The broad objective of this project is to analyze at the molecular level the regulatory mechanisms of altered RNA splicing that are controlled by the formation of pathological MBNL1 mega-complexes in myotonic dystrophy 1 (DM1) patient cells. The genetic defect in DM1 results in the production of mutant RNAs encoding expanded CUG tracts. Abnormally expanded CUG tracts have been shown to form aberrant mega-complexes that contain the alternative splice factor, MBNL1, within the nucleus. Several lines of evidence implicate the formation of these high molecular weight complexes in altered splicing of a subset of physiologically important RNAs and in the subsequent development of DM1 pathology in vivo. To determine the mechanism whereby formation of the MBNL1 mega-complexes alters the splice code in DM1 we propose to purify both normal MBNL1 complexes and the aberrant MBNL1 mega-complexes that develop in DM1 myoblasts. In complementary experiments the role of these complexes in dictating RNA splice site choice will be defined.
The Aims of this application are: 1. Purification and functional characterization of normal MBNL1 complexes in spliceosome assembly and RNA catalysis. 2. Purification of MBNL1 mega-complexes from DM1 myoblasts and definition of the mechanics of mega-complex formation in vivo. 3. Elucidation of the mechanisms by which formation of MBNL1 mega-complexes alters the splice code in DM1 myoblasts.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS060839-05
Application #
8197219
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Porter, John D
Project Start
2007-02-01
Project End
2014-11-30
Budget Start
2011-12-01
Budget End
2014-11-30
Support Year
5
Fiscal Year
2012
Total Cost
$498,174
Indirect Cost
$192,546
Name
University of Southern California
Department
Biochemistry
Type
Schools of Medicine
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Dansithong, Warunee; Jog, Sonali P; Paul, Sharan et al. (2011) RNA steady-state defects in myotonic dystrophy are linked to nuclear exclusion of SHARP. EMBO Rep 12:735-42
Paul, Sharan; Dansithong, Warunee; Jog, Sonali P et al. (2011) Expanded CUG repeats Dysregulate RNA splicing by altering the stoichiometry of the muscleblind 1 complex. J Biol Chem 286:38427-38
Dansithong, Warunee; Wolf, Cordula M; Sarkar, Partha et al. (2008) Cytoplasmic CUG RNA foci are insufficient to elicit key DM1 features. PLoS One 3:e3968