Novel proteins with binding specificity for DNA CTG repeats and RNA CUG repeats: implications for myotonic dystrophy
Novel proteins with binding specificity for DNA CTG repeats and RNA CUG repeats: implications for myotonic dystrophyL. T. Timchenko, N. A. Timchenko1, C. T. Caskey2 and R. Roberts*
Department of Medicine, Section of Cardiology, Baylor College of Medicine, Houston, TX 77030, USA, 1Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA and 2Merck Research Laboratories, Sumneytown Pike, West Point, PA 19486, USA
Received September 6, 1995;Revised and Accepted October 21, 1995
While an unstable CTG triplet repeat expansion is responsible for myotonic dystrophy, the mechanism whereby this genetic defect induces the disease remains unknown. To detect proteins binding to CTG triplet repeats, we performed bandshift analysis using as probes double-stranded DNA fragments having CTG repeats [ds(CTG)6-10] and single-stranded oligonucleotides having CTG repeats ss(CTG)8 or RNA CUG triplet repeats (CUG)8. The source of protein was nuclear and cytoplasmic extracts of HeLa cells, fibroblasts and myotubes. Proteins binding to the double-stranded DNA repeat [ds(CTG)6-10], were inhibited by nonlabeled ds(CTG)6-10, but not by a non-specific DNA fragment (USF/AD-ML). Another protein binding to ssCTG probe and RNA CUG probe was inhibited by nonlabeled (CTG)8 and (CUG)8. Nonlabeled oligos with different triplet repeat sequences, ss(CAG)8 or ss(CGG)8, did not inhibit binding to the ss(CTG)8 probe. However, when labeled as probes, the (CAG)8 and (CGG)8 bound to proteins distinct from the CTG proteins and binding was inhibited by nonlabeled (CAG)8 or (CGG)8 respectively. The protein binding only to the RNA repeat (CUG)8 was inhibited by nonlabeled (CUG)8 but not by nonlabeled single- or double-stranded CTG repeats. Furthermore, the CUG-BP exhibited no binding to an RNA oligonucleotide of triplet repeats of the same length but having a different sequence, CGG. The CUG binding protein was localized to the cytoplasm, whereas dsDNA binding proteins were localized to the nuclear extract. Thus, several trinucleotide binding proteins exist and their specificity is determined by the triplet sequence. The novel protein, CUG-BP, is particularly interesting since it binds to triplet repeats known to be present in myotonin protein kinase mRNA which is responsible for myotonic dystrophy.
Extensive expansion of a triplet of base pairs repeated in tandem in the human genome has been shown to be responsible for several neuromuscular diseases (1 -13 ). In some of these disorders, loss of gene expression suggests triplet repeat expansion affects the efficiency of transcription (14 -16 ), or translation (17 ). The chromosomal locus l9q, genetically linked to myotonic dystrophy, has a region of extensive repeats of the triplet CTG (6 -11 ). The number of repeats correlates with both the presence and severity of disease (18 -22 ). The extended triplet repeat region is 3' to the alleged responsible gene, myotonin protein kinase (Mt-PK). Results of studies assessing the expression of Mt-PK in patients with disease are inconclusive, some showing decreased expression of Mt-PK mRNA (23 -28 ), others increased Mt-PK mRNA expression (29 ). The molecular weight of Mt-PK was shown initially to be 52-55 kDa (23 ,28 ), however, recently we identified a full-length isoform of Mt-PK with a molecular weight of 72 kDa (30 ) which corresponds to the size of a protein translated from the first AUG codon in the frame and possesses serine specific kinase activity (30 ). Whiting et al. have also described the existence of high molecular weights Mt-PK isoforms (31 ). Wang et al. demonstrated increased efficiency of nucleosome formation at the region of CTG triplet expansion (32 ,33 ) which may repress transcription. The role of the triplet repeat in transcription has been further questioned by the data of Krahe et al. (34 ). It has also been suggested that the CTG repeat expansion in patients with DM dramatically alters the ability of the mutant mRNA to be processed into poly(A)+ mRNA (35 ) or results in abnormal transfer of Mt-PK transcripts from nucleus to the cytoplasm. This derives from the observation that increased levels of the enlarged Mt-PK transcript are present in the nucleus of cells from DM patients (36 ). Contradictory data on steady-state levels of Mt-PK mRNA has provided the impetus to explore the possibility that the CTG expansion exerts its dilatory effects not on the Mt-PK but on the gene immediately upstream of Mt-PK (37 ).
