Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs.
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.
Yuan
Yuan
Y
Department of Molecular Genetics and Microbiology and the Genetics Institute, University of Florida, College of Medicine, Gainesville, FL, USA.
Compton
Sarah A
SA
Sobczak
Krzysztof
K
Stenberg
Myrna G
MG
Thornton
Charles A
CA
Griffith
Jack D
JD
Swanson
Maurice S
MS
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D020862:GC Rich Sequence; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D009223:Myotonic Dystrophy / Q000235:genetics; D012322:RNA Precursors / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D019680:Trinucleotide Repeat Expansion; D020107:Troponin T / Q000235:genetics
D012322:RNA Precursors; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D020107:Troponin T
Nucleic Acids Res. 2000 Jan 15;28(2):416-23
10606638
Neuromuscul Disord. 2007 Mar;17(3):242-7
17306536
EMBO J. 2000 Sep 1;19(17):4439-48
10970838
Science. 2000 Sep 8;289(5485):1769-73
10976074
Curr Opin Struct Biol. 2001 Feb;11(1):53-8
11179892
Nucleic Acids Res. 2001 Jul 1;29(13):2766-71
11433021
Science. 2001 Aug 3;293(5531):864-7
11486088
Science. 2001 Sep 7;293(5536):1836-9
11546874
Hum Mol Genet. 2001 Sep 15;10(19):2165-70
11590133
Hum Mol Genet. 2002 Apr 1;11(7):805-14
11929853
Biochem Soc Trans. 2002 Nov;30(Pt 6):945-52
12440952
Nucleic Acids Res. 2003 Oct 1;31(19):5469-82
14500809
Genes Dev. 2003 Oct 15;17(20):2526-38
14522950
Nature. 2003 Nov 6;426(6962):96-100
14603324
Science. 2003 Dec 12;302(5652):1978-80
14671308
Ann Neurol. 2003 Dec;54(6):760-8
14681885
Hum Mol Genet. 2004 Mar 1;13(5):495-507
14722159
Nat Struct Mol Biol. 2004 Mar;11(3):257-64
14981510
Eur J Biochem. 2004 Apr;271(7):1258-65
15030475
Prog Mol Subcell Biol. 2004;35:57-77
15113079
Nat Biotechnol. 2004 May;22(5):535-46
15122293
Brain. 2004 Aug;127(Pt 8):1868-77
15231584
EMBO J. 2004 Aug 4;23(15):3103-12
15257297
J Mol Biol. 1996 Jun 14;259(3):337-48
8676373
J Biol Chem. 1997 Dec 5;272(49):31079-85
9388259
EMBO J. 1998 Dec 15;17(24):7505-13
9857205
Nucleic Acids Res. 1999 Sep 1;27(17):3534-42
10446244
Hum Mol Genet. 2004 Dec 15;13(24):3079-88
15496431
J Biol Chem. 2005 Feb 18;280(7):5773-80
15546872
FEBS J. 2005 May;272(9):2109-17
15853796
FEBS J. 2005 May;272(9):2118-31
15853797
J Cell Sci. 2005 Jul 1;118(Pt 13):2923-33
15961406
Science. 2005 Sep 2;309(5740):1514-8
16141059
Circ Res. 2005 Nov 25;97(11):1152-5
16254211
Differentiation. 2006 Mar;74(2-3):65-80
16533306
Hum Mol Genet. 2006 Jul 1;15(13):2087-97
16717059
Proc Natl Acad Sci U S A. 2006 Aug 1;103(31):11748-53
16864772
Nat Genet. 2006 Sep;38(9):1066-70
16878132
RNA. 2000 Jan;6(1):79-87
10668800