG-Tnnt3/au, were generated by site-
directed mutatgenesis using MSS2129/MSS2130,
MSS3131/3132 and MSS3163/MSS3164, respectively.
Wild-type pSG5-Tnnt3 (100 ng) (with 125 ng of each of
the primers) was subjected to the following PCR reaction:
948C30s,508C 1 min, 728C 8 min, 20 cycles using Pfu
DNA polymerase (Stratagene). After DpnI digestion,
the PCR product was transformed into DH10B and
mutants were identified by plasmid DNA sequencing.
Using pSG5-Tnnt3 as a template, PCR fragments
generated from primer pairs MSS1865/MSS1879 and
MSS1884/MSS1866 were TOPO-cloned into pCR4-
TOPO (Invitrogen) to make pTOPO-T5.1 and pTOPO-
T5.45, respectively.
Recombinant protein preparation
For the preparation of recombinant proteins, BL21(DE3)
RP containing pGEX-6P-1-MBNL1 or pGEX-6P-1-
MBNL1-N were grown to OD
600
= 0.5 followed
by induction with 1 mM IPTG for 2 h at 308C. Cells
were collected and resuspended in lysis buffer containing
25 mM Tris–Cl, pH 8.0, 0.5 M NaCl, 10 mM imidazole,
2mM b-mercaptoethanol, 2 mg/ml lysozyme, 10 mg/ml
DNase I, 5% glycerol, 0.1% Triton X-100 supplemented
with protease inhibitors. The cell suspension was incu-
bated on ice for 30 min with stirring prior to sonication
and centrifugation at 12 000g. For protein purifi-
cation, Ni-NTA-Sepharose (Amersham/GE Healthcare,
Piscataway, NJ, USA) (12 ml) was incubated with the
supernatant for 1 h at 48C and washed three times with
40 ml of wash buffer containing 25 mM Tris–Cl, pH 8.0,
0.5 M NaCl, 20 mM imidazole, 0.1% Triton X-100,
Nucleic Acids Research, 2007, Vol. 35, No. 16 5475
followed by three 10 ml elutions in 25 mM Tris–Cl, pH 8.0,
0.5 M NaCl, 250 mM imidazole, 0.1% Triton X-100.
Subsequently, b-mercaptoethanol was added (10 mM
final concentration) to the eluate, which was incubated
with 2 ml glutathione-Sepharose (Amersham) for 1 h at
48C. After three washes (10 ml each) of buffer (WB)
containing 25 mM Tris–Cl, pH 8.0, 300 mM NaCl, 5 mM
b-mercaptoethanol, 0.1% Triton X-100, the glutathione-
Sepharose beads were incubated with 4 ml WB containing
40 U of PreScission protease (Amersham) at 48C over-
night. The supernatant was collected following brief
centrifugation and concentrated to 1–8 mg/ml.
Chemical and enzymatic analysis of RNA structures
Transcription reactions were carried out in a 50 ml volume
which contained 2 mg of each DNA template, 1 mM
rNTPs, 3.3 mM guanosine, 60 U of ribonuclease inhibitor
RNase Out (Invitrogen), 200 U of T7 RNA polymerase
(Ambion, Austin, TX, USA), 10 mM DTT, 40 mM Tris-
HCl (pH 7.9), 6 mM MgCl
2
, 2 mM spermidine, 10 mM
NaCl. The reaction was performed at 378C for 2 h, the
transcript was then purified on a denaturing 10%
polyacrylamide gel and subsequently 50-end-labeled with
T4 polynucleotide kinase and [g
32
P]ATP (3000 Ci/mmol).
The labeled RNA was re-purified by electrophoresis on a
denaturing 10% polyacrylamide gel. Prior to structure
probing, the labeled RNA was subjected to a denatura-
tion/renaturation procedure in a reaction buffer contain-
ing 50 mM Tris–HCl (pH 8.0), 60 mM KCl, 15 mM NaCl,
2 mM MgCl
2
by heating the sample at 908C for 1 min and
slowly cooling to 258C. The RNA sample was then mixed
with either a 25-fold molar excess of recombinant MBNL1
in 50 mM Tris–HCl, pH 8.0, 60 mM KCl, 15 mM NaCl,
2 mM MgCl
2
, 2% glycerol, 0.5 mM DTT, 50 mg/ml BSA,
or with buffer only (control) and incubated 20 min at
258C. The final concentration of (CUG)
54
was 20 nM and
MBNL1 was 500 nM. Under these reaction conditions
>95% of RNA was bound with protein as revealed by
filter-binding assays. Additional control samples were
prepared by mixing RNA with MBNL1 protein previously
denatured by heating at 758C for 2 min. Limited RNA
digestion was initiated by mixing 5 ml of the RNA or
RNA/protein sample (25 000 c.p.m.) with 5 ml of a probe
solution containing either lead ions or ribonuclease T1 in
reaction buffer. The reactions were performed at 258C for
20 min and stopped by adding 20 volumes of 1TE buffer
followed by phenol/chloroform extraction. Precipitated
RNAs were dissolved in a denaturation solution (7.5 M
urea and 20 mM EDTA with dyes). To determine the
cleavage sites, the products of RNA fragmentation were
separated on 10% polyacrylamide gels containing 7.5 M
urea, 90 mM Tris-borate buffer and 2 mM EDTA, along
with the products of alkaline hydrolysis and limited T1
nuclease digestion of the same RNA. The alkaline
hydrolysis ladder was generated by the incubation of the
labeled RNA in formamide containing 0.5 mM MgCl
2
at
1008C for 10 min. The partial T1 ribonuclease digestion of
RNAs was performed under semi-denaturing conditions
(10 mM sodium citrate, pH 5.0; 3.5 M urea) with 0.2 U/ml
of the enzyme during incubation at 558C for 10 min.
Electrophoresis was performed at 1800 V (gel dimensions,
30/50 cm). The products of the structure probing reactions
were visualized by PhosphorImaging (Storm; Molecular
Dynamics, Sunnyvale, CA, USA) and analyzed by
ImageQuant 5.2 (Molecular Dynamics).
Protein–protein interactions
For yeast two-hybrid analysis, pGBKT7-MBNL1
together with one of the pGADT7 constructs (pGADT7-
T Antigen, pGADT7-MBNL1, pGADT7-MBNL11-N,
pGADT7-MBNL1-C) were transformed into yeast strain
AH109. In addition, pGBKT7-p53 and pGADT7-T
Antigen (Clontech) were co-transformed into AH109 as
a negative control. Double transformants were selected on
SD/-Trp/-Leu plates. Expression of the myc-tagged GAL4
DNA-binding domain (BD) and HA-tagged GAL4
activation domain (AD) fusion proteins were confirmed
by immunoblot analysis using mAb 9E10 and 16B12,
respectively. To test for protein interactions, transfor-
mants were streaked onto SD/-Trp/-Leu/-His incubated at
308C for 3–4 days and scored for growth.
