Biology subjects

Wang, E. T.

Publications and source records attributed to Wang, E. T..

13 recordsLinked to original sources

bioRxiv2026

Muscleblind-like proteins dimerize by forming disulfide bonds to regulate alternative splicing and pathogenic RNA foci formation

Muscleblind-like (MBNL) RNA-binding proteins (RBPs) possess modular domains that mediate regulation of alternative splicing and RNA localization. Myotonic Dystrophy Type 1 is a CTG repeat expansion disorder where MBNL is sequestered into intranuclear RNA foci, impairing its function. Previous studies found that MBNL self-associates through its exon 7, but the nature of this interaction is not well understood. We identified a cysteine in MBNL1 exon 7 that enables dimerization through formation of an intermolecular disulfide bond. We likewise demonstrate that MBNL2 dimerizes by forming disulfide bonds between multiple cysteines in its carboxy-terminus. Nucleocytoplasmic fractionation revealed a greater proportion of MBNL1 dimer in the nucleus, suggesting a nuclear function for the MBNL1 dimer. We investigated a connection between MBNL1 dimerization and MBNL1-mediated regulation of alternative splicing. To accomplish this, we mutated the MBNL1 cysteine in question to alanine (C325A) and performed RNAseq. We uncovered novel splicing events sensitive to MBNL1 dimerization. We also found that MBNL1 C325A, when co-expressed with expanded CTG repeats, produces smaller, more numerous foci, suggesting a role for the MBNL1 dimer in maintaining foci integrity. These results provide insight into biological and pathological mechanisms of MBNL1 dimerization and suggest other RBPs might similarly dimerize to regulate function. GRAPHICAL ABSTRACT

molecular biology↗

bioRxiv2025

Metformin decreases RAN proteins, rescues splicing abnormalities and improves behavioral phenotypes in SCA8 BAC mice

Spinocerebellar ataxia type 8 is one member of a larger group of dominantly inherited, debilitating neurological diseases caused by CTG*CAG expansions for which there are no effective disease-targeting treatments. RAN translation, which was discovered in SCA8, has previously been shown to occur across CAG and CUG expansion transcripts, making treatments that work for SCA8 potentially relevant to a much broader group of diseases, including SCA1, 2, 3, 6, 7, 12, Huntingtons Disease (HD) Fuchs endothelial corneal dystrophy (FECD), and myotonic dystrophy type 1 (DM1). The FDA-approved drug, metformin, has been previously shown to reduce RAN protein levels in cells overexpressing SCA8 CAG repeats. Here we show, using SCA8 BAC transgenic mice, that metformin treatment improves ambulatory performance, including rotarod, DigiGait, and open field measures. At the molecular level, metformin-treated mice show reduced RAN protein levels and improved splicing abnormalities without changing the levels of the expanded RNAs. Metformin-treated mice also show decreased neuroinflammation with reduced levels of astrogliosis and reduced numbers of activated microglia. Taken together, these data provide strong support for testing FDA-approved metformin in clinical trials for SCA8 and potentially the broader group of CAG*CTG repeat expansion disorders. One Sentence SummaryMetformin improves behavior, neuropathological and molecular phenotypes in SCA8 BAC transgenic mice.

neuroscience↗

bioRxiv2025

Myonuclear domain-associated and central nucleation-dependent spatial restriction of dystrophin protein expression in a novel DMD mouse model

The restoration of uniformly-distributed dystrophin protein expression is an important consideration for the development of advanced therapeutics for Duchenne muscular dystrophy (DMD). To explore this concept, we generated a novel genetic mouse model (mdx52-Xist{Delta}hs) that expresses variable, and non-uniformly distributed, dystrophin protein from birth as a consequence of skewed X-chromosome inactivation. mdx52-Xist{Delta}hs myofibers are heterokaryons containing a mixture of myonuclei expressing either wild-type or mutant dystrophin alleles in a mutually exclusive manner, resulting in dystrophin protein being spatially restricted to corresponding dystrophin-expressing myonuclear domains. This phenotype models the situation in female DMD carriers, and dystrophic muscle in which dystrophin has been incompletely restored by partially-effective experimental therapeutics. Total dystrophin expression increased in aged (60-week-old) mdx52-Xist{Delta}hs mice relative to 6-week-old adults, suggestive of an accumulation of dystrophin-expressing myonuclei through positive selection, although this was insufficient to resolve sarcolemmal dystrophin patchiness. Nevertheless, compared to mice expressing no dystrophin, non-uniformly-distributed dystrophin was protective against pathology-related muscle turnover in an expression-level-dependent manner in both adult and aged mdx52-Xist{Delta}hs mice. Systematic classification of isolated mdx52-Xist{Delta}hs myofibers revealed profound differences associated with central nucleation, with dystrophin found to be translationally repressed in centrally-nucleated myofibers and myofiber segments. These findings have important implications for the development of dystrophin restoration therapies.