In our attempts to explore other mechanisms whereby expanded triplet repeats may induce disease, we identified a group of proteins that exhibit binding specific for CTG triplet repeats whether they be double- or single-stranded. In addition, an RNA binding protein (CUG-BP) was identified in the cytoplasm of HeLa cells which was specific for the RNA sequence, (CUG)8, and did not bind to single- or double-stranded DNA CTG repeats. There are at least two previous reports of triplet DNA binding proteins (38 ,39 ). In this study we have identified several other novel trinucleotide binding proteins and, in particular, a novel protein which binds specifically to the RNA triplet repeat (CUG)8. The latter protein has particular implications for the pathogenesis of myotonic dystrophy.
Plasmid DNA containing the myotonin protein kinase cDNA sequence (pRMK) and human genomic DNA were amplified to generate double-stranded CTG repeats as probes (Fig. 1 A). The DNA fragment from plasmid pRMK had six (CTG) repeats, referred to as dsCTG-1 and that from human genomic DNA (normal allele) had 10 (CTG) repeats referred to as dsCTG-2. These fragments were labeled with 32P and incubated with nuclear extracts from HeLa cells. Separation by electrophoretic mobility shift assay showed two major DNA-protein complexes (dsI and dsII) and one minor complex (dsIII) bound to each probe (Fig. 1 B and C). Detection of complex dsIII required prolonged exposure. The prior addition of 100-fold excess of nonlabeled DNA fragments dsCTG-1 and dsCTG-2 competitively inhibited the formation of the DNA-proteins complexes indicating that the protein binding was specific for dsCTG-1 and dsCTG-2 (Fig. 1 B). We also used double-stranded USF/Ad-ML DNA (40 ) as a potential competitor. An addition of 100-fold excess of USF/Ad-ML did not affect the binding of nuclear proteins to dsCTG-1 (data not shown). Nuclear protein extracts from human fibroblasts, myotubes and HeLa cells were incubated with the dsCTG-l probe and bandshift assays showed (Fig. 1 C) complexes dsI and dsII of similar intensity in each preparation. Formation of dsIII complex was registered only in HeLa nuclear extracts. Extracts from the cytoplasm of HeLa cells exhibited no binding to the dsCTG-1 probe (data not shown).
To determine if there are proteins that bind to single stranded (ss) DNA repeats, a synthetic DNA oligonucleotide made of CTG repeats was radiolabeled and used as a probe for bandshift analysis. Given that (CTG)8 represents GC rich DNA (Fig. 3 A), two triplet repeat sequences [ss(CAG)8 and ss(CGG)8] were selected as probes to determine whether binding reflects specific interaction with the CTG sequence or simply binding to GC rich regions. Two DNA/protein complexes (major ssI and minor ssII) were observed after incubation of the ss(CTG)8 probe with whole cell HeLa protein extracts (Fig. 3 B). Binding was inhibited by the addition of nonlabeled ssCTG DNA (30 ng) indicating binding was specific (Fig. 3 B). The same preparation of whole HeLa cell protein extracts incubated with labeled ss(CAG)8 and ss(CGG)8 resulted in the formation of two (CAG)8 DNA/protein complexes (Fig. 3 B) and four (CGG)8 DNA/protein complexes respectively (Fig. 3 B). Unlabelled ss(CGG)8 and (CAG)8 completely inhibited binding indicating specificity (data not shown).
A synthetic RNA oligonucleotide (CUG)8 was labeled by the kinase method and used as a probe for bandshift analysis with HeLa whole cell extract. Two RNA/protein complexes (Fig. 5 A) were detected by bandshift analysis after incubation of the (CUG)8 probe with the HeLa whole cell extract. The addition of 100 ng of nonlabeled RNA (CUG)8 completely inhibited binding of the labeled probe to either complex (Fig. 5 A). One of the RNA/protein complexes migrated with a mobility similar to the ssCTG/ssCRRP complex. Incubation of the extract with nonlabeled ss(CTG)8 or nonlabeled (CUG)8 inhibited the formation of this complex indicating that the same protein is involved in the formation of DNA/protein and RNA/protein complexes (Fig. 4 B and Fig. 6 B). Thus, the specificity of ssCRRP for DNA (CTG) repeats and for RNA (CUG) repeats was confirmed with both ss(CTG)8 and (CUG)8 probes. The other RNA/protein complex with different mobility than that of the (CUG)8/(ssCRRP) complex was inhibited by the nonlabeled (CUG)8 oligo and was not inhibited by the nonlabeled DNA (CTG)8 oligo (Fig. 6 B). The protein with exclusive binding specificity to CUG triplet repeat was referred to as CUG-BP. To further determine the specificity of CUG-BP we selected a triplet repeat RNA oligo having a different sequence, namely, (CGG)8. The addition of 100-fold excess of nonlabeled (CGG)8 RNA competitor did not affect the binding of CUG binding protein (CUG-BP) to the (CUG)8 probe, indicating specific binding (data not shown). Results shown in Figure 5 B indicate the binding activity of ssCRRP was observed preferentially in cytoplasmic preparations with minimal binding detected in the nuclear extract. In contrast, the RNA CUG-BP complex showed high intensity in the cytoplasmic extract (Fig. 5 C), but was not detectable in the nuclear extract. To verify the purity of the separation of nuclei from cytoplasm, the probe, USF/Ad-ML specific for the nuclear upstream stimulatory factor (USF) (40 ) was incubated with the same nuclear and cytoplasm preparations. USF binding was detected as expected only in the nuclear extract and not in the cytoplasm (Fig. 5 D), confirming good separation of nuclear and cytoplasmic fractions. Thus, the proteins binding to the (CUG)8 RNA probe are localized to the cytoplasm.
