Stop codon readthrough proteins are unstable and recruit specific protein quality control machinery
(A) Expression of readthrough reporter protein in C. elegans muscle cells. Left: reporter constructs YFP-UTR, allowing readthrough into the unc-54 UTR, and YFP-STOP used as control. Right: fluorescence microscopy images of animals expressing these proteins. White boxes indicate head region (magnified and contrast adjusted) (exposure 4ms). Arrow heads point to inclusions of YFP-UTR.
(B) Immunoblot analysis of lysates from adult worms expressing YFP-UTR and YFP-STOP using anti-GFP antibody, revealing the destabilization of YFP-UTR. α-Tubulin served as loading control (n = 3).
(C) Fluorescence microscopy images of wild-type (WT) and Δrpn-10 worms expressing YFP-UTR (exposure 100 ms).
(D) Volcano plot representation of label-free proteome analysis of YFP pull-down fractions from worm lysates as in (B). Components of the BAG6 complex, proteasomes, TRiC/CCT chaperonin, and molecular chaperones, including sHSPs and HSP-1, are significantly enriched on YFP-UTR. Selected proteins are annotated. See also Figure S1 and Table S1A.