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.
|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|