cell biology↗

bioRxiv2023

Myospreader improves gene editing in skeletal muscle by myonuclear propagation

Successful CRISPR/Cas9-based gene editing in skeletal muscle is dependent on efficient propagation of Cas9 to all myonuclei in the myofiber. However, nuclear-targeted gene therapy cargos are strongly restricted to their myonuclear domain of origin. By screening nuclear localization signals and nuclear export signals, we identify "Myospreader", a combination of short peptide sequences that promotes myonuclear propagation. Appending Myospreader to Cas9 enhances protein stability and myonuclear propagation in myoblasts and myofibers. AAV-delivered Myospreader dCas9 better inhibits transcription of toxic RNA in a myotonic dystrophy mouse model. Furthermore, Myospreader Cas9 achieves higher rates of gene editing in CRISPR reporter and Duchenne muscular dystrophy mouse models. Myospreader reveals design principles relevant to all nuclear-targeted gene therapies and highlights the importance of the spatial dimension in therapeutic development.

cell biology↗

bioRxiv2022

Muscleblind-like proteins use modular domains to localize RNAs by riding kinesins and docking to membranes

RNA transport and local translation provide spatial control of gene expression, and RNA binding proteins (RBPs) act as critical adapters in this multi-step process. Muscleblind-like (MBNL) RNA binding proteins, implicated in myotonic dystrophy and cancer, localize RNAs to myoblast membranes and distal neurites through unknown mechanisms. We found that MBNL forms motile and anchored granules in neurons and myoblasts, and selectively associates with kinesins Kif1b and Kif1c through its zinc finger (ZnF) domains. Other RBPs with similar ZnFs also associate with these kinesins, implicating a motor-RBP specificity code. Live cell imaging and fractionation revealed that membrane anchoring is mediated through the unstructured carboxy-terminal tail of MBNL1. Both kinesin- and membrane-recruitment functions were reconstituted using MBNL-MS2 coat protein fusions. This approach, termed RBP Module Recruitment and Imaging (RBP-MRI), decouples RNA binding, kinesin recruitment, and membrane anchoring functions, while also establishing general strategies for studying multi-functional, modular domains of RBPs.

cell biology↗

bioRxiv2021

Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells

Cas13 is a unique family of CRISPR endonucleases exhibiting programmable binding and cleavage of RNAs and is a strong candidate for eukaryotic RNA knockdown in the laboratory and the clinic. However, sequence-specific binding of Cas13 to the target RNA unleashes non-specific bystander RNA cleavage, or collateral activity, which may confound knockdown experiments and raises concerns for therapeutic applications. Although conserved across orthologs and robust in cell-free and bacterial environments, the extent of collateral activity in mammalian cells remains disputed. Here, we investigate Cas13d collateral activity in the context of an RNA-targeting therapy for myotonic dystrophy type 1, a disease caused by a transcribed long CTG repeat expansion. We find that when targeting CUGn RNA in HeLa and other cell lines, Cas13d depletes endogenous and transgenic RNAs, interferes with critical cellular processes, and activates stress response and apoptosis pathways. We also observe collateral effects when targeting other repetitive and unique transgenic sequences, and we provide evidence for collateral activity when targeting highly expressed endogenous transcripts. To minimize collateral activity for repeat-targeting Cas13d therapeutics, we introduce gRNA excision for negative-autoregulatory optimization (GENO), a simple strategy that leverages crRNA processing to control Cas13d expression and is easily integrated into an AAV gene therapy. We argue that thorough assessment of collateral activity is necessary when applying Cas13d in mammalian cells and that implementation of GENO illustrates the advantages of compact and universally robust regulatory systems for Cas-based gene therapies. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

bioRxiv2021

A comprehensive atlas of fetal splicing patterns in the brain of adult myotonic dystrophy type 1 patients

In patients with myotonic dystrophy type 1 (DM1), dysregulation of RNA-binding proteins like MBNL and CELF1 leads to alternative splicing of exons and is thought to induce a return to fetal splicing patterns in adult tissues, including the central nervous system (CNS). To comprehensively evaluate this, we created an atlas of developmentally regulated splicing patterns in the frontal cortex of healthy individuals and DM1 patients by combining RNA-seq data from BrainSpan, GTEx and DM1 patients. Thirty four splice events displayed an inclusion pattern in DM1 patients that is typical for the fetal situation in healthy individuals. The regulation of DM1-relevant splicing patterns could partly be explained by changes in mRNA expression of the splice regulators MBNL1, MBNL2 and CELF1. On the contrary, interindividual differences in splicing patterns between healthy adults could not be explained by differential expression of these splice regulators. Our findings lend transcriptome-wide evidence to the previously noted shift to fetal splicing patterns in the adult DM1 brain as a consequence of an imbalance in antagonistic MBNL and CELF1 activities. Our atlas serves as a solid foundation for further study and understanding of the cognitive phenotype in patients.

