Person:

Emerman, Amy Beth

Accurate chromosome segregation is essential for cell viability. The mitotic spindle is crucial for chromosome segregation, but much remains unknown about factors that regulate spindle assembly. Recent work implicates RNA in promoting proper spindle assembly independently of mRNA translation; however, the mechanism by which RNA performs this function is currently unknown. Here, we show that RNA regulates both the localization and catalytic activity of the mitotic kinase, Aurora-B (AurB), which is present in a ribonucleoprotein (RNP) complex with many mRNAs. Interestingly, AurB kinase activity is reduced in Xenopus egg extracts treated with RNase, and its activity is stimulated in vitro by RNA binding. Spindle assembly defects following RNase-treatment are partially rescued by inhibiting MCAK, a microtubule depolymerase that is inactivated by AurB-dependent phosphorylation. These findings implicate AurB as an important RNA-dependent spindle assembly factor, and demonstrate a translation-independent role for RNA in stimulating AurB.

Many 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.