Non-coding RNAs and ribonucleoprotein complexes in unicellular eukaryotes

非编码rna （ncRNAs）是一大类功能多样的细胞分子，通常不编码多肽产物。这些rna在称为RNPs的复合物中的蛋白质成分的帮助下发挥其功能。许多基因组，特别是在真核生物如人类中，提供了比蛋白质更多的DNA序列信息来指定ncrna的表达。然而，即使在研究更充分的生物体中，大多数ncRNA的详细功能仍然未知，并且在许多其他生物体中，ncRNA的组成和相关的蛋白质伙伴尚未被表征。因此，鉴定和确定ncrna和RNPs的功能是功能基因组学的重要领域；将DNA序列信息与生物功能联系起来，这是我研究计划的主要目标。我们将研究被称为原生生物的单细胞真核生物中的ncrna及其相关蛋白，原生生物构成了真核生物系统发育多样性的大部分。获得诺贝尔奖的发现源于首次在原生生物中表征RNA及其细胞功能，如自剪接内含子（自催化RNA）和端粒酶RNA，首次在四膜虫中发现。我们将主要研究三类ncRNA：小核仁rna （sno）、小核rna （sn）和微rna （mirna）。我们将主要研究两种原生生物，绿枝绿枝和兰第鞭毛虫。这些物种特别有趣，因为它们具有与ncRNA功能相关的不寻常的细胞特征，因为它们代表了原生生物基因组复杂性的极端；贾第鞭毛虫的基因组显著减少，而绿藻的原生生物基因组相对较大。最近，我们创建并测序了第一个来自薄叶草的广泛的ncRNA文库，使我们现在能够进行详细的生物信息学鉴定，然后对新的ncRNA进行功能表征，包括检查ncRNA的表达、定位、加工和RNP结构。剪接体是在剪接过程中从真核rna中去除中间序列（内含子）所需的大型RNP复合体。我们将描述贾第鞭毛虫和绿枝鞭毛虫的剪接和剪接体成分。这两种生物都含有不寻常的、很大程度上未被探索过的、带有新型内含子的剪接系统。我们的研究也将集中在理解ncrna组装成rnp的特性上。我们将采用重组方法，将细菌细胞或体外产生的单个纯化重组成分结合在一起，并从绿藻和贾第鞭毛虫细胞中纯化RNP复合物。我们的研究不仅将揭示ncRNA和RNP结构和功能的多样性，而且可能为其他模式真核生物和细菌的ncRNA功能提供新的见解。

Non-coding RNAs (ncRNAs) are a large and functionally diverse class of cellular molecules that typically do not code for polypeptide products. These RNAs exert their functions with the assistance of protein components in complexes called RNPs. Many genomes, particularly in eukaryotic organisms such as humans, devote much more DNA sequence information to specifying expression of ncRNAs than proteins. However, even in the more well-studied organisms the detailed function of most ncRNAs remains unknown and in many other organisms, the ncRNA composition and associated protein partners has not been characterized. Consequently, identifying and determining the functions of ncRNAs and RNPs are important areas of functional genomics; relating DNA sequence information to biological function, a primary objective of my research program. We will study ncRNAs and their associated proteins in the single-celled eukaryotes called protists, organisms that constitute much of the eukaryotic phylogenetic diversity. Nobel Prize-winning discoveries resulted from first characterizing RNAs and their cellular functions in protists, such as self-splicing introns (autocatalytic RNA) and telomerase RNA, first identified in Tetrahymena. Three classes of ncRNA that we will primarily investigate are the small nucleolar (sno) RNAs, small nuclear (sn) RNAs and micro (miRNAs). We will mainly study them in two protists, Euglena gracilis and Giardia lamblia. These species are particularly interesting because they have unusual cellular features related to ncRNA function and because they represent extremes in protist genome complexity; significant genome reduction in Giardia vs relatively large protist genome size in Euglena. Recently we created and sequenced the first extensive ncRNA libraries from E. gracilis allowing us to now perform detailed bioinformatic identification followed by functional characterization of new ncRNAs, including examining ncRNA expression, localization, processing, and RNP structure. The spliceosome is a large RNP complex required for the removal of intervening sequences (introns) from eukaryotic RNAs in a process called splicing. We will characterize splicing and spliceosome components in both Giardia and Euglena. Both organisms contain unusual and largely unexplored splicing systems with novel types of introns. Our research will also focus on understanding properties of the assembly of ncRNAs into RNPs. We will employ both a reconstitution approach where we combine individual purified recombinant components produced in bacterial cells or in vitro, and also purify RNP complexes from Euglena and Giardia cells. Cumulatively, our research will not only reveal the diversity of ncRNA and RNP structure and function, but also may provide new insights into ncRNA function in other model eukaryotes and bacteria.