bioinformatics↗

bioRxiv2021

Sex differences in microRNA expression in first and third trimester human placenta

Maternal and fetal pregnancy outcomes related to placental function vary based on fetal sex, which may be the result of sexually dimorphic epigenetic regulation of RNA expression. We identified sexually dimorphic miRNA expression throughout gestation in human placentae. Next-generation sequencing was used to identify miRNA expression profiles in first and third trimester uncomplicated pregnancies using tissue obtained at chorionic villous sampling (n=113) and parturition (n=47). Sequencing and differential expression (DE) analysis identified 432 mature miRNAs expressed in the first trimester female, 425 in the first trimester male, 400 in the third trimester female, and 508 in the third trimester male placenta (baseMean >10). Of these, 11 sexually dimorphic (FDR<0.05, baseMean >10) miRNAs were identified in the first and 4 miRNAs were identified in the third trimester, including miR-361-5p, significant in both trimesters, all upregulated in females. Across gestation, 207 miRNAs were DE across gestation, common to both females and males, miR-4483, the most DE across gestation. There were twice as many female-specific differences across gestation as male-specific (44 miRNAs vs 21 miRNAs), indicating that miRNA abundance across human gestation is sexually dimorphic. Pathway enrichment analysis identified significant pathways that were differentially regulated in first and third trimester as well as across gestation. This work provides the normative sex dimorphic miRNA atlas in first and third trimester, as well as the sex independent and sex specific placenta miRNA atlas across gestation which may be used to identify biomarkers of placental function and direct functional studies investigating placental sex differences. Summary SentenceSex dimorphism in miRNA expression is more pronounced in first compared to third trimester placenta, and there are twice as many female-specific gestational differences, indicating miRNA abundance across human gestation is also sexually dimorphic.

developmental biology↗

bioRxiv2021

Microtubule-based Transport is Essential to Distribute RNA and Nascent Protein in Skeletal Muscle

While the importance of RNA localization in highly differentiated cells is well appreciated, basic principles of RNA localization in skeletal muscle remain poorly characterized. Here, we develop a method to detect single RNA molecules and quantify localization patterns in skeletal myofibers, and we uncover a critical and general role for directed transport of RNPs in muscle. We find that RNAs are localized and translated along cytoskeletal filaments, and we identify the Z-disk as a biological hub for RNA localization and protein synthesis. We show that muscle development triggers complete reliance on the lattice-like microtubule network to transport RNAs and that disruption of microtubules leads to striking accumulation of RNPs and nascent protein around myonuclei. Our observations suggest that active transport may be globally required to distribute RNAs in highly differentiated cells and reveal fundamental mechanisms relevant to myopathies caused by perturbations to RNPs, microtubules, and the nuclear envelope. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/433059v2_ufig1.gif" ALT="Figure 1"> View larger version (88K): org.highwire.dtl.DTLVardef@1d801ddorg.highwire.dtl.DTLVardef@625623org.highwire.dtl.DTLVardef@54230org.highwire.dtl.DTLVardef@13c95be_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2020

Transcriptome Alterations in Myotonic Dystrophy Frontal Cortex

Myotonic dystrophy (dystrophia myotonica, DM) is caused by expanded CTG/CCTG microsatellite repeats, leading to multi-systemic symptoms in skeletal muscle, heart, gastrointestinal, endocrine, and central nervous systems (CNS), among others. For some patients, CNS issues can be as debilitating or more so than muscle symptoms; they include hypersomnolence, executive dysfunction, white matter atrophy, and neurofibrillary tangles. Although transcriptomes from DM type 1 (DM1) skeletal muscle have provided useful insights into pathomechanisms and biomarkers, limited studies of transcriptomes have been performed in the CNS. To elucidate underlying causes of CNS dysfunction in patients, we have generated and analyzed RNA-seq transcriptomes from the frontal cortex of 21 DM1 patients, 4 DM type 2 (DM2) patients, and 8 unaffected controls. One hundred and thirty high confidence splicing changes were identified, most occurring exclusively in the CNS and not in skeletal muscle or heart. Mis-spliced exons were found in neurotransmitter receptors, ion channels, and synaptic scaffolds, and we identified an alternative exon in GRIP1 that modulates association with kinesins. Splicing changes exhibited a gradient of severity correlating with CTG repeat length, as measured by optical mapping of individual DNA molecules. All individuals studied, including those with modest splicing defects, showed extreme somatic mosaicism, with a subset of alleles having >1000 CTGs. Analyses of gene expression changes showed up-regulation of genes transcribed in microglia and endothelial cells, suggesting neuroinflammation, and downregulation of genes transcribed in neurons. Gene expression of RNAs encoding proteins detectable in cerebrospinal fluid were also found to correlate with mis-splicing, with implications for CNS biomarkers of disease severity. These findings provide a framework for future mechanistic and therapeutic studies of CNS issues in DM.

