Selective inhibition of MBNL1�CCCUG interaction by small molecules toward potential therapeutic agents for myotonic dystrophy type 2 (DM2)

Myotonic dystrophy type 2 (DM2) is an incurable neuromuscular disease caused by expanded CCUG repeats that may exhibit toxicity by sequestering the splicing regulator MBNL1. A series of triaminotriazine- and triaminopyrimidine-based small molecules (ligands 1–3) were designed, synthesized and tested as inhibitors of the MBNL1–CCUG interaction. Despite the structural similarities of the triaminotriazine and triaminopyrimidine units, the triaminopyrimidine-based ligands bind with low micromolar affinity to CCUG repeats (K_d ∼ 0.1–3.6 µM) whereas the triaminotriazine ligands do not bind CCUG repeats. Importantly, these simple and small triaminopyrimidine ligands exhibit both strong inhibition (K_i ∼ 2 µM) of the MBNL1–CCUG interaction and high selectivity for CCUG repeats over other RNA targets. These experiments suggest these compounds are potential lead agents for the treatment of DM2.     

Muscleblind-like 1 interacts with RNA hairpins in splicing target and pathogenic RNAs

The MBNL and CELF proteins act antagonistically to control the alternative splicing of specific exons during mammalian postnatal development. This process is dysregulated in myotonic dystrophy because MBNL proteins are sequestered by (CUG)_n and (CCUG)_n RNAs expressed from mutant DMPK and ZNF9 genes, respectively. While these observations predict that MBNL proteins have a higher affinity for these pathogenic RNAs versus their normal splicing targets, we demonstrate that MBNL1 possesses comparably high affinities for (CUG)_n and (CAG)_n RNAs as well as a splicing target, Tnnt3. Mapping of a MBNL1-binding site upstream of the Tnnt3 fetal exon indicates that a preferred binding site for this protein is a GC-rich RNA hairpin containing a pyrimidine mismatch. To investigate how pathogenic RNAs sequester MBNL1 in DM1 cells, we used a combination of chemical/enzymatic structure probing and electron microscopy to determine that MBNL1 forms a ring-like structure which binds to the dsCUG helix. While the MBNL1 N-terminal region is required for RNA binding, the C-terminal region mediates homotypic interactions which may stabilize intra- and/or inter-ring interactions. Our results provide a mechanistic basis for dsCUG-induced MBNL1 sequestration and highlight a striking similarity in the binding sites for MBNL proteins on splicing precursor and pathogenic RNAs.     

MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1

The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families—muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins—are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5′ end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins.     

The four Zn fingers of MBNL1 provide a flexible platform for recognition of its RNA binding elements

Background  

Muscleblind-like 1 (MBNL1) is an alternative splicing factor containing four CCCH Zinc fingers (ZnFs). The sequestration of MBNL1 by expanded CUG and CCUG repeats is a major component in causing myotonic dystrophy. In addition to binding the structured expanded CUG and CCUG repeats; previous results suggested that MBNL1 binds single-stranded RNAs containing GC dinucleotides.     

PEX13 is required for thermogenesis of white adipose tissue in cold-exposed mice

Non-shivering thermogenesis (NST) is a heat generating process controlled by the mitochondria of brown adipose tissue (BAT). In the recent decade, ‘functionally’ acting brown adipocytes in white adipose tissue (WAT) has been identified as well: the so-called process of the ‘browning’ of WAT. While the importance of uncoupling protein 1 (UCP1)-oriented mitochondrial activation has been intensely studied, the role of peroxisomes during the browning of white adipocytes is poorly understood. Here, we assess the change in peroxisomal membrane proteins, or peroxins (PEXs), during cold stimulation and importantly, the role of PEX13 in the cold-induced remodeling of white adipocytes. PEX13, a protein that originally functions as a docking factor and is involved in protein import into peroxisome matrix, was highly increased during cold-induced recruitment of beige adipocytes within the inguinal WAT of C57BL/6 mice. Moreover, beige-induced 3 T3-L1 adipocytes and stromal vascular fraction (SVF) cells by exposure to the peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone showed a significant increase in mitochondrial thermogenic factors along with peroxisomal proteins including PEX13, and these were confirmed in SVF cells with the beta 3 adrenergic receptor (β3AR)-selective agonist CL316,243. To verify the relevance of PEX13, we used the RNA silencing method targeting the Pex13 gene and evaluated the subsequent beige development in SVF cells. Interestingly, siPex13 treatment suppressed expression of thermogenic proteins such as UCP1 and PPARγ coactivator 1 alpha (PGC1α). Overall, our data provide evidence supporting the role of peroxisomal proteins, in particular PEX13, during beige remodeling of white adipocytes.     

