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Analysis of the role of RNA silencing protein 1 (Rsp1) in the biogenesis of ~23-24 nt sRNAs in Tetrahymena thermophila
Date Permissions Signed
Date of Award
Department or Program Affiliation
Master of Science (MS)
Lee, Suzanne R.
RNA interference (RNAi) pathways regulate a variety of biological processes, including normal cell growth and development, through the action of protein-RNA complexes containing small RNAs (sRNAs). Our research focused an RNAi pathway in the ciliated unicellular eukaryote Tetrahymena thermophila. This pathway produces ~23-24 nucleotide (nt) sRNAs through the action of RNA-dependent RNA polymerase (RdRP) complexes (complexes termed RdRCs) and their interaction with an RNA nuclease called Dicer 2 (Dcr2). The accumulation of sRNAs also requires a protein called RNA Silencing Protein 1 (Rsp1) which associates with a subset of RdRC proteins. In this study, we first sought to learn more about the potential function and evolutionary conservation of Rsp1 by examining its sequence. Our results indicate Rsp1 may have structural similarity to RNA polymerases, including RdRPs, but lacks the conserved catalytic residues for RNA synthesis. We also identified Rsp1-like predicted proteins in other Tetrahymena species, but no clear homologs in more distantly related organisms.
Second, we tested three hypotheses for why Rsp1 is required for sRNA accumulation: 1) Rsp1 stabilizes the precursor RNA transcripts that are later processed into sRNA, 2) Rsp1 is necessary for the accumulation of RdRC proteins, and 3) Rsp1 is necessary for correct assembly of RdRCs. Our experimental results indicate that Rsp1 does not appear to regulate sRNA biogenesis by regulating the levels of sRNA precursors or RdRC proteins levels. Instead, purification of RdRCs revealed that in strains lacking Rsp1, RdRCs cannot be recovered. This suggests that RdRCs are disrupted somehow in the absence of Rsp1.
RNAi, RNA, RNA dependent RNA polymerases, RdRP, Tetrahymena thermophila, protein, biogenesis pathway
Western Washington University
Subject – LCSH
Tetrahymena; RNA interference; Life--Origin
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Ernest, Samantha Elizabeth O'Keffe Neff, "Analysis of the role of RNA silencing protein 1 (Rsp1) in the biogenesis of ~23-24 nt sRNAs in Tetrahymena thermophila" (2020). WWU Graduate School Collection. 951.