Expanded CUG Repeats Dysregulate RNA Splicing by Altering the Stoichiometry of the Muscleblind 1 Complex

Abstract

To understand the role of the splice regulator muscleblind 1 (MBNL1) in the development of RNA splice defects in myotonic dystrophy I (DM1), we purified RNA-independent MBNL1 complexes from normal human myoblasts and examined the behavior of these complexes in DM1 myoblasts. Antibodies recognizing MBNL1 variants (MBNL1 CUG ), which can sequester in the toxic CUG RNA foci that develop in DM1 nuclei, were used to purify MBNL1 CUG complexes from normal myoblasts. In normal myoblasts, MBNL1 CUG bind 10 proteins involved in remodeling ribonucleoprotein complexes including hnRNP H, H2, H3, F, A2/B1, K, L, DDX5, DDX17, and DHX9. Of these proteins, only MBNL1 CUG colocalizes extensively with DM1 CUG foci (>80% of foci) with its partners being present in <10% of foci. Importantly, the stoichiometry of MBNL1 CUG complexes is altered in DM1 myoblasts, demonstrating an increase in the steady state levels of nine of its partner proteins. These changes are recapitulated by the expression of expanded CUG repeat RNA in Cos7 cells. Altered stoichiometry of MBNL1 CUG complexes results from aberrant protein synthesis or stability and is unlinked to PKC␣ function. Modeling these changes in normal myoblasts demonstrates that increased levels of hnRNP H, H2, H3, F, and DDX5 independently dysregulate splicing in overlapping RNA subsets. Thus expression of expanded CUG repeats alters the stoichiometry of MBNL1 CUG complexes to allow both the reinforcement and expansion of RNA processing defects. Myotonic dystrophy I (DM1)4 is a multi-system disorder resulting from the expansion of a CTG repeat sequence in the 3Ј-untranslated region of DMPK, located on chromosome 19 (1-4). A key molecular consequence of expanded CUG repeat expression is the abnormal splicing of physiologically important RNAs. Specifically, abnormal splicing of the chloride channel (ClC-1) and the insulin receptor (IR) RNAs have been implicated in the development of myotonia and insulin resistance, respectively, in DM1 patients (5-8). In DM1 cells, expression of expanded CUG repeats results in the formation of aberrant nuclear CUG RNA aggregates or foci (9). The significance of nuclear CUG aggregate formation in the development of aberrant splicing and DM1 pathology is reflected in a recent study, which demonstrates that antisense RNA-mediated silencing of the mutant DMPK RNA and the consequent decrease of CUG foci allow the correction of splice defects in DM1 animal models (10). Significantly, CUG focus formation requires the binding of the alternative splice factor, muscleblind 1 (MBNL1) to the expanded CUG repeat expansions (11, 12). The importance of the aberrant MBNL1-CUG interaction in DM1 is underscored by the use of pentamidine and morpholino antisense oligonucleotides that dislodge MBNL1 from expanded CUG RNA, restore free MBNL1 levels, and rescue splice defects in DM1 mouse models (13,14). Consistent with these observations, inactivation of Mbnl1 in mice recapitulates a large fraction of the splice defects and several key features of DM1 pathology (15,16). Thus...

Nuclear ARVCF Protein Binds Splicing Factors and Contributes to the Regulation of Alternative Splicing

“…However, in higher eukaryotes, alternative splicing expands the complexity of the proteome in different tissues (56,57), and this is achieved through complex networks of protein-RNA interactions (58 -60). All three ARVCF-interacting proteins were reported to be involved in alternative splicing of specific genes (37,45,59,61). Upon knockdown of ARVCF, alternative splicing of several genes was significantly altered in our study.…”

“…Aberrant RNA processing by alternative splicing of genes related to schizophrenia had been frequently observed in the past (69,70), and this was correlated with the pathophysiology of the disorder. In future studies, it will be clarified whether ARVCF contributes to malfunctions in alternative splicing that may lead to aberrant pre-mRNA splicing events giving rise to neurological disorders, such as schizophrenia, or other human diseases, such as myotonic dystrophy or cancer (33,61,71,72).…”

Reduction of toxic RNAs in myotonic dystrophies type 1 and type 2 by the RNA helicase p68/DDX5

“…Recent analysis of the proteins bound to CUG repeats showed that p68 binds to CUG foci and modulates splicing activity of MBNL1 (14). It appears that p68 levels are increased in immortalized cultured DM1 myoblasts (17). However, p68 levels were reduced in DM1 biopsied muscle (Fig.…”

RNA-Binding Protein Misregulation in Microsatellite Expansion Disorders

“…HnRNP H protein levels are also elevated in DM1 myoblasts and hnRNP H or CELF1 overexpression leads to the formation of a repressor complex that inhibits splicing of insulin receptor (IR) exon 11 (Paul et al, 2006). HnRNP H exists in a complex with MBNL1 and 9 other proteins (hnRNP H2, H3, F, A2/B1, K, L, DDX5, DDX17, and DHX9) in normal myoblast extracts but the stoichiometry of these complexes is altered in DM1 extracts (Paul et al, 2011). Staufen1 (STAU1) is a double-stranded (ds)RNA-binding protein that is also mis-regulated in DM1 skeletal muscle (Ravel-Chapuis et al, 2012).…”