To test for MBNL1–MBNL1 interactions in mamma-
lian cells, HEK293T cells were transfected with 5 mg
of pcDNA3-V5-MBNL1 alone as a control or 5 mgof
pcDNA3-V5-MBNL1 together with either 5 mgof
pcDNA3-MBNL1mycHis or pcDNA3-MBNL1-N-
mycHis. Cells were harvested 20–24 h post-transfection
by trypsinization followed by neutralization in media
contain 10% fetal bovine serum and two washes in 50 mM
Tris–HCl, pH 7.4, 150 mM NaCl. Cell pellets were
resuspended in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl,
0.1% IGEPAL with protease inhibitors, and sonicated on
ice (3 5 s). Cell debris was removed by centrifugation at
16 100gfor 10 min at 48C. Cleared lysates were treated
with 200 mg/ml RNase A for 20 min on ice (17) followed by
another 10 min centrifugation. Cleared lysates were mixed
with Dynabeads coupled to Protein A (Invitrogen)
precoated with rabbit anti-V5 polyclonal antibody
(Novus, Littleton, CO, USA) and incubated at 48C for
2 h. Dynabeads were washed three times with IPP150
buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1%
IGEPAL) and once with 50 mM Tris–HCl, pH 7.4,
150 mM NaCl. Proteins were dissociated from the beads
by heating at 958C for 2 min in 1SDS–PAGE sample
buffer. Proteins (50% of the immunoprecipitated proteins,
2.5% of the input) were separated on 12.5% SDS–PAGE
gels. Immunoblotting was performed using mAb 9E10
(1:1000) or mAb anti-V5 (1:1000, AbD Serotec).
Electron microscopy
The CUG
136
RNA, transcribed from pBC-CTG136
plasmid (R. Osborne, University of Rochester), was
incubated with MBNL1 protein at molar ratio of 1:2.5
or 1:10 in buffer containing 16 mM HEPES, 2 mM
magnesium acetate, 0.16 mM EDTA, 0.4 mM DTT,
1 mM ATP, 50 mM potassium acetate and 16% glycerol
for 30 min at 308C. The resulting complexes were fixed in
0.6% glutaraldehyde for 5 min at room temperature and
subsequently passed over a 2 ml column containing Bio-
Gel A-5M (Bio-Rad, Hercules, CA, USA) equilibrated
5476 Nucleic Acids Research, 2007, Vol. 35, No. 16
with 0.01 M Tris (pH 7.6) and 0.1 mM EDTA to remove
free protein and fixatives. Protein–RNA enriched frac-
tions were incubated with 2.5 mM spermidine and
adsorbed on to glow charged carbon coated copper grids
and dehydrated in a series of ethanol washes of 25, 50,
75 and 100% ethanol each for 5 min at room temperature.
Samples were air dried prior to rotary shadow casting with
tungsten. Protein–RNA complexes were visualized using a
FEI Tecnai 12 electron microscope (FEI, Hillsboro, OR)
at an accelerating voltage of 40 kV and images were
captured using a 4K 4K Gatan CCD camera using plate
film or Gatan digital image capturing software (Gatan,
Pleasanton, CA, USA). Plate film negatives were scanned
using an Imacon scanner and supporting software
(Imacon, Redmond, WA). Images were photographed at
a magnification of 52K.
Photocrosslinking
To prepare whole cell lysates for cross-linking, HEK293T
cells were grown in 10 cm plates and transfected with 10 mg
of pcDNA3-CUGBP1mycHis, pcDNA3-MBNL1mycHis
or pcDNA3-MBNL1-NmycHis. Cells were trypsinized
and then neutralized in media containing 10% FBS
followed by two additional PBS washes. Cells were then
resuspended in 250 ml of 20 mM HEPES–KOH (pH 8.0),
100 mM KCl, 0.1% IGEPAL and protease inhibitors,
sonicated and lysates centrifuged (13 200 r.p.m., 10 min,
48C). Glycerol was added to the supernatants to a final
concentration of 20%.
RNAs for photocrosslinking were uniformly labeled
with 40 mCi each of (a-
32
P)-GTP and (a-
32
P)-UTP
(800 Ci/mmol) in the presence of 0.5 mM ATP and CTP,
0.02 mM GTP and UTP. Cross-linking was performed by
incubating 0.1 pmol RNA with 15 ml of HEK293T whole
cell lysate in 25 ml reactions containing 16 mM HEPES–
KOH (pH 8.0), 65 mM potassium glutamate, 2 mM
Mg(OAC)
2
, 0.4 mM DTT, 0.16 mM EDTA, 20 mM
creatine phosphate, 2 mM ATP and 16% glycerol (final
concentration). Reactions were incubated at 308C for
15 min, transferred to pre-chilled PCR caps on ice and
photocrosslinked in Stratalinker (Stratagene, La Jolla,
CA, USA) for 2.5 min (three times) with a 3 min interval
between each irradiation. Samples were digested with 5 mg
of RNase A for 20 min at 378C and immunopurified using
the anti-myc monoclonal antibody 9E10 pre-coated
protein A Sepharose (Amersham). Purified proteins were
fractionated on 12.5% SDS–PAGE gels followed by
autoradiography.
Filter-binding assays
Uniformly labeled RNA was prepared as described
previously (9). Calibration of the non-specific retention
rate of the nitrocellulose filter was performed by incubat-
ing 0.01–0.1 nM RNA at 308C for 30 min in BB [50 mM
Tris-HCl (pH 8.0), 40 mM KCl, 20 mM potassium
glutamate, 15 mM NaCl, 0.5 mM DTT, 0.05 U/ul
RNasin (Promega)] followed by filtration through a Bio-
Dot (BioRad) apparatus containing a sandwich of
nitrocellulose (BioRad) and Hybond-N plus (Amersham)
membranes followed by a single wash step with the
same buffer. The membranes were UV-cross-linked, air
dried and exposed to a phosphorimager screen. Non-
specific retention on the nitrocellulose membrane was
undetectable. Binding reactions were set up in the same
buffer with 5 pM RNA and 3.13 10
12
Mto
1.02 10
7
M of MBNL1/41-His, and incubated at 308C
for 30 min. Each reaction was applied to the Bio-Dot
apparatus followed by one wash with binding buffer.
Membranes were processed as described above and signals
quantified using ImageQuant TL (Amersham). Standard
deviations were calculated based on three independent
experiments and apparent dissociation constants were
calculated using a one-site binding model and GraphPad
Prism (v3.00) software.
