Nucleic Acids Research Advance Access published online on August 15, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm601
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Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs
1Department of Molecular Genetics and Microbiology and the Genetics Institute, University of Florida, College of Medicine, Gainesville, FL, 2Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC and 3Department of Neurology, University of Rochester Medical Center, Rochester, NY, USA
*To whom correspondence should be addressed. Tel: +1 352 273 8076; Fax: +1 352 273 8284; Email: mswanson{at}ufl.edu
Received April 30, 2007. Revised July 18, 2007. Accepted July 24, 2007.
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 binding, 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.
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