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.

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

RNA binding proteins (RBPs) act as critical facilitators of spatially regulated gene expression. Muscleblind-like (MBNL) proteins, implicated in myotonic dystrophy and cancer, localize RNAs to myoblast membranes and neurites through unknown mechanisms. We find that MBNL forms motile and anchored granules in neurons and myoblasts, and selectively associates with kinesins Kif1bα and Kif1c through its zinc finger (ZnF) domains. Other RBPs with similar ZnFs associate with these kinesins, implicating a motor-RBP specificity code. MBNL and kinesin perturbation leads to widespread mRNA mis-localization, including depletion of Nucleolin transcripts from neurites. Live cell imaging and fractionation reveal that the unstructured carboxy-terminal tail of MBNL1 allows for anchoring at membranes. An approach, termed RBP Module Recruitment and Imaging (RBP-MRI), reconstitutes kinesin- and membrane-recruitment functions using MBNL-MS2 coat protein fusions. Our findings decouple kinesin association, RNA binding, and membrane anchoring functions of MBNL while establishing general strategies for studying multi-functional, modular domains of RBPs.

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

CRISPR-Cas13 RNA endonucleases show promise for programmable RNA knockdown. However, sequence-specific binding of Cas13 unleashes non-specific bystander RNA cleavage, or collateral activity, raising concerns for experiments and therapeutic applications. Although robust in cell-free and bacterial environments, collateral activity in mammalian cells remains disputed. We investigate Cas13d collateral activity in a therapeutic context for myotonic dystrophy type 1, caused by a transcribed CTG repeat expansion. We find that, when targeting CUGn RNA in mammalian cells, Cas13d depletes endogenous and transgenic RNAs, interferes with critical cellular processes, and activates stress response and apoptosis. Collateral effects also occur when targeting abundant endogenous transcripts. To minimize collateral activity for repeat-targeting approaches, we introduce GENO, an adeno-associated virus-compatible strategy that leverages guide RNA processing to control Cas13d expression. We argue that thorough assessment of collateral activity is necessary when applying Cas13 in mammalian cells and that GENO illustrates advantages of compact regulatory systems for Cas-based gene therapies.

Small-molecule targeted recruitment of a nuclease to cleave an oncogenic RNA in a mouse model of metastatic cancer.

As the area of small molecules interacting with RNA advances, general routes to provide bioactive compounds are needed as ligands can bind RNA avidly to sites that will not affect function. Small-molecule targeted RNA degradation will thus provide a general route to affect RNA biology. A non-oligonucleotide-containing compound was designed from sequence to target the precursor to oncogenic microRNA-21 (pre-miR-21) for enzymatic destruction with selectivity that can exceed that for protein-targeted medicines. The compound specifically binds the target and contains a heterocycle that recruits and activates a ribonuclease to pre-miR-21 to substoichiometrically effect its cleavage and subsequently impede metastasis of breast cancer to lung in a mouse model. Transcriptomic and proteomic analyses demonstrate that the compound is potent and selective, specifically modulating oncogenic pathways. Thus, small molecules can be designed from sequence to have all of the functional repertoire of oligonucleotides, including inducing enzymatic degradation, and to selectively and potently modulate RNA function in vivo.

Aberrant Myokine Signaling in Congenital Myotonic Dystrophy.

Myotonic dystrophy types 1 (DM1) and 2 (DM2) are dominantly inherited neuromuscular disorders caused by a toxic gain of function of expanded CUG and CCUG repeats, respectively. Although both disorders are clinically similar, congenital myotonic dystrophy (CDM), a severe DM form, is found only in DM1. CDM is also characterized by muscle fiber immaturity not observed in adult DM, suggesting specific pathological mechanisms. Here, we revealed upregulation of the interleukin-6 (IL-6) myokine signaling pathway in CDM muscles. We also found a correlation between muscle immaturity and not only IL-6 expression but also expanded CTG repeat length and CpG methylation status upstream of the repeats. Aberrant CpG methylation was associated with transcriptional dysregulation at the repeat locus, increasing the toxic RNA burden that upregulates IL-6. Because the IL-6 pathway is involved in myocyte maturation and muscle atrophy, our results indicate that enhanced RNA toxicity contributes to severe CDM phenotypes through aberrant IL-6 signaling.

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. Reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that deactivated Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences; targeting the non-template strand was more effective. 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.

A Defective mRNA Cleavage and Polyadenylation Complex Facilitates Expansions of Transcribed (GAA)n Repeats Associated with Friedreich's Ataxia.

Expansions of microsatellite repeats are responsible for numerous hereditary diseases in humans, including myotonic dystrophy and Friedreich's ataxia. Whereas the length of an expandable repeat is the main factor determining disease inheritance, recent data point to genomic trans modifiers that can impact the likelihood of expansions and disease progression. Detection of these modifiers may lead to understanding and treating repeat expansion diseases. Here, we describe a method for the rapid, genome-wide identification of trans modifiers for repeat expansion in a yeast experimental system. Using this method, we found that missense mutations in the endoribonuclease subunit (Ysh1) of the mRNA cleavage and polyadenylation complex dramatically increase the rate of (GAA)n repeat expansions but only when they are actively transcribed. These expansions correlate with slower transcription elongation caused by the ysh1 mutation. These results reveal an interplay between RNA processing and repeat-mediated genome instability, confirming the validity of our approach.