Gel shift assays
Gel shift assays were performed using modifications of
previously described protocols (18,19). RNA was uni-
formly labeled with 40 mCi (a-
32
P)-GTP or UTP (800 Ci/
mmol) in the presence of 0.5 mM ATP, 0.5 mM CTP,
0.02 mM GTP, 0.02 mM UTP and purified using a 5%
denaturing gel containing 8 M urea. Prior to use, purified
RNA was heated at 658C for 5 min in 50 mM Tris–HCl
(pH 8.0), 40 mM KCl, 20 mM potassium glutamate,
15 mM NaCl, 0.5 mM DTT, 0.5 U/ml SUPER-asin
(Ambion) following by renaturation at RT. Reactions
(20 ml) were assembled with 0.1 nM RNA and 0–256 nM
protein in 50 mM Tris–HCl, pH 8.0, 40 mM KCl, 20 mM
KGlutamate, 15 mM NaCl, 15% glycerol, 0.5 mM DTT,
20 mg/ml acetylated BSA. Alternatively, RNAs were
heated using a higher salt concentration in the presence
of magnesium (50 mM Tris–HCl (pH 8.0), 60 mM KCl,
120 mM potassium glutamate, 20 mM NaCl, 2 mM
magnesium acetate, 0.5 mM DTT, 0.5 U/ml SUPER-asin
(Ambion) and then reactions were performed in 50 mM
Tris-HCl (pH 8.0), 60 mM KCl, 120 mM potassium
glutamate, 20 mM NaCl, 2 mM magnesium acetate,
0.5 mM DTT, 20 mg/ml acetylated BSA. After incubation
at 308C for 30 min, reactions were immediately loaded
onto a 4% polyacrylamide gel (80:1) containing 0.5 mM
DTT and 5% glycerol which had been pre-run at 150 V
for 1–2 h at 48C. Gels were run in 0.5TBE (pH 8.3) at
200 V for 2 h, fixed and dried prior to autoradiography.
RESULTS
MBNL1 interacts with an intronic stem-loop structure
upstream of the Tnnt3 fetal exon
A recent mapping study identified several MBNL1-
binding sites containing a core element within human
cardiac troponin T (cTNT/TNNT2) intron 4 pre-mRNA
immediately upstream of developmentally regulated exon
5 (8). Although the major MBNL1-binding sites in
chicken cTNT are positioned downstream of exon 5,
alignment of the human- and chicken-binding sites
revealed a hexanucleotide consensus motif (50-YGCUU/
GY-30). Neither the human- or chicken-binding sites were
located in regions predicted to form secondary structures.
Together with prior observations that MBNL proteins
are sequestered by CUG
exp
and CCUG
exp
hairpins in
Nucleic Acids Research, 2007, Vol. 35, No. 16 5477
ribonuclear foci in DM cells, this finding suggests that the
MBNL proteins bind to both ssRNA and dsRNA
structural motifs.
To determine if MBNL1 recognizes primarily ssRNA
targets in other splicing precursors, we first mapped the
MBNL1-binding site on fast skeletal muscle troponin T
(Tnnt3) RNAs. This pre-mRNA was selected because
earlier studies demonstrated that Tnnt3 fetal (F) exon
splicing is particularly sensitive to MBNL1 levels in vitro
and in vivo (16,20). Mapping was performed using a
photocrosslinking protocol in which 293T cells were trans-
fected with protein expression plasmids encoding myc-
tagged versions of either CUGBP1, full-length MBNL1
(MBNL1 FL) or the MBNL1 N-terminal region
(MBNL1 N) which contains the four CCCH motifs respon-
sible for RNA binding (21). Following transfection,
cell lysates were incubated with radiolabeled Tnnt3
RNAs encompassing exons 7–9 or different 500–645
nucleotide (nt) subregions designated T1-6 (Figure 1A),
photocrosslinked with UV-light and digested with RNase
A. Protein–RNA complexes were then immunopurified
with the anti-myc monoclonal antibody (mAb) 9E10 and
resolved by SDS–PAGE. Because these studies indicated
that only Tnnt3 T5 RNA (500 nt) cross-linked to both
MBNL1 FL and MBNL1 N proteins, this region was
further subdivided into T5.1–T5.45. Interestingly, only
T5.45 RNA (200 nt) cross-linked to CUGBP1 and
MBNL1 proteins while T5.1 (125 nt), and the other
subregions (T5.2 and T5.3, data not shown), did not
(Figure 1B). In agreement with prior studies, CUGBP1
failed to cross-link to a CUG
exp
, (CUG)
54
, while both
MBNL1 FL and MBNL1 N did (9).
To further delineate MBNL1-binding sites on Tnnt3,
we analyzed MBNL1 cross-linking to a several subregions
of T5.45. One region was particularly interesting because
it contains a 50-CGCU-30motif which is conserved in the
MBNL1-binding site in human cTNT/TNNT2 (8).
Interestingly, this motif is located within a predicted
18-nt stem-loop structure (Figure 2A). We first confirmed
the existence of this Tnnt3 18-nt hairpin structure using
chemical and enzymatic structure probing (Figure 2A).
Cross-linking assays were then used to test whether wild-
type and mutant Tnnt3/98 RNA (a 98-nt subregion
encompassing 83-nt of the 30end of intron 8 and 15-nt
of the F exon) was recognized by both CUGBP1 and
MBNL1 (Figure 2B). Concurrently, wild-type or mutant
Tnnt3 minigenes were transfected into C2C12 cells to
assay whether Tnnt3 splicing remained responsive to
MBNL1 overexpression (Figure 2C). These assays were
performed using C2C12 myoblasts since they showed a
higher default level of Tnnt3 F exon skipping compared to
293T cells (Figure 2C, left panel, lane 1 and data not
shown). Cross-linking assays confirmed that wild-type
Tnnt3/98 (Figure 2B, left panel) RNA was recognized by
both MBNL1 and CUGBP1. Correspondingly, the spli-
cing pattern of the wild-type Tnnt3 minigene was not
altered upon CUGBP1 overexpression (Figure 2C, left
panel, compare lanes 1 and 2) while F exon exclusion was
enhanced by MBNL1-mycHis (Fig. 2C, left panel, lane 3),
consistent with our previous study (20).
Mutations in the 18-nt hairpin positioned just upstream
of F exon (Figure 2A) were generated to test whether it
contains an MBNL1-binding site. The relative position of
this hairpin in the F exon 30splice site region is significant
since a potential binding site for the essential splicing
factor U2AF, which binds preferentially to U-rich tracts,
lies just upstream of this stem-loop structure and this GC-
rich hairpin contains a pyrimidine mismatch reminiscent
of the structure of CUG
exp
and CCUG
exp
RNA hairpins.