Increasing cAMP levels of preadipocytes by cyanidin-3-glucoside treatment induces the formation of beige phenotypes in 3T3-L1 adipocytes

Obesity is a serious health problem and a major risk factor for the onset of several diseases such as heart disease, diabetes, stroke and cancer. The conversion of white adipocytes to brown-like adipocytes, also called beige or brite adipocytes, by pharmacological and dietary compounds has gained attention as an effective treatment for obesity. Cyanidin-3-glucoside (Cy3G), a polyphenolic compound contained in black soybean, blueberry and grape, has several antiobesity effects. However, there are no reports on the role of Cy3G in the induction of differentiation of preadipocytes to beige adipocytes and corresponding phenotypes. Here, the formation of beige adipocyte phenotypes following treatment with Cy3G was evaluated using 3T3-L1 adipocytes. Cy3G induced phenotypic changes to white adipocytes, such as increased multilocular lipid droplets and mitochondrial content. Additionally, the expression of mitochondrial genes (TFAM, SOD2, UCP-1 and UCP-2), UCP-1 protein and beige adipocyte markers (CITED1 and TBX1) in 3T3-L1 adipocytes was increased by Cy3G. Furthermore, Cy3G promoted preadipocyte differentiation by up-regulating of C/EBPβ through the elevation of the intracellular cAMP levels. These results indicated that Cy3G elevates the intracellular cAMP levels, which induces beige adipocyte phenotypes. This is the first report on the effect of Cy3G on induction of differentiation of preadipocytes into beige adipocyte phenotypes.     

Dose-Dependent Regulation of Alternative Splicing by MBNL Proteins Reveals Biomarkers for Myotonic Dystrophy

Alternative splicing is a regulated process that results in expression of specific mRNA and protein isoforms. Alternative splicing factors determine the relative abundance of each isoform. Here we focus on MBNL1, a splicing factor misregulated in the disease myotonic dystrophy. By altering the concentration of MBNL1 in cells across a broad dynamic range, we show that different splicing events require different amounts of MBNL1 for half-maximal response, and respond more or less steeply to MBNL1. Motifs around MBNL1 exon 5 were studied to assess how cis-elements mediate the MBNL1 dose-dependent splicing response. A framework was developed to estimate MBNL concentration using splicing responses alone, validated in the cell-based model, and applied to myotonic dystrophy patient muscle. Using this framework, we evaluated the ability of individual and combinations of splicing events to predict functional MBNL concentration in human biopsies, as well as their performance as biomarkers to assay mild, moderate, and severe cases of DM.     