Myotonic dystrophy: disease repeat range, penetrance, age of onset, and relationship between repeat size and phenotypes.

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease primarily characterized by myotonia and progressive muscle weakness. The pathogenesis of DM involves microsatellite expansions in noncoding regions of transcripts that result in toxic RNA gain-of-function. Each successive generation of DM families carries larger repeat expansions, leading to an earlier age of onset with increasing disease severity. At present, diagnosis of DM is challenging and requires special genetic testing to account for somatic mosaicism and meiotic instability. While progress in genetic testing has been made, more rapid, accurate, and cost-effective approaches for measuring repeat lengths are needed to establish clear correlations between repeat size and disease phenotypes.

Antisense transcription of the myotonic dystrophy locus yields low-abundant RNAs with and without (CAG)n repeat.

The unstable (CTG·CAG)n trinucleotide repeat in the myotonic dystrophy type 1 (DM1) locus is bidirectionally transcribed from genes with terminal overlap. By transcription in the sense direction, the DMPK gene produces various alternatively spliced mRNAs with a (CUG)n repeat in their 3' UTR. Expression in opposite orientation reportedly yields (CAG)n-repeat containing RNA, but both structure and biologic significance of this antisense gene (DM1-AS) are largely unknown. Via a combinatorial approach of computational and experimental analyses of RNA from unaffected individuals and DM1 patients we discovered that DM1-AS spans >6 kb, contains alternative transcription start sites and uses alternative polyadenylation sites up- and downstream of the (CAG)n repeat. Moreover, its primary transcripts undergo alternative splicing, whereby the (CAG)n segment is removed as part of an intron. Thus, in patients a mixture of DM1-AS RNAs with and without expanded (CAG)n repeat are produced. DM1-AS expression appears upregulated in patients, but transcript abundance remains very low in all tissues analyzed. Our data suggest that DM1-AS transcripts belong to the class of long non-coding RNAs. These and other biologically relevant implications for how (CAG)n-expanded transcripts may contribute to DM1 pathology can now be explored experimentally.

Distal Alternative Last Exons Localize mRNAs to Neural Projections.

Spatial restriction of mRNA to distinct subcellular locations enables local regulation and synthesis of proteins. However, the organizing principles of mRNA localization remain poorly understood. Here we analyzed subcellular transcriptomes of neural projections and soma of primary mouse cortical neurons and two neuronal cell lines and found that alternative last exons (ALEs) often confer isoform-specific localization. Surprisingly, gene-distal ALE isoforms were four times more often localized to neurites than gene-proximal isoforms. Localized isoforms were induced during neuronal differentiation and enriched for motifs associated with muscleblind-like (Mbnl) family RNA-binding proteins. Depletion of Mbnl1 and/or Mbnl2 reduced localization of hundreds of transcripts, implicating Mbnls in localization of mRNAs to neurites. We provide evidence supporting a model in which the linkage between genomic position of ALEs and subcellular localization enables coordinated induction of localization-competent mRNA isoforms through a post-transcriptional regulatory program that is induced during differentiation and reversed in cellular reprogramming and cancer.

Characterization of FUS mutations in amyotrophic lateral sclerosis using RNA-Seq.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting in severe muscle weakness and eventual death by respiratory failure. Although little is known about its pathogenesis, mutations in fused in sarcoma/translated in liposarcoma (FUS) are causative for familial ALS. FUS is a multifunctional protein that is involved in many aspects of RNA processing. To elucidate the role of FUS in ALS, we overexpressed wild-type and two mutant forms of FUS in HEK-293T cells, as well as knocked-down FUS expression. This was followed by RNA-Seq to identify genes which displayed differential expression or altered splicing patterns. Pathway analysis revealed that overexpression of wild-type FUS regulates ribosomal genes, whereas knock-down of FUS additionally affects expression of spliceosome related genes. Furthermore, cells expressing mutant FUS displayed global transcription patterns more similar to cells overexpressing wild-type FUS than to the knock-down condition. This observation suggests that FUS mutants do not contribute to the pathogenesis of ALS through a loss-of-function. Finally, our results demonstrate that the R521G and R522G mutations display differences in their influence on transcription and splicing. Taken together, these results provide additional insights into the function of FUS and how mutations contribute to the development of ALS.

Molecular classification of thyroid nodules using high-dimensionality genomic data.