We first generated a 10-nt deletion within the 18-nt region
to eliminate this hairpin (Figure 2A, deleted nucleotides in
gray). As predicted, this mutant showed an impairment of
both MBNL1 cross-linking (Figure 2B, 10 mutant) and
F exon skipping promoted by MBNL1 overexpression
(Figure 2C, 10 mutant). Indeed, the 10 deletion
eliminated F exon skipping in cells transfected with the
Tnnt3 minigene alone or with CUGBP1-mycHis together
with the minigene (Figure 2C, 10 mutant, lanes 1–2)
B
control
CUGBP1
MBNL1 FL
MBNL1 N
37
25
50
T5.45 T5.1 (CUG)54
A
T1 T2 T3 T4 T5 T6
T5.3
T5.1 T5.2
T5.45
F
Tnnt3
E7 E8 F E 9
crosslink
protein 37
25
50
control
CUGBP1
MBNL1 FL
MBNL1 N
control
CUGBP1
MBNL1 FL
MBNL1 N
full-length (FL, 1-382)
N-terminus (N, 1-253)
MBNL1
Figure 1. MBNL1 cross-links to both splicing precursor and patho-
genic RNAs. (A) Mapping of MBNL1-binding sites on Tnnt3.
Illustration shows the Tnnt3 genomic region between exons 7 (E7)
and 9 (E9) (thin lines, introns; black boxes, exons) and the subregions
corresponding to the RNAs tested for MBNL1 cross-linking (thick
lines, T1–6; thin lines, T5.1–T5.3; green line, T5.45). (B) Interaction
sites for MBNL1 and CUGBP1 on Tnnt3 pre-mRNA are positioned in
a region which includes the F exon and upstream intron 8. HEK293T
cells were transfected with myc-His tagged CUGBP1, MBNL1 FL or
MBNL1 N followed by incubation of the corresponding cell lysates
with
32
P-labeled Tnnt3 T5.45, T5.1 or (CUG)
54
RNAs and subsequent
UV-light induced cross-linking and RNase A treatment. Labeled
proteins were detected by SDS–PAGE and autoradiography (upper
panels, cross-link) while protein levels in the lysates are shown
by immunoblotting with the anti-myc mAb 9E10 (lower panel, protein).
The primary structures of MBNL1 FL and MBNL1 N are illustrated
below the immunoblot with the four CCCH motifs highlighted
(shaded boxes).
5478 Nucleic Acids Research, 2007, Vol. 35, No. 16
suggesting loss of MBNL1 binding and enhanced spliceo-
some recruitment to the F exon region.
The cross-linking and splicing results obtained with the
10 mutant indicated that the 18-nt hairpin was a binding
site for MBNL1 and that MBNL1–hairpin interactions
might promote F exon skipping. Because both sequence
and structural elements could contribute to efficient
MBNL1 binding, a double C!G and U!G mutant
was generated that eliminated the C-C mismatch in
the 18-nt hairpin, which also increases the stability
Ci Pb S1 T1 F T
g
g
c
g
u
g
c
c
u
gc
c
u
c
u
g
c
u
g
u
u
c
c
a
u
g
u
g
g
c
c
a
c
u
g
c
u
g
c
ug
u
g
u
g
g
c
c
u
c
u
gcac
u
u
c
u
g
c
a
g
c
u
g
c
g
u
g
g
c
u
u
u
u
u
ucuugc
a
u
g
u
g
c
g
c
u
ugu
gcccacaccau
g
a
a
g
C
A
U
G
C
U
G
U
C
G
C
C
G
A
G
GAGGAGCGGGAGGAGGAG
AGGA AG
A
A
5′
3′
10
20
30
40
50
60
80
100
110
120
130
140
F exon
18-nt stem-loop
18-nt stem-loop
G52
G38
G60
G70
G94
G107
G26
G125
S1 nuclease
T1 RNase
70
lead ions
G82
g
g
C
B
F
Tnnt3 9F8
F
+
−F
mock
CUGBP1
MBNL1
mock
CUGBP1
MBNL1
wild type ∆10 mutant
wild type
mock
CUGBP1
MBNL1
mock
CUGBP1
MBNL1
∆10 mutant
mock
CUGBP1
100
150
MBNL1
gg mutant
mock
CUGBP1
MBNL1
50
gg mutant
A
a
u
au mutant
mock
CUGBP1
MBNL1
au mutant
mock
CUGBP1
MBNL1
Figure 2. MBNL1 recognizes a RNA hairpin upstream of the Tnnt3 fetal exon. (A) Cleavage pattern (left) of the 50-end labeled Tnnt3 151-nt
transcript (a 50truncated form of the 200 nt T5.45 RNA) encompassing the fetal (F) exon 30splice site (110-nt of intron 8, 41-nt of F exon) obtained
with use of three structure probes. Lanes are: Ci, incubation control or no probe added; Pb, lead ions (0.25, 0.5mM); S1, S1 nuclease (1, 2 U/ml and
1 mM ZnCl
2
was present in each reaction); T1, RNase T1 (0.5, 1 U/ml); F, formamide (statistical ladder); T, guanine-specific ladder. The sequences
forming the 18-nt stem-loop structure are also indicated. Also illustrated (right) is the proposed secondary structure model of the 151-nt transcript.
The cleavage sites are indicated for each probe used and the figure inset shows the probe designations and cleavage intensity classification. The F
exon sequence is marked in upper case and intron 8 in lower case. The positions of the G, A and U substitutions in the 18-nt stem-loop are also
indicated. (B) Photocrosslinking analysis indicates reduced MBNL1, but not CUGBP1, binding to the Tnnt3 10, gg and au mutants in contrast to
wild-type RNA. Photocrosslinking analysis was performed as described in Figure 1 using the same lysates (protein loading controls shown in
Figure 1B) except only MBNL1 FL (MBNL1) protein was used. (C) Tnnt3 F exon skipping is impaired in the 10 and au mutants compared to wild
type while F exon inclusion is eliminated in the gg double mutant. C2C12 cells were co-transfected with either a wild type, 10, gg or au point
mutant splicing reporter plasmid and a protein expression plasmid for either CUGBP1mycHis or MBNL1mycHis (full-length protein only).
32
P-labeled splicing products, which included (+F) or excluded (F) the Tnnt3 F exon (black box), were detected by RT-PCR, using primers
positioned in Tnnt3 exons 8 and 9 (open boxes with arrows), followed by gel electrophoresis.
