Abstract
In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.
Highlights
In the majority of eukaryotes, one ribosomal protein (RP) of each subunit is encoded as a linear fusion with an N-terminal ubiquitin (Ub)
When inspecting the dataset for candidate FUBI processing enzymes, we found three DUBs to confidently interact with the FUBI-eS30 constructs, namely USP16, USP10, and USP36, all of which belong to the ubiquitinspecific protease (USP) family
We have investigated how processing of the exceptional ribosomal protein eS30 that is synthesized as a fusion with the ubiquitin-like protein FUBI, is coordinated with the biogenesis of 40S ribosomal subunits
Summary
In the majority of eukaryotes, one ribosomal protein (RP) of each subunit is encoded as a linear fusion with an N-terminal ubiquitin (Ub). The ribosomal proteins eS31 (RPS27A) and eL40 (RPL40) of the small and large ribosomal subunit, respectively, are synthesized as Ub-RP precursor proteins (Baker and Board, 1991; Kirschner and Stratakis, 2000; Lund et al, 1985), similar to their yeast homologs (FernandezPevida et al, 2016; Finley et al, 1989; Lacombe et al, 2009; Martın-Villanueva et al, 2020).
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