Biogenesis, Function and Turnover of Noncoding RNAs

我们实验室的长期目标是了解非编码rna的功能，细胞如何识别和降解有缺陷和不需要的rna，以及无法降解这些rna如何影响细胞功能并导致人类疾病。我们工作的一个重点是一类丰富的核糖核蛋白（RNPs），被称为Ro60 RNPs，广泛存在于动物细胞和许多细菌中。发现其主要蛋白成分环状ro60kda自身抗原，是因为它是系统性红斑狼疮和干燥综合征患者自身抗体的重要临床靶点。在所有被检测的生物体中，Ro60结合被称为Y rna的非编码rna。通过研究细菌中的Ro60 RNPs，我们发现了ncRNA的一个新作用，即将蛋白质辅助因子拴在效应蛋白上以改变其功能。具体来说，我们发现细菌Ro60同源物被Y RNA拴在一个环状核糖核酸酶上，形成一个新的双环RNA降解机器。我们目前正在努力确定Ro60 RNPs在哺乳动物细胞中的功能。作为这项工作的一部分，我们使用CRISPR来产生缺乏一种或两种小鼠Y rna的小鼠胚胎干细胞。尽管有报道称Y rna对动物细胞的DNA复制至关重要，但缺乏这两种Y rna的小鼠胚胎干细胞分裂正常。然而，缺乏Y RNA的细胞Ro60水平降低，我们可以通过在这些细胞中表达Y RNA来弥补这一缺陷。我们还证明了Ro60调节Ro60的亚细胞位置，并且Y rna将Ro60连接到不同的蛋白质上以产生专门的RNPs。总之，这些数据揭示了Y rna的功能与其Ro60伙伴的功能密切相关。在第二个重点中，我们描述了RNA监视途径在哺乳动物细胞生理学中的作用。在这里，我们最近的一项成就是我们发现被称为RNA外泌体的多核酶复合体抑制人类胚胎干细胞的分化。我们发现外泌体部分通过降解编码FOXH1的前mrna来抑制分化，FOXH1是中胚层形成的关键转录因子，是中胚层和内胚层的前体。这些研究揭示了RNA降解在维持人类胚胎干细胞多能性中的重要性。

The long-term goals of our laboratory are to understand how noncoding RNAs function, how cells recognize and degrade defective and unneeded RNAs, and how failure to degrade these RNAs affects cell function and contributes to human disease. One focus of our work is an abundant class of ribonucleoproteins (RNPs), known as Ro60 RNPs, which are widespread in animal cells and present in many bacteria. The major protein component, the ring-shaped Ro 60 kDa autoantigen, was discovered because it is a clinically important target of autoantibodies in patients with systemic lupus erythematosus and Sjogren's syndrome. In all organisms examined, Ro60 binds noncoding RNAs called Y RNAs. By studying Ro60 RNPs in bacteria, we uncovered a novel role for ncRNA, that of tethering a protein cofactor to an effector protein to alter its function. Specifically, we discovered that a bacterial Ro60 ortholog was tethered by Y RNA to a ring-shaped ribonuclease, forming a new double-ringed RNA degradation machine. We are currently working to define the functions of Ro60 RNPs in mammalian cells. As part of this effort, we used CRISPR to generate mouse embryonic stem cells lacking one or both of the two mouse Y RNAs. Despite reports that Y RNAs are essential for DNA replication in animal cells, mouse embryonic stem cells lacking both Y RNAs divided normally. However, cells lacking Y RNAs had reduced levels of Ro60, a defect that we could complement by expressing Y RNA in these cells. We also demonstrated that Ro60 regulates the subcellular location of Ro60 and that Y RNAs tether Ro60 to diverse proteins to create specialized RNPs. Together, these data reveal that the functions of Y RNAs are closely connected to those of their Ro60 partner. In a second focus, we are characterizing the roles of RNA surveillance pathways in mammalian cell physiology. Here, a recent accomplishment was our discovery that the multinuclease complex known as the RNA exosome represses differentiation of human embryonic stem cells. We showed that the exosome restrains differentiation in part by degrading pre-mRNAs encoding FOXH1, a transcription factor crucial for formation of mesendoderm, the precursor to both mesoderm and endoderm. These studies revealed the importance of RNA degradation in maintaining human embryonic stem cell pluripotency.