Processing of the Ribosomal Ubiquitin-Like Fusion Protein FUBI-eS30/FAU is Required for 40S Maturation and Depends on USP36

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.

MALDI-TOF Mass Spectrometry for interrogating ubiquitin enzymes

“…In fact, most ubiquitin-like conjugating enzymes and pathways resemble those involved in ubiquitylation allowing for similar MALDI-TOF MSbased detection strategies. A further niche of investigation is the identification of DUBs able to process ubiquitin-like domains that are integrated within longer substrates, such as in ribosome biogenesis and function (van den Heuvel et al, 2021).…”