Sam68 Regulates S6K1 Alternative Splicing during Adipogenesis

The requirement for alternative splicing during adipogenesis is poorly understood. The Sam68 RNA binding protein is a known regulator of alternative splicing, and mice deficient for Sam68 exhibit adipogenesis defects due to defective mTOR signaling. Sam68 null preadipocytes were monitored for alternative splicing imbalances in components of the mTOR signaling pathway. Herein, we report that Sam68 regulates isoform expression of the ribosomal S6 kinase gene (Rps6kb1). Sam68-deficient adipocytes express Rps6kb1-002 and its encoded p31S6K1 protein, in contrast to wild-type adipocytes that do not express this isoform. Sam68 binds an RNA sequence encoded by Rps6kb1 intron 6 and prevents serine/arginine-rich splicing factor 1 (SRSF1)-mediated alternative splicing of Rps6kb1-002, as assessed by cross-linking and immunoprecipitation (CLIP) and minigene assays. Depletion of p31S6K1 with small interfering RNAs (siRNAs) partially restored adipogenesis of Sam68-deficient preadipocytes. The ectopic expression of p31S6K1 in wild-type 3T3-L1 cells resulted in adipogenesis differentiation defects, showing that p31S6K1 is an inhibitor of adipogenesis. Our findings indicate that Sam68 is required to prevent the expression of p31S6K1 in adipocytes for adipogenesis to occur.     

Single-molecule study of the CUG repeat–MBNL1 interaction and its inhibition by small molecules

Effective drug discovery and optimization can be accelerated by techniques capable of deconvoluting the complexities often present in targeted biological systems. We report a single-molecule approach to study the binding of an alternative splicing regulator, muscleblind-like 1 protein (MBNL1), to (CUG)_{n = 4,6} and the effect of small molecules on this interaction. Expanded CUG repeats (CUG^exp) are the causative agent of myotonic dystrophy type 1 by sequestering MBNL1. MBNL1 is able to bind to the (CUG)_n–inhibitor complex, indicating that the inhibition is not a straightforward competitive process. A simple ligand, highly selective for CUG^exp, was used to design a new dimeric ligand that binds to (CUG)_n almost 50-fold more tightly and is more effective in destabilizing MBNL1–(CUG)₄. The single-molecule method and the analysis framework might be extended to the study of other biomolecular interactions.     

Milk fat globule membrane and its component phosphatidylcholine induce adipose browning both in vivo and in vitro

The functional induction of brown-like adipocytes in white adipose tissue (WAT) provides a defense against obesity. The aim of this study was to analyze the effects of milk fat globule membrane (MFGM) and its component phosphatidylcholine (PC) on the brown remodeling of WAT. Male C57BL/6 J mice were fed a high-fat diet (HFD) for 8 weeks and then fed HFD for another 8 weeks with MFGM. In vitro studies were performed in C3H10T1/2 pluripotent stem cells, 3T3-L1 pre-adipocytes and differentiated inguinal WAT stromal vascular cells (SVCs) to determine the role of MFGM and PC on the formation of brown-like adipocytes. MFGM decreased fasting glucose and serum insulin levels in HFD-fed mice. MFGM improved glucose tolerance and insulin sensitivity, and induced browning of inguinal WAT. MFGM and its component PC stimulated transformation of brown-like adipocytes in C3H10T1/2 pluripotent stem cells, 3T3-L1 adipocytes and SVCs by increasing the protein expression of UCP1, PGC-1α, PRDM16 as well as the mRNA expression of other thermogenic genes and beige cell markers. MFGM and PC also increased mitochondrial DNA (mtDNA) copy number, mitochondrial density and oxygen consumption rate and up-regulated the mRNA expression of mitochondria-biogenesis-related genes in vitro. PPARα inhibitor GW6471 treatment or knockdown of PPARα using lentivirus-expressing shRNA inhibited the PC-induced increase in the protein expression of UCP1, PGC-1α and PRDM16 in C3H10T1/2 pluripotent stem cells and 3T3-L1 adipocytes, indicating the potential role of PPARα in PC-mediated brown-like adipocyte formation. In conclusion, MFGM and milk PC induced adipose browning, which has major protective effects against obesity and metabolic dysfunction.     

