Fbxo42 promotes the degradation of Ataxin-2 granules to trigger terminal Xbp1 signaling

ABSTRACT: In Drosophila melanogaster, Fbxo42 acts downstream of Unfolded Protein Response activation but upstream of the apoptotic machinery. Fbxo42 associates through its N-terminal F-box domain with SkpA, a component of the RBX-SCF E3 ubiquitin ligase complex. Hence, in order to uncover Fbxo42 substrates, we conducted a label free quantitative proteomics experiment based on the bioUb strategy. First, transgenic flies expressing full-length FLAG-HA-Fbxo42, FLAG-HA-Fbxo42 or FLAG-Fblx7 were crossed with bioUb flies, a strain containing ubiquitin (Ub) with a short biotinylatable motif and BirA (biotin ligase), and GMR-GAL4, to drive the expression of the constructs in the Drosophila eye. Then, total protein was extracted and ubiquitylated proteins were purified by pulldowns using a high-capacity streptavidin resin from triplicate samples. Eluted proteins were fractionated by SDS-PAGE, gel lanes were cut into slices and subjected to in-gel trypsinization. Finally, generated peptides were analysed is a Q Exactive mass spectrometer and acquired raw data files were processed with the MaxQuant software using the internal search engine Andromeda and tested against the UniProt database filtered for Drosophila melanogaster. Among all the proteins that were more ubiquitinated upon Fbxo42 overexpression in comparison with FbxI7 overexpression, and consequently could be considered as putative Fbxo42 substrates, we focused on Ataxin-2, a RNA binding protein that participates in the formation of ribonucleoproteoin granules. Additional experiments demonstrated that Fbxo42 promotes the ubiquitylation and degradation of Ataxin-2. Upon ER-stress, Fbxo42 is recruited to Ataxin-2 granules where Xbp1 is sequestered and degrades Ataxin-2, allowing the translation of Xbp1 mRNA, to trigger cell death during the terminal stages of UPR activation.

INSTRUMENT(S):

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

SUBMITTER: Mikel Azkargorta  

LAB HEAD: NEREA OSINALDE

PROVIDER: PXD065621 | Pride | 2025-07-01

REPOSITORIES: Pride