Nucleic Acids Research, 2007, Vol. 35, No. 16 5479
of this stem-loop, and furthermore substituted a G for a U
in the loop. These mutations are not predicted to alter the
overall folding pattern of Tnnt3/98. This double point (gg)
mutant showed considerably reduced MBNL1 cross-
linking compared to wild-type Tnnt3 (Figure 2B) con-
firming that this region was an MBNL1-binding site.
Interestingly, a similar decrease in MBNL1 cross-linking
activity was also observed for the single C!G stem
substitution mutant (data not shown). While loss of
MBNL1 binding should promote F exon splicing, inclu-
sion activity was completely eliminated (Figure 2C, gg
mutant). Loss of F exon splicing activity could result from
enhanced hairpin stability, or an increase in the purine
content of the polypyrimidine (Py) region upstream of the
F exon 30splice site, and subsequent impairment of
spliceosome assembly. Interestingly, another double
mutant (G!A and C!U), which preserved the predicted
18-nt stem-loop structure while reducing the GC content
in the stem, also showed reduced MBNL1 cross-linking
compared to wild type (Figure 2B, au mutant) while
MBNL1-induced F exon skipping activity was reduced
similar to the 10 mutant (Figure 2C, au mutant).
Overall, these studies indicated that MBNL1 prefers to
bind to a GC-rich stem-loop containing a pyrimidine
mismatch in a normal splicing target.
Similar affinities of MBNL1 for splicing precursor and
pathogenic RNAs
According to the RNA sequestration model, pathogenic
RNAs outcompete normal RNA-binding targets for
MBNL1 leading to loss of MBNL-mediated regulation
of alternative splicing during postnatal development.
However, it is not clear why MBNL1 accumulates on
DM1 and DM2 expansion RNAs in ribonuclear foci. The
most straightforward explanation is that MBNL1 has a
higher affinity for DM pathogenic RNAs compared to its
physiological RNA splicing targets. Since the cross-linking
of MBNL1 to the Tnnt3 F exon 30splice site region and
CUG
exp
RNAs appeared to be comparable (Figure 1B),
we determined the relative affinities of MBNL1 for
(CUG)
54
, (CAG)
54
and Tnnt3/5.45 using recombinant
MBNL1 protein in filter binding and gel shift assays.
The MBNL1 proteins used for this study were either
MBNL1 FL or the C-terminal truncation mutant
MBNL1 N. For the MBNL1 FL preparation, 60% of
the purified protein was full length while the MBNL1 N
protein preparation was homogeneous as determined by
Coomassie blue staining and immunoblot analysis
(Figure 3A). Because MBNL1 shows a temperature-
dependent binding profile in cell extracts in the absence
of ATP (data not shown), all recombinant protein-binding
studies were performed at 308C to maximize binding while
minimizing RNA degradation. As anticipated, MBNL1
FL showed a high affinity in the filter-binding assay for
(CUG)
54
(K
d
= 5.3 0.6 nM), but it also showed high
affinities for Tnnt3 5.45 (Tnnt5.45) (K
d
= 6.6 0.5 nM)
and (CAG)
54
(K
d
= 11.2 1.5 nM) (Figure 3B). In con-
trast, Tnnt3/T5.1 RNA, which did not cross-link to
MBNL1 (Figure 1B), bound poorly. The similarities in
the binding affinities of MBNL1 for (CUG)
54
and
(CAG)
54
accounts for the prior observation that over-
expression of either of these repeat RNAs in COS-M6 cells
results in the formation of nuclear foci that colocalize with
GFP-MBNL1 (22). Relatively weak cooperativity was
noted for interactions between MBNL1 and (CUG)
54
,
(CAG)
54
and Tnnt3 T5.45 RNAs (Hill coefficients of
1.62 0.21, 1.67 0.27 and 1.40 0.14, respectively).
Comparable affinities were obtained when MBNL1–
RNA complexes were analyzed by gel shift analysis
(Figure 3C). Incubation of full-length MBNL1 (MBNL1
FL) with Tnnt3/T5.45 generated several protein–RNA
complexes resolved by the polyacrylamide gel whereas
significant binding to Tnnt3/T5.1 was only detectable at
256 nM and only gel excluded complexes were observed.
Similar complexes were also formed with MBNL1 N
although the truncated protein had a higher affinity for
Tnnt3/T5.1 RNA and formed fewer gel-resolved com-
plexes with Tnnt3/T5.45 and (CUG)
54
(Figure 3C).
Incubation of MBNL1 with (CUG)
54
also resulted in the
formation of several major complexes at protein concen-
trations (4–16 nM) near the K
d
determined by filter
binding. At higher MBNL1 FL concentrations (564 nM)
the majority of the resulting MBNL1–(CUG)
54
com-
plexes migrated at, or near, the top of the gel. Similar gel
shift profiles were obtained when RNA–protein com-
plexes were formed in a higher salt buffer containing
magnesium (see Methods section) although all K
d
values
increased 4-fold. The striking similarity in the binding
affinities of MBNL1 for pathogenic and splicing pre-
cursor RNAs prompted us to re-examine the interaction
of this splicing factor with CUG
exp
RNA.
MBNL1 binds to the stem region of a pathogenic dsRNA
Muscleblind-like proteins were originally characterized as
nuclear factors which are recruited by CUG
exp
RNAs (9).
The predicted double-stranded nature of these pathogenic
RNAs has been validated by chemical and nuclease
mapping, thermal denaturation and electron microscopy
(23–26). While the binding of MBNL proteins to CUG
exp
RNA is proportional to the predicted stem length, there is
currently no direct experimental evidence that MBNL
binds directly to the CUG
exp
stem or that the RNA
structure remains in the hairpin configuration following
MBNL binding. Therefore, we initially used chemical and
enzymatic structure probing of labeled RNAs to identify
MBNL1-binding sites on (CUG)
54
RNA. RNAs were
50end-labeled, subjected to either lead ion (Pb)-induced
hydrolysis or RNase T1 digestion in the absence or
presence of recombinant MBNL1, and the products were
fractionated on denaturing polyacrylamide gels. As shown
previously, lead ions cleave both ssRNA and relaxed
dsRNA structures which yielded a uniform ladder that
increased in intensity with increasing lead concentration
(Figure 4, left panel). As anticipated, addition of MBNL1
inhibited strand cleavage in a concentration-dependent
manner and densitometry analysis failed to show sig-
nificant regional differences in the cleavage pattern by
MBNL1 suggesting uniform binding of this protein
throughout the stem region. In contrast to lead, RNase
T1 prefers to cleave after G nucleotides in single-stranded
5480 Nucleic Acids Research, 2007, Vol. 35, No. 16
regions. Thus, incubation with RNase T1 resulted in
strong cleavage at the terminal loop (Figure 4, right panel,
G26–G29). Interestingly, terminal loop cleavage was
unaffected by MBNL1 addition while stem cleavage was
uniformly inhibited. We conclude that MBNL1 interacts
primarily with the stem region of CUG
exp
RNAs.
