Buratowski, StephenBlower, MichaelEmerman, Amy Beth2015-07-172015-052015-05-182015Emerman, Amy Beth. 2015. Molecular Details and Functional Analysis of RNA Binding by ESCRT-II. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.http://nrs.harvard.edu/urn-3:HUL.InstRepos:17465322Many RNAs show distinct localization patterns in cells with enrichment at particular subcellular sites or organelles. RNA localization is a highly conserved process that both spatially and temporally controls gene expression. A common mechanism to selectively sort RNAs within the cell involves recognition of cis-acting sequences on the RNA by trans-acting RNA-binding proteins. Recently, the ESCRT-II complex was identified as a novel trans-acting factor required for the localization of bicoid mRNA in Drosophila oocytes. ESCRT-II was previously uncharacterized as an RNA-binding complex but has a well-established role in multivesicular body formation and receptor downregulation. Recent studies have revealed links between endosomes and RNA regulatory pathways, and the dual roles of ESCRT-II in both cellular processes suggest that it will be an important factor to better understand as an RNA-binding complex. However, bicoid is the only identified direct RNA target of ESCRT-II, and whether ESCRT-II’s role in RNA localization is conserved in other organisms is unclear. Here we report that the role of ESCRT-II in RNA regulation is conserved in Xenopus eggs. We found that ESCRT-II interacts with hundreds of RNAs in Xenopus eggs, and we characterized the molecular details of this interaction. Using a UV-crosslinking approach, we show that ESCRT-II binds directly to RNA through the subunit Vps25. Furthermore, by performing CLIP-seq, we found that ESCRT-II recognizes a polypurine motif. Selective binding of the polypurine motif through Vps25 can be recapitulated in vitro by multiple binding assays using purified components. Furthermore, ESCRT-II interacts with a subset of RNAs that are enriched on the mitotic spindle, and we provide preliminary evidence that ESCRT-II may be involved in localizing RNAs to the mitotic spindle. Consistent with previous reports, we found that ESCRT-II localizes to the centrosome in Xenopus tissue culture cells and to exogenous centrosomes added to egg extract. Our results suggest that the role of ESCRT-II in RNA regulation is conserved and shed light on an unexpected link between the cellular systems that control endosomal sorting and RNA localization.application/pdfenBiology, MolecularBiology, GeneralBiology, CellThesis or Dissertation2017-05-01