OBJECTIVE: We set out to develop a molecular test that distinguishes benign and malignant thyroid nodules using fine-needle aspirates (FNA). DESIGN: We used mRNA expression analysis to measure more than 247,186 transcripts in 315 thyroid nodules, comprising multiple subtypes. The data set consisted of 178 retrospective surgical tissues and 137 prospectively collected FNA samples. Two classifiers were trained separately on surgical tissues and FNAs. The performance was evaluated using an independent set of 48 prospective FNA samples, which included 50% with indeterminate cytopathology. RESULTS: Performance of the tissue-trained classifier was markedly lower in FNAs than in tissue. Exploratory analysis pointed to differences in cellular heterogeneity between tissues and FNAs as the likely cause. The classifier trained on FNA samples resulted in increased performance, estimated using both 30-fold cross-validation and an independent test set. On the test set, negative predictive value and specificity were estimated to be 96 and 84%, respectively, suggesting clinical utility in the management of patients considering surgery. Using in silico and in vitro mixing experiments, we demonstrated that even in the presence of 80% dilution with benign background, the classifier can correctly recognize malignancy in the majority of FNA samples. CONCLUSIONS: The FNA-trained classifier was able to classify an independent set of FNAs in which substantial RNA degradation had occurred and in the presence of blood. High tolerance to dilution makes the classifier useful in routine clinical settings where sampling error may be a concern. An ongoing multicenter clinical trial will allow us to validate molecular test performance on a larger independent test set of prospectively collected thyroid FNAs.

Biomechanical forces in atherosclerosis-resistant vascular regions regulate endothelial redox balance via phosphoinositol 3-kinase/Akt-dependent activation of Nrf2.

Local patterns of biomechanical forces experienced by endothelial cells (ECs) in different vascular geometries appear to play an essential role in regulating EC function and determining the regional susceptibility to atherosclerosis, even in the face of systemic risk factors. To study how biomechanical forces regulate EC redox homeostasis, an important pathogenic factor in atherogenesis, we have cultured human ECs under 2 prototypic arterial shear stress waveforms, "atheroprone" and "atheroprotective," which were derived from 2 distinct vascular regions in vivo that are typically "susceptible" or "resistant" to atherosclerosis. We demonstrate that atheroprotective flow decreases EC intracellular redox level and protects ECs against oxidative stress-induced injury. To identify the molecular mechanisms that control this cellular response, we examined several major oxidative/antioxidative pathways and found that atheroprotective flow upregulated certain antioxidant genes and strongly activated the transcription factor Nrf2. Using a strategy of small interfering RNA inhibition of Nrf2 expression combined with genome-wide transcriptional profiling, we determined the downstream targets of Nrf2 activation and identified Nrf2 as a critical determinant for the changes in endothelial redox balance exerted by atheroprotective flow. In addition, we showed that atheroprotective flow activates Nrf2 via the phosphoinositol 3-kinase/Akt pathway, and this activation occurs differentially in atherosclerosis-resistant and atherosclerosis-susceptible regions of the mouse aorta. Taken together, our data demonstrate that hemodynamic forces present in atherosclerosis-resistant and -susceptible regions of the vasculature differentially regulate EC redox state and antioxidant potential. These alterations in redox homeostasis are primarily the result of the phosphoinositol 3-kinase/Akt-dependent activation of Nrf2 and its downstream transcriptional targets.

Gene expression changes evoked in a venous segment exposed to arterial flow.

OBJECTIVE: This study was conducted to characterize the coordinated molecular changes evoked in the structure and composition of the wall of a venous segment when exposed to fistula flow. METHODS: An arteriovenous shunt was created in adult C57BL/6J mice. Remodeled veins and contralateral control jugular veins were isolated 7 days after surgery. Total RNA was isolated, linearly amplified, and the transcriptional profiles of this early adaptive response were obtained by microarray analysis. Histologic and immunohistochemical analyses were performed on remodeled veins and control veins isolated on days 1, 3, 5, and 7 after surgery to further examine distinct spatial and temporal aspects of this early process. RESULTS: There were 131 significantly upregulated and 165 downregulated genes in the remodeled vein compared with the control jugular vein. Genes involved in extracellular matrix reorganization were highly upregulated. Movat's pentachrome staining revealed ground substance on day 3 that was not observed on day 5. The appearance of elastin fibers was first observed on day 7. Morphometric analysis demonstrated maximum wall thickness on day 3. Immunohistochemical analysis revealed the presence of tenascin-C, thrombospondin, lysyl oxidase, and osteopontin in different cell types at different time points throughout the first week after surgery. CONCLUSION: Major changes in the organization of the extracellular matrix occur during the early response of venous remodeling. Elastin, tenascin-C, thrombospondin, lysyl oxidase, and osteopontin are expressed within the wall of the remodeling vein resulting in the de novo formation of an extracellular matrix scaffold that may be part of a critical adaptation program being evoked to allow the vessel to cope with its new biomechanical environment. CLINICAL RELEVANCE: The Kidney Dialysis Outcomes Quality Initiative has proposed the construction of arteriovenous fistulas as the primary vascular access for hemodialysis. As the vein is exposed to arterial flow, the vein wall dilates and a vascular remodeling process is triggered. With continued exposure, intimal hyperplasia occurs at the anastomosis that in many cases leads to failure. However, the molecular mechanisms by which the outflow vein remodels into a mature fistula remain incompletely understood. By investigating venous remodeling in a fistula model, candidate genes important for the remodeling process are discovered and their functional significance examined. Thus, the identification of relevant genes involved in this process should provide insight into arteriovenous fistula maturation and may suggest novel approaches for achieving higher patency rates.