Visualization of MBNL1–CUG
exp
complexes
In a previous study, we reported that the MBNL splicing
antagonist, CUGBP1, binds to out-of-register ssCUG
repeats at the base of CUG
exp
RNA hairpins but not the
A-form helical region while the dsRBD protein TRBP
associates with the stem region (25). To confirm that
MBNL1 is a stem-binding protein, electron microscopy
(EM) was performed using the full-length MBNL1
protein. Purified (CUG)
136
was examined following
direct absorption to thin carbon foils, dehydration and
tungsten shadowing. In contrast to ssRNA, (CUG)
136
RNA formed rod-like segments as described previously
for (CUG)
130
(Figure 5A–C). To examine the structures of
MBNL1–RNA complexes, MBNL1 was incubated with
RNAs at two different RNA:protein molar ratios (1:2.5 or
1:10) and subsequently prepared for EM. In the presence
of RNA, purified MBNL1 formed a ring-shaped structure
with a prominent central cavity and for (CUG)
136
–
MBNL1 complexes incubated at a ratio of 1:2.5, 70%
of the RNAs were bound by one of these MBNL1 rings
(Figure 5D–F). At higher protein levels (1:10), free
(CUG)
136
RNA was rarely detectable (6.4% of the
RNAs in the field) while >90% of the RNAs were
bound by two or more MBNL1 rings (Figure 5G and H).
Also shown is a representative field of dsCUG RNAs and
MBNL1–(CUG)
136
complexes (Figure 5I, RNA:protein is
1:2.5). Although the majority of MBNL1 rings were
associated with RNA under our RNA assembly condi-
tions, a few free rings were visualized in the background in
the absence of associated (CUG)
136
helices suggesting that
MBNL1 may form a ring structure independent of RNA.
We failed to visualize rings by negative staining suggesting
that this structure is disrupted by the acidic conditions of
the negative staining protocol. Interestingly, when
MBNL1 N proteins were incubated with (CUG)
136
, ring
C
B
0
10
20
30
40
50
60
70
80
90
100
(CUG)54
(CAG)54
Tnnt3/5.1
Tnnt3/5.45
% RNA bound
RNA
0
MBNL1
well
(CUG)54
Tnnt5.1Tnnt5.45
MBNL1
10−12 10−11 10−10 10−910−810−7
37
75
50
MBNL1 FL
MBNL1 N
A
382
270
253
Coomassie immunoblot
MBNL1 FL
MBNL1 N
*
MBNL1 FL
MBNL1 N
RNA
well
0MBNL1
0
[MBNL1 FL, M]
Figure 3. MBNL1 binds to pathogenic and splicing precursor RNAs with similar affinities. (A) Purified recombinant MBNL1 proteins. Coomassie-
stained gels (left panels) and immunoblots (right panels) of either full-length or N-terminal proteins. Illustration shows the primary structures (line;
C
3
H motifs are shown as shaded boxes) indicating the full-length (382 aa) and N-terminal (253 aa) MBNL1 proteins together with a C-terminal
truncated protein (270 aa) generated during expression in E. coli and an unknown 75 kDa protein (asterisk). (B) Nitrocellulose filter-binding analysis
of MBNL1 FL binding to (CUG)
54
(red square), (CAG)
54
(orange triangle), Tnnt3 5.1(T5.1, blue cross) and Tnnt3 5.45 (T5.45, green circle) RNAs.
(C) Gel shift analysis of MBNL1 FL and MBNL1 N binding to Tnnt3 5.45, 5.1 or (CUG)
54
. The positions of the free RNA (bracket), the gel origin
(well) and MBNL1 FL and N concentrations (triangle, lanes are 0, 0.25, 1, 4, 16, 64, 256 nM) are indicated.
Nucleic Acids Research, 2007, Vol. 35, No. 16 5481
structures were observed although ring size was much less
uniform and there was considerably less stacking of
MBNL1 N rings compared to MBNL1 FL (data not
shown). This observation prompted us to examine
whether the C-terminal region was important for
MBNL1 homotypic interactions.
MBNL1 self-interaction mediated by the C-terminal region
A number of studies have demonstrated that MBNL1
accumulates in ribonuclear foci together with pathogenic
RNAs (13,27–31). While additional proteins might also
bind to CUG
exp
and CCUG
exp
RNAs, the number of
ribonuclear foci in DM myoblasts declines significantly
following loss of MBNL1 suggesting that this protein is
required for the formation and/or maintenance of these
unusual nuclear structures (15). Since many RNA-binding
proteins function as components of large multi-subunit
complexes (32) and some of these proteins self-interact via
their auxiliary or non-RNA-binding regions (33), we
tested the possibility that MBNL1 proteins self-associate
using the yeast two-hybrid system. Although the amino
terminal region of MBNL1 contains all four C
3
H motifs
and is responsible for protein–RNA interactions (21), very
little is known about the function of the C-terminal region.
The MBNL1 FL protein showed strong homotypic
interactions in this system (Figure 6A). Although
MBNL FL failed to interact with the MBNL1 N-terminal
region (1–264), interactions between the full-length
protein and C-terminal region (239–382) were readily
detectable. This C-terminal region does not contain any
known RNA binding, or other, structural motifs.
To confirm that MBNL1 homotypic interactions
occurred in a mammalian cell context, 293T cells were
co-transfected with plasmids which expressed either
V5-MBNL1 FL alone, V5-MBNL1 FL and MBNL1
FL-myc or V5-MBNL1 FL and MBNL1 N-myc. Twenty-
four hours following transfection, V5-tagged MBNL1 was
immunopurified from cell lysates using an anti-V5 anti-
body and the precipitates were then immunoblotted using
either anti-myc or anti-V5 antibodies. In agreement with
the two-hybrid analysis, the full-length V5 and myc-
tagged proteins were associated in vivo while the
N-terminal MBNL1 region (MBNL1 N-myc) failed to
co-immunopurify with V5-MBNL1 FL (Figure 6B).
Interactions between V5-MBNL1 FL and MBNL1 N-
myc were not mediated by RNA tethering since treatment
of the cell lysate with RNase A did not affect the amount
of MBNL1 N-myc in the V5-MBNL1 FL immunopreci-
pitate. Although it is possible that MBNL1 interactions
could be mediated by other nuclear factors, our demon-
stration that purified MBNL1 forms a ring-like structure
argues against this interpretation. We conclude that
MBNL1–MBNL1 interactions occur in vivo and these
interactions are mediated by the C-terminal region.
