Mechanisms of Telomerase RNA Processing and Formation of Ribonucleoprotein Particles in Saccharomyces cerevisiae

近年来，在真核细胞的细胞质中发现了几种含有rna的亚区室。有人认为，这些隔室是液体的，表现得像水包油乳液中的油。直到今天，人们还不明白为什么RNA加工需要这样的区隔化。从进化的角度来看，RNA体的一些成分非常古老，这些结构被认为是早期RNA世界的残余，可能在脂质双层封闭生命出现之前代表了单独的反应空间。有趣的是，Sm或类似Sm的蛋白质存在于目前所描述的大多数RNA小体中。这些蛋白质形成环状结构；然而，它们的分子功能尚不清楚。酿酒酵母的端粒酶RNA TLC1作为与sm环相关的非编码RNA的模型。在许多方面，TLC1成熟类似于剪接体snRNPs的产生。虽然snRNP组装已经在高等真核生物中得到了广泛的研究，但在酵母中对该过程的了解较少，并且在几个重要方面似乎与后生动物途径不同。在哺乳动物细胞中，sm复合物的组装发生在细胞质中，可能在一个专门的RNA室中。虽然体外组装是自发发生的，但这个过程是高度调节的，并且涉及各种额外的因素，然而，其中许多因素在酵母中并不保守。我将利用酵母TLC1研究酿酒酵母的sm环组装。为了确定装配室，我将结合生物化学和高分辨率显微镜对Sm蛋白和相关rna进行研究。此外，TLC1的纯化将允许鉴定蛋白质成分，并有助于推断加工机制。利用酿酒酵母的实验可处理性，我的研究将澄清Sm-RNA加工的重要方面，并将有助于阐明RNA加工室的作用，其普遍性和重要性刚刚开始显现。

Recently, several RNA-containing sub-compartments have been discovered in the cytoplasm of eukaryotic cells. It has been suggested that these compartments are liquid and behave like oil in an oil-in-water emulsion. To this day, it is not understood why RNA processing requires such compartmentalization. Evolutionarily, some components of RNA bodies are very old, and these structures have been proposed to be remnants of the early RNA world that may have represented separate reaction spaces before the rise of lipid-bilayer enclosed life. Interestingly, Sm or Sm-like proteins are found in most RNA bodies described so far. These proteins form ring-like structures; however, their molecular function remains unclear. TLC1, the telomerase RNA of S. cerevisiae, serves as a model for non-coding RNAs that associate with the Sm-ring. In many respects, TLC1 maturation resembles the generation of spliceosomal snRNPs. While snRNP assembly has been studied extensively in higher eukaryotes, the process is less well understood in yeast, and seems to differ from the metazoan pathway in several important aspects. In mammalian cells, assembly of the Sm-complex takes place in the cytoplasm, potentially in a specialized RNA compartment. Although assembly occurs spontaneously in vitro, the process is highly regulated and involves various additional factors, many of which are, however, not conserved in yeast.I will use yeast TLC1 to study Sm-ring assembly in S. cerevisiae. To identify the assembly compartment, I will combine biochemistry with high-resolution microscopy on Sm proteins and associated RNAs. In addition, purification of TLC1 will allow the identification of protein components and will help to infer processing mechanisms. Using the experimental tractability of S. cerevisiae, my research will clarify important aspects of Sm-RNA processing and will help to shed light on the role of RNA processing compartments, the ubiquity and importance of which is just beginning to emerge.