Dinucleotide and trinucleotide sequences repeated in tandem referred to as microsatellites occur every 5000-10 000 bp throughout the human genome and because of the marked variation in the number of repeats in each satellite are used almost exclusively today as markers in genetic linkage analysis to map chromosomal loci (41 ). The recent finding that extended repeats can induce several neuromuscular and degenerative diseases (1 -13 ) has intensified the research for the inherent dysfunction. Myotonic dystrophy is particularly puzzling in that the triplet repeat occurs in the 3' non-protein coding region of the gene (6 -11 ). The existence of proteins specific for trinucleotide repeats particularly the novel proteins (ssCRRP and CUG-BP) which bind the RNA CUG repeats sequence found in the mRNA of the gene for myotonic dystrophy has obvious implications. Reports suggest the mRNA of the myotonin kinase gene is transcribed (23 -29 ), however, one hypothesis is that the 3' end of the mature myotonin protein kinase mRNA is improperly processed (35 ). It is reasonable to speculate that the (CUG) RNA binding proteins may be needed for stability in the processing of the mRNA or for the attachment of the poly A tail. Since ssCRRP exhibited dual binding activity to ssCTG and RNA CUG repeats, we speculate that it may be involved in two possible events: (i) ssCRRP, found primarily in the cytoplasm, may participate in translation or the regulation of Mt-PK mRNA stability, or (ii) under some conditions ssCRRP, since a minimal amount was also detected in the nuclear extract, may translocate to nuclei and play some role in the transcription of Mt-PK gene or in the processing of the pre-mRNA transcript.
Whether the CTG or CUG binding proteins play a role in the pathogenesis of myotonic dystrophy is unknown but it now becomes important to characterize and determine the function of these specific proteins. Two previous reports have described binding proteins to DNA triplet repeat sequences (38 ,39 ). We confirm the previous reports showing proteins with specific binding for the DNA repeat sequences of CAG, CGG and have described several other novel proteins. The novel protein, CUG-BP, is particularly interesting since it binds specifically and exclusively to the RNA triplet repeat, CUG, which is the triplet repeat present in multiple copies alleged to be responsible for myotonic dystrophy. It is highly likely that these proteins have specific functions and evolved in conjunction with the evolution of these trinucleotide tandem repeats. It is reasonable to speculate that disease or alteration in function will occur if these proteins are in some way mutated or impaired. A new class of proteins are now available to be explored both in terms of their possible functional role in transcription and/or translation and their potential role of inducing diseases or altered physiological states as a result of mutations.
The following DNA oligonucleotides were used as profiles ss(CTG)8, 5'-CTGCTGCTGCTGCTGCTGCTGCTG-3', ss(CAG)8, 5'-CAGCAGCAGCAGCAGCAGCAGCAG-3' and ss(CGG)8, 5'-CGGCGGCGGCGGCGGCGGCGGCGG-3'. The RNA oligonucleotides were synthesized in the Nucleic Acids Core Laboratory of the Department of Molecular and Human Genetics, Baylor College of Medicine or in Oligo's Etc. Double-stranded DNA fragments dsCTG-1 and dsCTG-2 were synthesized by PCR from plasmid pRMK or human genomic DNA respectively with primers 5'-GCTCGAAGGGTCCTTGTAGCCGGGAATG-3' and 5'-GAAAGAAAGAAATGGTGCTGTGATCCCCC-3'.