DISCUSSION
MBNL1 targets similar binding motifs in splicing precursor
and pathogenic RNAs
A number of studies have demonstrated that CUG
exp
and
CCUG
exp
RNAs sequester MBNL proteins in nuclear
RNA foci of DM cells (9,13,15,27–31,34,35). Loss of
MBNL1 function by sequestration correlates with the
inhibition of alternative splicing regulation for a specific
set of developmentally regulated exons that are mis-
spliced in DM tissues (8,15,20,36). Moreover, Mbnl1
knockout mice develop many of the characteristic features
associated with DM disease, including myotonia and
subcapsular cataracts, while adeno-associated virus
(AAV)-mediated Mbnl1 overexpression in transgenic
− MBNL1
+MBNL FL
+MBNL1 N
−MBNL1
+MBNL1
G10
Pb
G20
G30
G40
G50
G1G10G20G30
T1
G1
Ci
Pb
FT
Ci
Pb
−MBNL1
+MBNL1
Ci
T1
FT
Ci
T1
Figure 4. MBNL1 binds throughout the dsCUG stem. Structural
analysis of 50-end labeled (CUG)
54
transcript (20 nM) either in the
presence (+) or absence () of 500 nM recombinant MBNL1 and
either lead ion (0.25 or 0.5 mM, lanes Pb) or ribonuclease T1 (0.5 or
1U/ml, lanes T1); incubation control (no probe added, lane Ci);
formamide (lane F); guanine-specific ladder obtained with RNase T1
digestion (lane T). The positions of selected G residues are shown along
the T1 ladder (G-residues of the corresponding CUG repeat are
indicated). Below the gel panels is the color-coded densitometric
analysis (ImageQuant) of cleavage patterns for the CUG stem obtained
with lead ion (Pb) and T1 ribonuclease (T1) in the presence of MBNL1
FL (green), MBNL1 N (red) or in the absence of protein (blue).
5482 Nucleic Acids Research, 2007, Vol. 35, No. 16
mice expressing a CUG
exp
in skeletal muscle reverses
myotonia and DM-associated mis-splicing (16,20).
Although there is considerable evidence for this
muscleblind loss-of-function model for DM pathogenesis,
the molecular basis for MBNL1 sequestration by CUG
exp
and CCUG
exp
RNAs has not been elucidated. Since
mutant RNA expansions must compete with normal pre-
mRNA, and possibly mRNA, binding sites for MBNL1
recruitment, effective MBNL1 sequestration might occur
if the affinity of this protein for CUG
exp
and CCUG
exp
RNAs is greater than for its normal splicing targets. The
binding analysis presented here does not support this
conjecture since MBNL1 also possesses relatively high
affinities for CAG
exp
and Tnnt3 precursor RNAs. Indeed,
these binding studies provide an explanation for the
formation of MBNL1-containing ribonuclear foci in cells
overexpressing CAG
exp
(22). Additionally, mapping of a
binding site to a stem-loop structure in Tnnt3 intron 8 just
upstream of the F exon indicates that RNA recognition by
MBNL proteins involves a common interaction mode for
both pathogenic and normal pre-mRNAs: recognition of
GC-rich hairpins containing pyrimidine mismatches.
What is the physiological significance of the binding
preference of MBNL1 for RNA stem-loop structures? In
this study, we provide evidence that MBNL1 is a
developmentally regulated splicing regulator which acts
as an intronic splicing repressor by recognizing a stem-
loop near the Tnnt3 F exon 30splice site, possibly resulting
in the stabilization of this secondary structure and
interference with U2AF recruitment. Based on this
observation, we speculate that MBNL1 recognizes similar
intronic stem-loop structures adjacent to the 50splice sites
of other MBNL1-regulated fetal exons to inhibit U1
snRNP recruitment or MBNL1 may stabilize interactions
between introns flanking alternative exons resulting in
RNA looping and increased fetal exon skipping.
The importance of RNA secondary structures in
inherited disease and alternative splicing has been
Figure 5. Visualization of dsCUG and MBNL1–dsCUG complexes. Electron microscopy of either free (CUG)
136
RNAs (A–C) or MBNL1–(CUG)
136
complexes (D–I) at a protein:RNA ratio of either 2.5:1 (D–F, I) or 10:1 (G–H). As reported previously, purified dsCUG RNAs, which were directly
adsorbed onto thin carbon foils followed by dehydration and rotary shadow casting with tungsten, are elongated rod-shaped structures (25). For
analysis of complexes, MBNL1 protein and (CUG)
136
RNA were incubated together at 308C, fixed with glutaraldehyde, passed over a gel filtration
column and adsorbed on copper grids for rotary shadowing. Under these conditions, MBNL1 has a distinct ring-shaped structure. Size bars (white
line) are 40 nm (H) or 50 nM (I).
Nucleic Acids Research, 2007, Vol. 35, No. 16 5483
previously highlighted in frontotemporal dementia with
parkinsonism linked to chromosome 17 (FTDP-17) which
is caused by mutations in the MAPT gene encoding the
microtubule-associated protein tau (37). Some FTDP-17
mutations destabilize a predicted stem-loop structure,
which forms between the 30end of exon 10 and the 50end
of the downstream intron, resulting in an increase in U1
snRNP recruitment and E10 inclusion. Interestingly,
MAPT exon 10 skipping increases in the DM brain
suggesting that MBNL1 promotes exon 10 inclusion
during splicing (20). The MBNL1-binding preferences
shown in this study suggest that this factor may also
function as a splicing activator by recognizing RNA stem-
loop structures in novel exonic and/or intronic splicing
enhancers or by blocking splicing silencer elements by
stabilizing overlapping RNA secondary structures.
Another interesting question arose when we mapped the
MBNL1-binding site on Tnnt3 to an 18-nt stem-loop
structure. Our previous study showed that MBNL1 in
HeLa nuclear extract cross-links strongly to (CUG)
74
and
(CUG)
97
but not to (CUG)
<20
(9). Based on this
observation, we proposed that below a certain length
threshold (<20 repeats) the dsCUG helix was unstable in
the cell extract and ssCUG was not a binding site
for MBNL1. The results reported here support that
proposal and demonstrate that MBNL1 is primarily
a dsRNA-binding protein which recognizes relatively
short GC-rich hairpins if the overall RNA secondary
structure is stabilized by additional sequence interactions.