genetics↗

bioRxiv2019

A Toxic RNA Catalyzes the Cellular Synthesis of Its Own Inhibitor, Shunting It to Endogenous Decay Pathways

Myotonic dystrophy type 2 (DM2) is a genetically defined muscular dystrophy caused by a toxic expanded repeat of r(CCUG) [heretofore (CCUG)exp], harbored in intron 1 of CHC-Type Zinc Finger Nucleic Acid Binding Protein (CNBP) pre-mRNA. This r(CCUG)exp causes DM2 via a gain-of-function mechanism that results in three hallmarks of its pathology: (i) binding to RNA-binding proteins (RBPs) that aggregate into nuclear foci; (ii) sequestration of muscleblind-like-1 (MBNL1) protein, a regulator of alternative pre-mRNA splicing, leading to splicing defects; and (iii) retention of intron 1 in the CNBP mRNA. Here, we find that CNBP intron retention is caused by the r(CCUG)exp-MBNL1 complex and can be rescued by small molecules. We studied two types of small molecules with different modes of action, ones that simply bind and ones that can be synthesized by a r(CCUG)exp-templated reaction in cells, that is the RNA synthesizes its own drug. Indeed, our studies completed in DM2 patient-derived fibroblasts show that the compounds disrupt the r(CCUG)exp-MBNL1 complex, reduce intron retention, subjecting the liberated intronic r(CCUG)exp to native decay pathways, and rescue other DM2-associated cellular defects. Collectively, this study shows that small molecules can affect RNA biology by shunting toxic transcripts towards native decay pathways.\n\nHIGHLIGHTSO_LIIntron retention in RNA repeat expansions can be due to repeats binding to proteins\nC_LIO_LISmall molecules that bind RNA repeats and inhibit protein binding can trigger decay\nC_LIO_LIA toxic RNA repeat can catalyze the synthesis of its own inhibitor on-site\nC_LIO_LIOn-site drug synthesis most potently affects disease biology\nC_LI\n\neTOC BLURBThe most common way to target RNA is to use antisense oligonucleotides to target unstructured RNAs for destruction. Here, we show for the first time that small molecules targeting structured, disease-causing RNAs can shunt them towards native decay pathways by affecting their processing.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC=\"FIGDIR/small/741926v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (21K):\norg.highwire.dtl.DTLVardef@1ff5d1forg.highwire.dtl.DTLVardef@3adccborg.highwire.dtl.DTLVardef@75caf0org.highwire.dtl.DTLVardef@15e58f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2019

The maternal-fetal interface of successful pregnancies and impact of fetal sex using single cell sequencing

The first trimester is a critical window of maternal-fetal communication for pregnancy. Therefore, we characterized crosstalk in ongoing human pregnancies at 11-13 weeks gestation. RNA-sequencing of matched maternal decidua and placenta identified 818 receptors and 3502 ligands, including 126 differentially expressed receptor-ligand pairs. Using single cell RNA-sequencing to further dissect placenta heterogeneity, we identified five major cell types (trophoblasts, stromal cells, hofbauer cells, antigen presenting cells and endothelial cells) with unique crosstalk at the maternal-fetal interface. We identified seven unique trophoblast subclusters, including new subtypes that transition into the terminal cell types, extra-villous trophoblasts and syncytiotrophoblasts. As fetal sex impacts pregnancy, we analyzed sex differences in each cell type and identified differences in immune cell function. TGF{beta}1, {beta}-estradiol, and dihydrotestosterone emerge as upstream regulators of sexually dimorphic genes in a cell type specific manner. Thus, the fetal contribution at the maternal-fetal interface is cell and sex specific.

developmental biology↗

bioRxiv2017

Impeding transcription of expanded microsatellite repeats by deactivated Cas9

Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs endothelial corneal dystrophy, and C9orf72-ALS/FTD. Eliminating or reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that a deactivated form of the Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction in the abundance of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, and C9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.

molecular biology↗