Leptin deficiency impairs adipogenesis and browning response in mouse mesenchymal progenitors

Although phenotypically different, brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT) are able to produce heat through non-shivering thermogenesis due to the presence of mitochondrial uncoupling protein 1 (UCP1). The appearance of thermogenically active beige adipocytes in iWAT is known as browning. Both brown and beige cells originate from mesenchymal stem cells (MSCs), and in culture conditions a browning response can be induced with hypothermia (i.e. 32 °C) during which nuclear leptin immunodetection was observed. The central role of leptin in regulating food intake and energy consumption is well recognised, but its importance in the browning process at the cellular level is unclear. Here, immunocytochemical analysis of MSC-derived adipocytes established nuclear localization of both leptin and leptin receptor suggesting an involvement of the leptin pathway in the browning response. In order to elucidate whether leptin modulates the expression of brown and beige adipocyte markers, BAT and iWAT samples from leptin-deficient (ob/ob) mice were analysed and exhibited reduced brown/beige marker expression compared to wild-type controls. When MSCs were isolated and differentiated into adipocytes, leptin deficiency was observed to induce a white phenotype, especially when incubated at 32 °C. These adaptations were accompanied with morphological signs of impaired adipogenic differentiation. Overall, our results indicate that leptin supports adipocyte browning and suggest a potential role for leptin in adipogenesis and browning.     

Mechanistic determinants of MBNL activity

Muscleblind-like (MBNL) proteins are critical RNA processing factors in development. MBNL activity is disrupted in the neuromuscular disease myotonic dystrophy type 1 (DM1), due to the instability of a non-coding microsatellite in the DMPK gene and the expression of CUG expansion (CUG^exp) RNAs. Pathogenic interactions between MBNL and CUG^exp RNA lead to the formation of nuclear complexes termed foci and prevent MBNL function in pre-mRNA processing. The existence of multiple MBNL genes, as well as multiple protein isoforms, raises the question of whether different MBNL proteins possess unique or redundant functions. To address this question, we coexpressed three MBNL paralogs in cells at equivalent levels and characterized both specific and redundant roles of these proteins in alternative splicing and RNA foci dynamics. When coexpressed in the same cells, MBNL1, MBNL2 and MBNL3 bind the same RNA motifs with different affinities. While MBNL1 demonstrated the highest splicing activity, MBNL3 showed the lowest. When forming RNA foci, MBNL1 is the most mobile paralog, while MBNL3 is rather static and the most densely packed on CUG^exp RNA. Therefore, our results demonstrate that MBNL paralogs and gene-specific isoforms possess inherent functional differences, an outcome that could be enlisted to improve therapeutic strategies for DM1.     

MBNL binds similar RNA structures in the CUG repeats of myotonic dystrophy and its pre-mRNA substrate cardiac troponin T

Myotonic dystrophy (DM) is a genetic disorder with multisystemic symptoms that is caused by expression (as RNA) of expanded repeats of CTG or CCTG in the genome. It is hypothesized that the RNA splicing factor muscleblind-like (MBNL) is sequestered to the expanded CUG or CCUG RNAs. Mislocalization of MBNL results in missplicing of a subset of pre-mRNAs that are linked to the symptoms found in DM patients. We demonstrate that MBNL can bind short structured CUG and CCUG repeats with high affinity and specificity. Only 6 base pairs are necessary for MBNL binding: two pyrimidine mismatches and four guanosine-cytosine base pairs in a stem. MBNL also has a preference for pyrimidine mismatches, but many other mismatches are tolerated with decreased affinity. We also demonstrate that MBNL binds the helical region of a stem-loop in the endogenous pre-mRNA target, the cardiac troponin T (cTNT) pre-mRNA. The stem-loop contains two mismatches and resembles both CUG and CCUG repeats. In vivo splicing results indicate that MBNL-regulated splicing is dependent upon the formation of stem-loops recognized by MBNL. These results suggest that MBNL may bind all of its RNA substrates, both normal and pathogenic, as structured stem-loops containing pyrimidine mismatches.     