This conclusion provides a plausible resolution for the
apparently conflicting results that overexpression of a
GFP-DMPK 30-UTR (CTG)
5
transgene results in a DM
phenotype (14) while mice expressing the HSA
SR
(human
skeletal a-actin containing a 30-UTR with five CTG
repeats) transgene are normal (38). For the DMPK
30-UTR, the (CTG)
5
repeat is predicted to interact with
an upstream region to form a GC-rich stem interrupted by
several U-U and C-C mismatches while this repeat in the
HSA 30-UTR is located in sequential unpaired loops. We
postulate that this structural arrangement in the DMPK
30-UTR promotes MBNL1 sequestration when GFP-
DMPK 30-UTR (CTG)
5
RNA is overexpressed in
transgenic mice.
MBNL1 rings: interactions with CUG
exp
RNA and
potential relevance to DM disease
Filter binding and gel shift assays indicated that the
affinity of MBNL1 for Tnnt3/T5.45 lies between that of
(CUG)
54
and (CAG)
54
. While overexpression of either
CUG or CAG repeats induces the formation of nuclear
foci in cell culture, it is interesting to note that we did not
observe RNA foci formation following Tnnt3 minigene
overexpression. This observation argues that high affinity
MBNL1–RNA interactions together with abundant
expression of MBNL1 target RNAs is not sufficient for
ribonuclear foci formation. Of course, MBNL1 may be
cleared from splicing target, but not pathogenic, RNAs
during RNA processing and nuclear export or unusual
BD: p53
AD: TAntigen
BD: MBNL1
AD: TAntigen
BD: MBNL1
AD: MBNL1
BD: MBNL1
AD: MBNL1 (1-264)
BD: MBNL1
AD: MBNL1
(239-382)
1-382
MBNL1 protein
239-382
1-264
Interaction
+
_
+
full-length
C-terminal
N-terminal
37
A
αmyc
input
input
αV5
αV5
B
αV5
input
input
αV5
αV5
25
input
input
αV5
αV5 V5-MBNL1 FL
V5-MBNL1 FL
MBNL1 FL-myc
V5-MBNL1
MBNL1 N-myc
RNase
++ ++
++
+ + +
V5-MBNL1 FL
V5-MBNL1 FL
MBNL1 FL-myc
V5-MBNL1
MBNL1 N-myc
RNase
37
25
Figure 6. Self-association of MBNL1 proteins is mediated by the C-terminal region. (A) Two-hybrid analysis using yeast strains transformed with the
following binding domain (BD) and activation domain (AD) plasmids: (i) GAL4 DNA-binding domain (BD) plasmids with either p53 (activation
control) or full-length MBNL1(MBNL1); (ii) activation domain plasmids with either T-antigen (activation control), MBNL1 (residues 1–382), MBNL1
1–264 (N-terminal region) or MBNL 239–382 (C-terminal region). Functional interactions between the proteins expressed from the BD and AD plasmids
results in growth on the Trp
Leu
His
selection plate. (B) Co-immunopurification of MBNL1 requires the C-terminal region. HEK293T cells were
co-transfected for 24 h with plasmids expressing tagged versions of either full-length or N-terminal MBNL1 (V5-MBNL1 FL alone, co-transfected
V5-MBNL1 FL and MBNL1 FL-myc, co-transfected V5-MBNL1 FL and MBNL1 N-myc). Cell lysates were prepared and the V5-MBNL1 FL protein
immunopurified (aV5) followed by SDS–PAGE (50% of IP sample) and immunodetection of MBNL1 FL-myc or MBNL1 N-myc using mAb 9E10 (top
panel, input lanes represent 2.5% of total IP) or V5-MBNL1 FL (bottom panel, only the input lanes are shown).
5484 Nucleic Acids Research, 2007, Vol. 35, No. 16
interactions between MBNL proteins and CUG
exp
RNA
might drive foci formation. In support of the latter
possibility, we provide EM evidence that MBNL1 forms a
tandem ring structure when bound to CUG
exp
RNA.
The size of the rings is uniform with a diameter of 18 nm
and since the MBNL1 isoform employed for these studies
is 41 kDa, the ring structure must be an oligomeric
complex. At a protein:RNA ratio of 2.5:1, most CUG
exp
helices were bound by a single ring while the majority of
these hairpins were bound by at least two rings at a higher
protein:RNA ratio. It is not clear if the MBNL1 ring
contains a hole but if a central cavity exists it might allow
threading of dsRNA. When multiple rings were bound to
a single RNA molecule they appeared to be tandemly
stacked suggesting either a preferential ring-loading site or
potential ring–ring interactions. The latter possibility is
supported by our finding that MBNL1 self-interacts via its
C-terminal region both in the yeast two-hybrid system and
in mammalian cells. In contrast, the MBNL1 N-terminal
region encompassing the CCCH RNA-binding motifs fails
to interact with full-length MBNL1 although RNA-
binding activity is comparable to the full-length MBNL1
(data not shown). In this regard, it is interesting to note
that the rings formed using MBNL1 N, lacking the
C-terminal region, were less uniform in size and there was
less ring–ring stacking at higher protein:RNA ratios. A
similar situation has been noted for the Escherichia coli
protein Hfq (Host factor 1) which is a single-strand RNA-
binding protein involved in the translational regulation
and stability of several RNAs (39). As visualized in the
EM by negative staining, Hfq forms hexameric rings.
Similar to MBNL1, the RNA-binding activity of Hfq
resides in the N-terminal region and rings are still formed
by a C-terminal truncated protein although they are less
stable.
Does MBNL1 exist as a ring structure when bound to
its normal RNA splicing targets? To address this question,
we performed EM analysis of the MBNL1–Tnnt3/T5.45
complex. Although MBNL1 rings were observed, the
result was inconclusive due to the difficulty in visualizing
small and partially single-stranded Tnnt3/T5.45 RNA by
EM. However, MBNL1 forms large complexes with both
CUG and Tnnt3/T5.45 RNAs (Figure 3C) so it is possible
that MBNL1 forms a ring structure when bound to
splicing regulatory sites. Nevertheless, these normal
binding targets do not contain the tandemly arrayed
MBNL1-binding sites present on pathogenic CUG
exp
RNAs which we propose are essential for multiple ring
interactions, MBNL1 sequestration and ribonuclear foci
formation in DM cells.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We thank Keith Nykamp for help with the yeast
two-hybrid analysis, Rob Osborne for the gift of pBC-
CTG136, members of the Swanson lab for comments on
the manuscript and Andy Berglund for communicating
results prior to publication. This work was supported by
grants to M.S.S. from the NIH and the Paul D. Wellstone
Muscular Dystrophy Cooperative Research Center
(AR46799, NS48843) and a post-doctoral fellowship to
K.S. from the Foundation for Polish Science. The electron
microscopy studies were supported by NIH grants
GM31819 and ES013773 to J.D.G. Funding to pay the
Open Access publication charges for this article was
provided by NIH AR46799.
Conflict of interest statement. None declared.
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