Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy

Science

Volumn 280, Issue 5364, 1998, Pages 737-741

  Philips, Anne V ^a   Timchenko, Lubov T ^a   Cooper, Thomas A ^a  

^a BAYLOR COLLEGE OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

RIBONUCLEOPROTEIN;

ARTICLE; GENETIC ANALYSIS; GENETIC LINKAGE; HUMAN; MYOTONIC DYSTROPHY; NONHUMAN; PRIORITY JOURNAL; RNA SPLICING; SPLICEOSOME; TRANSCRIPTION REGULATION; TRINUCLEOTIDE REPEAT;

ALTERNATIVE SPLICING; CELL LINE; CELL NUCLEUS; EXONS; HUMANS; INTRONS; MUSCLE, SKELETAL; MUTATION; MYOTONIC DYSTROPHY; PHOSPHORYLATION; PROTEIN-SERINE-THREONINE KINASES; RECOMBINANT FUSION PROTEINS; RIBONUCLEOPROTEINS; RNA PRECURSORS; RNA, MESSENGER; RNA-BINDING PROTEINS; TRANSCRIPTION, GENETIC; TRANSFECTION; TRINUCLEOTIDE REPEATS; TROPONIN; TROPONIN T;

EID: 0032076126     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.280.5364.737     Document Type: Article

References (40)

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22. Normal human skeletal muscle cells were purchased from Clonetics (San Diego). DM skeletal muscle cells were from patient DM1 (3, 25). CUG-BP localizes in nuclei in this DM cell line (3, 7) and these cells have been shown to express reduced amounts of DM transcripts and protein (3, 26). Cells were maintained as described (9) and transfected by the calcium phosphate method. RNA was extracted 48 hours later for single transfections or 72 hours later for cotransfections with the CUG-BP expression vector. RT-PCR of minigene mRNA was carried out as in Fig. 2B except that RNA from one-quarter of a 60-mm plate of cells was used for reverse transcription, one-half of the cDNA was used for PCR, the annealing temperature was 68°C, and PCR was done with 25 cycles.
23. Normal and DM skeletal muscle cultures were maintained as proliferating myoblasts. The observation that mutations in the CUG-BP binding site have no effect on splicing in normal muscle cultures is probably due to the absence of coregulators that are present in differentiated striated muscle.
24. Data not shown.
25. The fact that the CUG → CAG mutations did not completely eliminate exon 5 inclusion in cTNT mRNA in muscle cells indicates that additional elements in the 702-nt genomic fragment are required for regulation of splicing.
26. 20-3′ containing 20 CTG trinucleotides flanked by compatible restriction sites (Sal I and Xho I). Oligonucleotides were phosphorylated, annealed, gel-isolated, and concatamerized by ligation in solution. Concatamers not in the head-to-tail orientation regenerate Sal I or Xho I sites and were eliminated by digestion with both enzymes. Concatamers were gel isolated and cloned into the Sal I-Xho I sites of sp72 (Promega). Large concatamers were "grown" in the plasmid by sequential cloning of smaller cloned concatamers into the Sal I site. The DM minigenes contain the cytomegalovirus enhancer/transcription initiation site fused to a DM genomic fragment containing exons 11 (exon position 38) to 15. Exon 15 is truncated at the position of the natural CTG repeats (exon position 376) by the synthetic CTG repeats of lengths indicated in Fig. 3E. Presumably the transcript utilizes a cryptic polyadenylation site within the downstream plasmid sequence. We found that expression from constructs that lacked the DM polyadenylation site was more consistent than that from constructs that contained the DM polyadenylation site (17).
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39. The sequence specificity of rMBP-CUG-BP binding was increased by a 10-min preincubation in HeLa cell nuclear extracts, which suggests that modification of the protein, most likely phosphorylation, is required for optimal activity.
40. We thank J. Seidman and L. Thierfelder for genomic clones containing hcTNT exon 5; C. Lo for excellent technical assistance; and B. Casey, D. Nelson, and S. Berget for critical comments on the manuscript. Supported by grants from the Muscular Dystrophy Association and the National Institutes of Health (HL45565 to T.A.C. and AR 44387 to L.T.T.).