Non-coding RNAs and ribonucleoprotein complexes in unicellular eukaryotes

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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.