HeLa and fibroblasts cultured cells were grown in MM medium as previously described (43 ). Human myotubes were grown as previously recommended (44 ). To prepare the protein extracts, cells were washed twice with PBS, pelleted at 1000 g and resuspended in a solution of 50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 1 mM dithiotreitol (DTT), 10 mM MgCl2, 20% glycerol, 0.1 M KCl, 1% Triton X-100, leupeptine (0.5 [mu]g/ml) and pepstatine (20 [mu]g/ml). The cells were incubated on ice for 5 min in this buffer and centrifuged for 5 min at 10 000 g at 4oC. The supernatant (whole cell protein extract) was collected and stored at -80oC.
Nuclear extracts were prepared as described (45 ). Cells were washed twice with PBS, pelleted and resuspended in the buffer of 10 mM Tris HCl, pH 7.6, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT. After 15 min of incubation on ice cells were homogenized by pulling them through a 23-gauge needle (eight strokes) and nuclei were pelleted at 10 000 g. The nuclei were resuspended in a buffer composed of 20 mM Tris-HCl, pH 7.6, 25% sucrose, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT and incubated for 30 min on ice. Nuclear extract (NE) was collected after centrifugation at 10 000 g and used immediately.
The dsCTG-l and dsCTG-2 DNA fragments were generated by PCR, purified by agarose gel and labeled by T4 DNA kinase with [32P-gamma]ATP. Single-stranded DNA or RNA oligonucleotides were labeled by T4 polynucleotide kinase with gamma [32P]ATP to a high specific activity. The binding reactions were performed in 10 [mu]l mixture containing 0.1-0.5 ng of 32P-labeled DNA or RNA probe, 10 [mu]g of NE or 20-40 [mu]g of WCE, 2 [mu]g of poly (dI-dC), 20 mM Tris-HCl, pH 7.6, 100 mM KCl, 5 mM MgCl2, 5 mM DTT and 10% glycerol. In the experiments with the (CUG)8 and (CGG)8 RNA probes, we used 2 [mu]g of total RNA isolated from HeLa cells as a nonspecific competitor. For single-stranded DNA probes, poly (dI-dC) was heated at 95oC for 10 min and incubated for 5 min on ice prior to use in the binding reaction. To determine binding specificity, 100-fold excess of nonlabeled probe was added to the binding reaction following the mixtures were incubated for 30 min at room temperature. Protein/DNA or protein/RNA complexes were separated from the free probe by 6 or 8% polyacrylamide gel as described (45 ).
Nuclear extracts (100 [mu]g) were electrophoresed on 0.1% SDS-12% polyacrylamide gel (45 ) and proteins were transferred to a nitrocellulose membrane (NitroPure, MSI). The membrane was blocked by prebinding in a solution of 5% dry milk, 10 mM Tris-HCl, pH 7.6 and 1 mM DTT for 1 h at room temperature. Binding was performed in a solution of 10 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 100 mM KCl, 1 mM DTT, 0.25% dry milk, 0.1 mM EDTA with 106 c.p.m. of 32P-labeled oligonucleotide/ml. After incubation for 1 h at room temperature, the membrane was washed with the binding buffer that did not contain 32P-labeled probe.
Fractionation of proteins was performed as described (46 ). One hundred micrograms of total protein extract were resolved by 0.1% SDS-12% polyacrylamide gel electrophoresis. After transfer to nitrocellulose (NitroPure) the proteins were fractionated by cutting the membrane across the lane into pieces that represent ~5-10 kDa each. Proteins from each piece of the membrane were eluted overnight in 100 [mu]l of renaturation buffer: 20 mM HEPES, pH 7.9, 50 mM KCl, 5 mM MgCl2, 10% glycerol, 1% Triton X 100 and 0.1 mg/ml BSA (fraction V) at 4oC. The eluted protein fractions were collected and stored at -80oC. Five microliters of each fraction were used in the bandshift assay. Mixed fractions analysis was performed by combining of 5 [mu]l from each of two fractions in a final volume of 20 [mu]l.
We greatly appreciate the editorial assistance of Dr Linda Bachinski and the secretarial assistance of Debora Weaver and Esther Yeager in the preparation of this manuscript and figures. This work is supported in part by grants from the National Heart, Lung and Blood Institute, Specialized Centers of Research (P50-HL54313-01), the National Institutes of Health Training Center in Molecular Cardiology (T32-HL07706) and the American Heart Association, Bugher Foundation Center for Molecular Biology (86-2216).
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