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原性和非肌原性的协调作用来发育和维持的。 生肌细胞在骨骼肌中通常观察到生肌细胞特性和功能的失调， 肌肉疾病面肩肱型肌营养不良症（FSHD）是第二常见的遗传性 肌营养不良症，并导致进行性肌无力，没有任何有效的治疗。许多 在FSHD中观察到细胞病因学，例如肌源性细胞（包括肌肉干细胞）的损失， 肌纤维和增加的纤维形成、脂肪形成和免疫细胞。FSHD最常见的形式是 由D4 Z4基因座的表观遗传去抑制引起的DUX 4基因的异常表达。Dux4 FSHD个体中的表达是区域性变化的，并且在骨骼肌组织中是高度散发的。值得注意的是， DUX 4表达由非编码RNA（ncRNA）诱导并具有与非编码RNA（ncRNA）相关的致病机制。 非编码RNA（包括miRNA、lncRNA、snoRNA和eRNA）是骨骼肌的关键调节因子 细胞的身份和功能在健康和疾病，并通过转录网络的调制作用。 由于缺乏ncRNA，因此缺乏对ncRNA网络和机制的全面了解 剖析技术。传统的单细胞和空间RNA测序技术优先检测 多聚腺苷酸化的蛋白质编码mRNA，并且由于它们缺乏多聚腺苷酸化的蛋白质编码mRNA，不能有效地捕获大多数ncRNA。 聚腺苷酸化在这项提案中，我们将应用一种新的RNA作图技术，称为STRS-HD， 能够高效、全面地检测总转录组，包括多聚腺苷酸化和 非腺苷酸化转录物，具有单细胞空间分辨率，以揭示整体ncRNA表达异质性 在骨骼肌内的不同细胞类型中。我们将利用这种新的空间总RNA测序方法， 在健康骨骼肌发生和FSHD发病机制中广泛地询问非编码RNA。目标1： 将实施这种总转录组学方法来研究非编码RNA如何影响细胞命运调控 成年小鼠骨骼肌再生。我们将探索细胞类型特异性ncRNA表达变异， 使用空间转录组学将ncRNA特征映射到空间分辨的细胞-细胞通信相互作用上 以提供对肌原性细胞命运的ncRNA调节的见解。在目标2中，我们应用这些方法来解决 使用经受DUX 4反义寡核苷酸的两种小鼠模型，ncRNA在FSHD病理学中如何变化 疗法我们将整合空间总转录组图谱与组织病理学，以揭示ncRNA决定因素， FSHD中生肌细胞规格改变和肌纤维损伤。在目标3中，我们将扩展STRS-HD 方法人类FSHD活检，并将ncRNA特征与未受影响的家族对照进行比较， 空间ncRNA映射到供体血浆中的无细胞RNA测序，以鉴定FSHD的新的总RNA生物标志物。 这些新的全转录组学技术将广泛适用于发育中ncRNA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.