Interaction of musleblind, CUG-BP1 and hnRNP H proteins in DM1-associated aberrant IR splicing

Paul, Sharan; Dansithong, Warunee; Kim, Dongho; Rossi, John J.; Webster, Nicholas J. G.; Comai, Lucio; Reddy, Sita

doi:10.1038/sj.emboj.7601296

Cited by 134 publications

(165 citation statements)

References 32 publications

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“…22. cDNA clones for hnRNP K (BC014980; Clone ID 4906241, catalogue number MHS1011-76638), DDX5 (BC016027; Clone ID 3528578, catalogue number MHS1011-169975), and DDX17 (BC000595; Clone ID 3345982, catalogue number MHS1011-59342) were purchased from Open Biosystems Inc. cDNA sequences for hnRNP H2 (NM_019597), hnRNP H3(2H9) (NM_012207), hnRNP F (NM_001098204), hnRNP L (NM_001005335), DHX9 (NM_001357), and hnRNP A2/B1 (NM_031243) derived from the NCBI DNA data base were used to design primers to PCR-amplify the coding sequences of these genes from a HeLa cell cDNA library.…”

Section: Dna Constructsmentioning

confidence: 99%

“…To ensure that the signals in the PCR analyses were not saturated, prior standardization experiments were carried out as described in Ref. 22. The percentage of exon inclusion (or exclusion) was calculated as [exon inclusion (or exclusion)/(exon inclusion ϩ exon exclusion)] ϫ 100.…”

Section: Pcr Analyses Of the Steady State Expression Levels Of Rna Anmentioning

confidence: 99%

“…Detection of CUG transcripts was carried out primarily as described (12,22). Briefly, DM1 myoblasts were plated on chamber slides overnight and then fixed in 4% paraformaldehyde/PBS for 20 min at room temperature.…”

Section: Fluorescence In Situ Hybridization (Fish)mentioning

confidence: 99%

“…To ensure that the signals in Western blot analysis were not saturated, prior standardization experiments were carried out as described in Ref. 22.…”

Section: Whole Cell Extracts and Subcellular Fraction Preparationsmentioning

confidence: 99%

“…The oligonucleotides were deprotected, and the complementary strands were annealed. The sequences of the siRNAs used in this study are: scrambled siRNA, 5Ј-GCGCGCUUUGUAGGAUUCGdTdT-3Ј; MBNL1, 5Ј-CACUGGAAGUAUGUAGAGAdTdT-3Ј (22); and PKC␣, 5Ј-AAAGGCUGAGGUUGCUGAUdTdT-3Ј (23).…”

Section: Cell Culturementioning

confidence: 99%

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

Paul

Dansithong

Jog

et al. 2011

Journal of Biological Chemistry

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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...

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Section: Dna Constructsmentioning

confidence: 99%

Section: Pcr Analyses Of the Steady State Expression Levels Of Rna Anmentioning

confidence: 99%

Section: Fluorescence In Situ Hybridization (Fish)mentioning

confidence: 99%

“…To ensure that the signals in Western blot analysis were not saturated, prior standardization experiments were carried out as described in Ref. 22.…”

Section: Whole Cell Extracts and Subcellular Fraction Preparationsmentioning

confidence: 99%

Section: Cell Culturementioning

confidence: 99%

See 3 more Smart Citations

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

Paul

Dansithong

Jog

et al. 2011

Journal of Biological Chemistry

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Myotonic Dystrophy and Developmental Regulation of RNA Processing

Thomas

Oliveira

Sznajder

et al. 2018

Comprehensive Physiology

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Myotonic dystrophy (DM) is a multisystemic disorder caused by microsatellite expansion mutations in two unrelated genes leading to similar, yet distinct, diseases. DM disease presentation is highly variable and distinguished by differences in age-of-onset and symptom severity. In the most severe form, DM presents with congenital onset and profound developmental defects. At the molecular level, DM pathogenesis is characterized by a toxic RNA gain-of-function mechanism that involves the transcription of noncoding microsatellite expansions. These mutant RNAs disrupt key cellular pathways, including RNA processing, localization, and translation. In DM, these toxic RNA effects are predominantly mediated through the modulation of the muscleblind-like and CUGBP and ETR-3-like factor families of RNA binding proteins (RBPs). Dysfunction of these RBPs results in widespread RNA processing defects culminating in the expression of developmentally inappropriate protein isoforms in adult tissues. The tissue that is the focus of this review, skeletal muscle, is particularly sensitive to mutant RNA-responsive perturbations, as patients display a variety of developmental, structural, and functional defects in muscle. Here, we provide a comprehensive overview of DM1 and DM2 clinical presentation and pathology as well as the underlying cellular and molecular defects associated with DM disease onset and progression. Additionally, fundamental aspects of skeletal muscle development altered in DM are highlighted together with ongoing and potential therapeutic avenues to treat this muscular dystrophy. © 2018 American Physiological Society. Compr Physiol 8:509-553, 2018.

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Insulin receptor isoforms: an integrated view focused on gestational diabetes mellitus

Westermeier

Sáez

Arroyo

et al. 2015

Diabetes Metabolism Res

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SummaryThe human insulin receptor (IR) exists in two isoforms that differ by the absence (IR-A) or the presence (IR-B) of a 12-amino acid segment encoded by exon 11. Both isoforms are functionally distinct regarding their binding affinities and intracellular signalling. However, the underlying mechanisms related to their cellular functions in several tissues are only partially understood. In this review, we summarize the current knowledge in this field regarding the alternative splicing of IR isoform, tissue-specific distribution and signalling both in physiology and disease, with an emphasis on the human placenta in gestational diabetes mellitus (GDM). Furthermore, we discuss the clinical relevance of IR isoforms highlighted by findings that show altered insulin signalling due to differential IR-A and IR-B expression in human placental endothelium in GDM pregnancies. Future research and clinical studies focused on the role of IR isoform signalling might provide novel therapeutic targets for treating GDM to improve the adverse maternal and neonatal outcomes.

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Interaction of musleblind, CUG-BP1 and hnRNP H proteins in DM1-associated aberrant IR splicing

Cited by 134 publications

References 32 publications

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

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

Myotonic Dystrophy and Developmental Regulation of RNA Processing

Insulin receptor isoforms: an integrated view focused on gestational diabetes mellitus

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