Mapping the non-coding RNA landscape in skeletal muscle health and disease

项目摘要 骨骼肌组织是通过肌源性和非肌源性的协调作用而发展和维持的 肌源性细胞。骨骼肌源性细胞特性和功能的失调是常见的现象 肌肉疾病。面肩臂肌营养不良症(FSHD)是第二常见的遗传性疾病 肌肉营养不良，导致进行性肌肉无力，没有任何有效的治疗方法。数不胜数 在FSHD中观察到细胞病因，如肌源性细胞的丢失，包括肌肉干细胞和 肌纤维，增加成纤维细胞、成脂细胞和免疫细胞。FSHD最常见的形式是 由D4Z4基因座的表观遗传抑制引起的DUX4基因的异常表达。DUX4 FSHD患者的表达具有地区性差异，并且在骨骼肌组织中高度分散。值得注意的是， DUX4的表达既受非编码RNA(NcRNAs)的诱导，又与其致病机制有关。 非编码RNA(包括miRNAs、lncRNAs、snoRNAs和eRNAs)是骨骼肌的关键调控因子 细胞在健康和疾病中的身份和功能，并通过转录网络的调节发挥作用。 由于缺乏ncRNA，对ncRNA网络和机制缺乏全面的了解 分析技术。传统的单细胞和空间RNA测序技术优先检测 多腺素化的、蛋白质编码的mRNAs，由于缺乏 多聚腺苷酸化。在这项提案中，我们将应用一种名为STRS-HD的新RNA映射技术，该技术是唯一的 能够有效和全面地检测总转录组，包括多腺苷和 非腺化转录本，单细胞空间分辨率揭示全球ncRNA表达的异质性 在骨骼肌内不同类型的细胞中。我们将利用这种新的空间总RNA测序方法来 广泛询问健康骨骼肌发生和FSHD发病机制中的非编码RNA。在目标1中，我们 将实施这种全转录方法来研究非编码RNA如何影响细胞命运调控 成年小鼠骨骼肌再生。我们将探索特定细胞类型的ncRNA表达变异和 使用空间转录切割技术将ncRNA特征映射到空间分辨的细胞间通信相互作用上 以深入了解ncRNA对肌源性细胞命运的调控。在目标2中，我们应用这些方法来解决 应用DUX4反义寡核苷酸诱导的两种小鼠FSHD病理模型中ncRNAs的变化 心理治疗。我们将结合空间总转录组图谱和组织病理学来揭示ncRNA决定因素。 改变FSHD的肌原细胞规格和肌纤维损伤。在目标3中，我们将扩展STRS-HD 探讨人类FSHD活检并将ncRNA特征与未受影响的家族性对照和对照进行比较 空间ncRNA映射到供体血浆中的无细胞RNA测序，以确定新的FSHD总RNA生物标志物。 这些新的全转录技术将广泛应用于发育中ncRNAs的研究。 以及骨骼肌和其他组织的疾病生物学。

Project Summary Skeletal muscle tissues are developed and maintained through the coordinated action of myogenic and non- myogenic cells. Dysregulation of myogenic cell identities and functions are commonly observed in skeletal muscle disease. Facioscapulohumeral muscular dystrophy (FSHD) is the second most common inherited muscular dystrophy and results in progressive muscle weakness without any effective therapies. Numerous cellular etiologies are observed in FSHD, such as loss of myogenic cells, including muscle stem cells and myofibers, and increased fibrogenic, adipogenic, and immune cells. The most common form of FSHD arises from aberrant expression of the DUX4 gene caused by epigenetic de-repression of the D4Z4 locus. DUX4 expression in FSHD individuals is regionally varied and highly sporadic within skeletal muscle tissue. Notably, DUX4 expression is both induced by and has pathogenic mechanisms related to noncoding RNAs (ncRNAs). Noncoding RNAs (including miRNAs, lncRNAs, snoRNAs, and eRNAs) are critical regulators of skeletal muscle cell identities and functions in health and diseases and act through modulation of transcriptional networks. Comprehensive understanding of ncRNA networks and mechanisms is lacking due to a paucity of ncRNA profiling technologies. Conventional single-cell and spatial RNA-sequencing technologies preferentially detect polyadenylated, protein-coding mRNAs, and do not efficiently capture most ncRNAs due to their lack of polyadenylation. In this proposal, we will apply a new RNA mapping technology called STRS-HD that is uniquely capable of efficiently and comprehensively detecting the total transcriptome, including both polyadenylated and non-adenylated transcripts, with single-cell spatial resolution to reveal global ncRNA expression heterogeneity in diverse cell types within skeletal muscles. We will leverage this new spatial total RNA-sequencing method to broadly interrogate noncoding RNAs in healthy skeletal myogenesis and in FSHD pathogenesis. In Aim 1, we will implement this total transcriptomic method to investigate how noncoding RNAs impact cell fate regulation adult skeletal muscle regeneration in mice. We will explore cell type-specific ncRNA expression variation and use spatial transcriptomics to map ncRNA features onto spatially resolved cell-cell communication interactions to provide insights into ncRNA regulation of myogenic cell fates. In Aim 2, we apply these methods to resolve how ncRNAs vary in FSHD pathologies using two mouse models subject to DUX4 anti-sense oligonucleotide therapy. We will integrate spatial total transcriptome maps with histopathology to reveal ncRNA determinants of altered myogenic cell specification and myofiber damage in FSHD. In Aim 3, we will extend the STRS-HD approach to human FSHD biopsies and compare ncRNA features to unaffected familial controls and contrast spatial ncRNA maps to cell-free RNA-sequencing in donor plasma to identify new total RNA biomarkers of FSHD. These new total transcriptomic technologies will be broadly applicable to the study of ncRNAs in developmental and disease biology of skeletal muscle and other tissues.