RNA-binding Protein Muscleblind-like 3 (MBNL3) Disrupts Myocyte Enhancer Factor 2 (Mef2) β-Exon Splicing

University of Washington Seattle, Seattle, Washington, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2010; 285(44):33779-87. DOI: 10.1074/jbc.M110.124255
Source: PubMed


Mammalian MBNL (muscleblind-like) proteins are regulators of alternative splicing and have been implicated in myotonic dystrophy, the most common form
of adult onset muscular dystrophy. MBNL3 functions as an inhibitor of muscle differentiation and is expressed in proliferating
muscle precursor cells but not in differentiated skeletal muscle. Here we demonstrate that MBNL3 regulates the splicing pattern
of the muscle transcription factor myocyte enhancer factor 2 (Mef2) by promoting exclusion of the alternatively spliced β-exon.
Expression of the transcriptionally more active (+)β isoform of Mef2D was sufficient to overcome the inhibitory effects of
MBNL3 on muscle differentiation. These data suggest that MBNL3 antagonizes muscle differentiation by disrupting Mef2 β-exon
splicing. MBNL3 regulates Mef2D splicing by directly binding to intron 7 downstream of the alternatively spliced exon in the
pre-mRNA. The RNA binding activity of MBNL3 requires the CX7CX4–6CX3H zinc finger domains. Using a cell culture model of myotonic dystrophy and myotonic dystrophy patient tissue, we have evidence
that expression of CUG expanded RNAs can lead to an increase in MBNL3 expression and a decrease in Mef2D β-exon splicing.
These studies suggest that elevating MBNL3 activity in myogenic cells could lead to muscle degeneration disorders such as
myotonic dystrophy.

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    • "In contrast, MBNL3 has been reported as a member of the family with unusual functions. MBNL3 antagonizes muscle differentiation by promoting exclusion of the alternatively spliced β-exon of Myocyte enhancer factor 2D (Mef2D) [23] and also by the inhibition of myogenesis by maintaining myoblasts in a proliferative state [24], [25]. As a result of this regulation a negative correlation exists between MBNL1 and MBNL3 expression levels in muscle during development when MBNL3 is mainly detected during embryonic development, but also transiently during injury-induced adult skeletal muscle regeneration [13], [26]. "
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    ABSTRACT: Muscleblind-like 3 (MBNL3) belongs to a family of RNA binding proteins that regulate alternative splicing. We have generated a set of monoclonal antibodies (MAbs) against mouse MBNL3, three of which do not cross-react with the other muscleblind-like (MBNL) proteins, MBNL1 and MBNL2. Epitope mapping revealed that MAbs P1C7, P1E7, SP1C2, and P2E6 recognize distinct, non-overlapping segments of the MBNL3 polypeptide sequence. Immunohistochemical staining of proliferating muscle precursor cells localized MBNL3 to the nucleus in a punctate pattern, characteristic of subcellular structures in the nucleus enriched in pre-messenger RNA splicing factors. Although MBNL3 did not co-localize with SC35 and PSP1 (widely used markers of splicing speckles and paraspeckles), the punctate localization pattern of MBNL3 within interchromatin regions of the nucleus is highly predictive of proteins involved in pre-mRNA processing. Monoclonal antibodies specific for mouse MBNL3 will facilitate further investigation of the expression pattern and unique functions of this splicing factor during development and in different adult mouse tissues.
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