Palmitoyl lactic acid induces adipogenesis and a brown fat-like phenotype in 3T3-L1 preadipocytes

Brown adipose tissue is specialized to generate heat by dissipating chemical energy and may provide novel strategies for obesity treatment in humans. Recently, advances in understanding the pharmacological and dietary agents that contribute to the browning of white adipose tissue have been made to alleviate obesity by promoting energy expenditure. Krill oil is widely used as a health supplement in humans. In this study, the components from krill oil that promote adipogenesis of 3T3-L1 cells were screened to reveal palmitoyl lactic acid (PLA) as a promoter of adipogenesis. The PLA-induced adipocytes contained large number of small lipid droplets. Moreover, similar to the peroxisome proliferator-activated receptor (PPAR)γ agonists, pioglitazone and rosiglitazone, PLA significantly enhances adipogenesis in the presence of dexamethasone compared with PLA alone. Treatment with PLA causes a brown fat-like phenotype in 3T3-L1 cells by enhanced expression of various brown/beige cell-specific genes, such as PR domain containing 16 (Prdm16) and peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (Pgc1a), as well as adiponectin gene. The expression profile of the brown/beige cell-specific genes induced by PLA was similar to that of the PPARγ agonist in 3T3-L1 cells. Our findings suggest that PLA induces a brown fat-like phenotype and, thus, likely has therapeutic potential in treating obesity.     

Analysis of exonic regions involved in nuclear localization, splicing activity, and dimerization of Muscleblind-like-1 isoforms

Muscleblind-like-1 (MBNL1) is a splicing regulatory factor controlling the fetal-to-adult alternative splicing transitions during vertebrate muscle development. Its capture by nuclear CUG expansions is one major cause for type 1 myotonic dystrophy (DM1). Alternative splicing produces MBNL1 isoforms that differ by the presence or absence of the exonic regions 3, 5, and 7. To understand better their respective roles and the consequences of the deregulation of their expression in DM1, here we studied the respective roles of MBNL1 alternative and constitutive exons. By combining genetics, molecular and cellular approaches, we found that (i) the exon 5 and 6 regions are both needed to control the nuclear localization of MBNL1; (ii) the exon 3 region strongly enhances the affinity of MBNL1 for its pre-mRNA target sites; (iii) the exon 3 and 6 regions are both required for the splicing regulatory activity, and this function is not enhanced by an exclusive nuclear localization of MBNL1; and finally (iv) the exon 7 region enhances MBNL1-MBNL1 dimerization properties. Consequently, the abnormally high inclusion of the exon 5 and 7 regions in DM1 is expected to enhance the potential of MBNL1 of being sequestered with nuclear CUG expansions, which provides new insight into DM1 pathophysiology.     

Tau exon 2 responsive elements deregulated in myotonic dystrophy type I are proximal to exon 2 and synergistically regulated by MBNL1 and MBNL2

The splicing of the microtubule-associated protein Tau is regulated during development and is found to be deregulated in a growing number of pathological conditions such as myotonic dystrophy type I (DM1), in which a reduced number of isoforms is expressed in the adult brain. DM1 is caused by a dynamic and unstable CTG repeat expansion in the DMPK gene, resulting in an RNA bearing long CUG repeats (n > 50) that accumulates in nuclear foci and sequesters CUG-binding splicing factors of the muscleblind-like (MBNL) family, involved in the splicing of Tau pre-mRNA among others. However, the precise mechanism leading to Tau mis-splicing and the role of MBNL splicing factors in this process are poorly understood. We therefore used new Tau minigenes that we developed for this purpose to determine how MBNL1 and MBNL2 interact to regulate Tau exon 2 splicing. We demonstrate that an intronic region 250 nucleotides downstream of Tau exon 2 contains cis-regulatory splicing enhancers that are sensitive to MBNL and that bind directly to MBNL1. Both MBNL1 and MBNL2 act as enhancers of Tau exon 2 inclusion. Intriguingly, the interaction of MBNL1 and MBNL2 is required to fully reverse the mis-splicing of Tau exon 2 induced by the trans-dominant effect of long CUG repeats, similar to the DM1 condition. In conclusion, both MBNL1 and MBNL2 are involved in the regulation of Tau exon 2 splicing and the mis-splicing of Tau in DM1 is due to the combined inactivation of both.