Highlights
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Cleavage-resistant ubiquitin-fused human ribosomal proteins (RPs) impair ribosome function
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Unprocessed, Ub-fused RPs are targeted for proteasomal degradation by the UFD pathway
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DUB activity-based probes for Ub-fused RPs identify Cezanne/OTUD7B and other DUBs
- Catalytic activity-independent DUB association with Ub-RPs prevents their degradation
Summary
In eukaryotes, each ribosomal subunit includes a ribosomal protein (RP) that is encoded as a fusion protein with ubiquitin (Ub). In yeast, each Ub-RP fusion requires processing by deubiquitylating enzymes (DUBs) to generate ribosome-assembly-competent RPs and to contribute to the cellular Ub pool. However, the mechanism by which DUBs in human cells process Ub-RP fusions remains unclear. Here, we discovered that Ub-RPs are substrates of the Ub-fusion degradation (UFD) pathway in human cells via lysine 29 and 48 (K29/K48)-specific ubiquitylation and proteasomal degradation. We identified a pool of DUBs that catalytically process Ub-RPs, as well as DUBs that physically occlude the interaction of Ub-RPs with UFD pathway Ub E3 ligases to prevent their degradation in a non-catalytic manner. Our results suggest that DUBs both process and stabilize Ub-RPs, whereas the UFD pathway regulates levels of Ub-RPs that cannot be fully processed by DUBs to fine-tune protein homeostasis.