Abstract
RNA-binding proteins (RBP) are critical regulators of gene expression. Recent studies have uncovered hundreds of mRNA-binding proteins that do not contain annotated RNA-binding domains and have well-established roles in other cellular processes. Investigation of these nonconventional RBPs is critical for revealing novel RNA-binding domains and may disclose connections between RNA regulation and other aspects of cell biology. The endosomal sorting complex required for transport II (ESCRT-II) is a nonconventional RNA-binding complex that has a canonical role in multivesicular body formation. ESCRT-II was identified previously as an RNA-binding complex in Drosophila oocytes, but whether its RNA-binding properties extend beyond Drosophila is unknown. In this study, we found that the RNA-binding properties of ESCRT-II are conserved in Xenopus eggs, where ESCRT-II interacted with hundreds of mRNAs. Using a UV cross-linking approach, we demonstrated that ESCRT-II binds directly to RNA through its subunit, Vps25. UV cross-linking and immunoprecipitation (CLIP)-Seq revealed that Vps25 specifically recognizes a polypurine (i.e. GA-rich) motif in RNA. Using purified components, we could reconstitute the selective Vps25-mediated binding of the polypurine motif in vitro Our results provide insight into the mechanism by which ESCRT-II selectively binds to mRNA and also suggest an unexpected link between endosome biology and RNA regulation.
Highlights
RNA-binding proteins (RBP) are critical regulators of gene expression
In support of the possibility that the GA-rich motif promotes binding to ESCRT-II, we found that GA-rich sequences were preferentially enriched in the ESCRT-II RNA immunoprecipitation (RIP)-Seq library compared with other ESCRT-II– bound transcripts (Fig. 3E and Table 2) (p Ͻ 1 ϫ 10Ϫ12 using a two-sided, unpaired t test)
We have demonstrated that ESCRT-II is a conserved RNAbinding protein that interacts with hundreds of different mRNAs, validating the novel observation that ESCRT-II binds to bicoid mRNA and is required for its localization in Drosophila [19]
Summary
The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit, Vps. Blower From the Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114 and the Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115
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