Abstract
Muscleblind-like-1 (MBNL1) is a splicing regulatory factor controlling the fetal-to-adult alternative splicing transitions during vertebrate muscle development. Its capture by nuclear CUG expansions is one major cause for type 1 myotonic dystrophy (DM1). Alternative splicing produces MBNL1 isoforms that differ by the presence or absence of the exonic regions 3, 5, and 7. To understand better their respective roles and the consequences of the deregulation of their expression in DM1, here we studied the respective roles of MBNL1 alternative and constitutive exons. By combining genetics, molecular and cellular approaches, we found that (i) the exon 5 and 6 regions are both needed to control the nuclear localization of MBNL1; (ii) the exon 3 region strongly enhances the affinity of MBNL1 for its pre-mRNA target sites; (iii) the exon 3 and 6 regions are both required for the splicing regulatory activity, and this function is not enhanced by an exclusive nuclear localization of MBNL1; and finally (iv) the exon 7 region enhances MBNL1-MBNL1 dimerization properties. Consequently, the abnormally high inclusion of the exon 5 and 7 regions in DM1 is expected to enhance the potential of MBNL1 of being sequestered with nuclear CUG expansions, which provides new insight into DM1 pathophysiology.
Highlights
Particles Maturation-Structure-Function, Molecular and Structural Enzymology, Unite Mixte de Recherche 7214 Nancy University-CNRS, Faculty of Sciences, BP 70239, 54506 Vandoeuvre-les-Nancy Cedex, France, the ʈUniversite Pierre et Marie
Molecular and cellular approaches, we found that (i) the exon 5 and 6 regions are both needed to control the nuclear localization of MBNL1; (ii) the exon 3 region strongly enhances the affinity of MBNL1 for its pre-mRNA target sites; (iii) the exon 3 and 6 regions are both required for the splicing regulatory activity, and this function is not enhanced by an exclusive nuclear localization of MBNL1;
Our results provide a better definition of (i) the respective roles of the amino acid sequences encoded by exons 3 and 5 in the RNA binding property, nuclear localization and splicing regulatory property of MBNL1; (ii) the involvement of the exon 6-encoded sequence in MBNL1 nuclear retention and in cellulo splicing regulatory activity; (iii) the identification of a possible role of the exon 7-encoded sequence in MBNL1 self-dimerization
Summary
Particles Maturation-Structure-Function, Molecular and Structural Enzymology, Unite Mixte de Recherche 7214 Nancy University-CNRS, Faculty of Sciences, BP 70239, 54506 Vandoeuvre-les-Nancy Cedex, France, the ʈUniversite Pierre et Marie. A vast majority of vertebrate pre-mRNAs is alternatively spliced, allowing the production of several protein isoforms from transcripts of a given gene [1]. The regulation of alternative splicing plays a major role in cell differentiation and in development and depends on the expression and activity of numerous splicing regulatory factors that are expressed differentially during development, according to the type of tissue. Defects in these alternative-splicing processes can contribute to pathogenesis, as demonstrated for a growing number of diseases, including neuromuscular diseases such as myotonic dystrophy type 1 (DM1)9 [2, 3]. Mutant transcripts bearing long-CUG repeats acquire unusual A-form doublestranded RNA structures [7], accumulate in the nucleus, and lead to small ribonucleoprotein inclusions, named foci [8] that sequester RNA-binding proteins such as Muscleblind-like 1
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