1. Evaluation Summary:

    This study has discovered deubiquitinase USP36 as the enzyme that processes FAU, a ribosomal protein precursor comprised of a fusion between ubiquitin-like protein FUBI and the ribosomal protein eS30. This is an important advance because correct processing is crucial for biogenesis of the 40S ribosomal subunit. Knowing the identity of the processing enzyme now opens this step in ribosome biogenesis to molecular and mechanistic analysis.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  2. Reviewer #1 (Public Review):

    Across different species, eS31 (RPS27A) and eL40 (RPL40) are encoded as ubiquitin (Ub)-ribosomal protein (RP) precursor proteins. These fusion proteins are enzymatically processed to liberate the two proteins from one another, but how this cleavage is linked with the assembly of the ribosome is not well understood. Human cells produce a third RP as a fusion protein between a ubiquitin-like protein called FUBI and the eS30 (RPS30). Only eS30 is incorporated into the mature ribosome small subunit. This study focuses on the processing of FUBI-eS30 and how disruption of this cleavage affects ribosome assembly.

    The authors generate wildtype and non-cleavable mutant transgenes of FUBI-eS30 and express them in two human cell lines. Expression of non-cleavable mutants results in rRNA processing defects and defects in late cytoplasmic 40S subunit maturation. Cell imaging-based assays provide evidence that eS30 is normally incorporated into nuclear pre-40S particles. With these findings in hand, the authors then use biochemical approaches to identify potential FUBI interacting proteases. These experiments led to the identification of three deubiquitinases: USP16, USP10 and USP36. RNAi depletion experiments indicated that loss of USP36 resulted in defects in FUBI-eS30 processing in vivo. Recombinant USP36 was capable of cleaving FUBI-eS30 in vitro, whereas a catalytically dead mutant of USP36 was not. Together these data support a model in which USP36 processes FUBI-eS30 to promote proper ribosome biogenesis in human cells.


    The manuscript is clearly written and provides solid evidence regarding the importance of FUBI-eS30 processing during ribosome biogenesis. The authors also identify USP36 as a likely candidate deubiquitinase needed for FUBI-eS30 cleavage in vivo. This paper will have broad appeal to those interested in ribosome biology, gene regulation, and protein deubiquitination.


    The non-cleavable form of FUBI-eS30 behaves as a dominant negative when over-expressed in cell lines, raising potential concerns regarding some interpretations of the data.

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  3. Reviewer #2 (Public Review):

    In eukaryotes there are two ribosomal proteins encoded with a ubiquitin fused to the N-terminus, which are subsequently cleaved during ribosome maturation by various deubiquitinases. Interestingly, in non-yeast eukaryotes (holozoans) there is an additional fusion ribosomal protein, eS30 that is fused to the ubiquitin-like protein, FUBI, termed FUBI-eS30. Aside from eS30's location on the mature 40S subunit, little is known about FUBI-eS30. Therefore, the authors' aimed to determine at what step of 40S maturation does FUBI cleavage occur, its importance, and identify the potential deubiquitinase(s) that perform this cleavage.

    The authors utilized non-cleavable FUBI-eS30 constructs to probe at which step(s) this cleavage occurs and its role in ribosome biogenesis through a variety of biochemical and immunofluorescence assays. Their results strongly pointed towards a role for FUBI-eS30 cleavage in late pre-40S maturation in the cytoplasm. Next, they worked to identify potential deubiquitinases that perform this cleavage by differential affinity purification of FUBI-eS30-StHA versus a non-cleavable mutant FUBI-eS30 followed by mass-spectrometry, hypothesizing that the protease that cleaves FUBI-eS30 will be able to bind the mutant construct and thus be enriched in that sample. While it is unclear how exhaustive this methodology was, two deubiquintinases were enriched in the mutant affinity purification, USP10 and USP36. Multiple in-vitro and in-vivo follow-up assays pointed towards USP36 and not USP10 as the protease that cleaves FUBI-eS30, however there are possibly other enzymes that perform this cleavage and the relevance of USP36 in ribosome maturation regulation was left unstudied.

    The authors performed extensive experiments to ensure any results obtained were rigorously tested and validated. They were able to more precisely identify the role of FUBI-eS30 in pre-40S maturation by testing the non-cleavable mutant's effect on several RBFs, as well as ruling out its role in pre-60S maturation. Once USP36 was identified, the authors used multiple depletion methods in cells to confirm its role in FUBI-eS30 regulation.

    While the experiments performed were for the most part extensive, the interpretation and analysis was lacking in some areas, primarily in the in-situ hybridization and immunofluorescence experiments (Figure 2E, 3, and 4) and in-vitro processing assay (Figure 7). There were no clear nuclear and nucleolar markers used for colocalization and no quantification was performed for the images. Additionally, claims were made in the main text in regards to the kinetics of the in-vitro USP36 assay with no quantification or analysis performed to this end.
    Overall, the authors strongly support their claim that FUBI-eS30 cleavage is necessary for proper pre-40S maturation and that USP36 is a deubiquitinase that is able to perform this reaction in cells and in-vitro. However, USP36's impact on pre-40S maturation remains to be understood and if there are additional enzymes that perform FUBI-eS30 cleavage in this context, especially considering USP36's already known multidimensional functions. The authors substantially connect their results into the context of the existing literature, providing a useful framework for future experimentation. This work offers a novel example of ribosome biogenesis regulation in eukaryotes that also speaks to potential cross-talk mechanisms between protein synthesis and degradation.

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  4. Reviewer #3 (Public Review):

    This study has investigated the cellular role and mechanism of processing of the FUBI-eS30 fusion protein named FAU. FUBI is a ubiquitin-like protein that is removed from FAU to produce the 40S ribosomal protein eS30. The timing, mechanism of processing, and biological importance are all unclear. Here, the authors exploit processing mutations of FAU in which the di-Gly cleavage site is mutated. Using these mutants in mammalian cells, they systematically characterise the likely timing of cleavage by analysing which 40S biogenesis intermediates accumulate. This leads the authors to suggest that FAU is processed relatively late, and that impairment of its processing results in a myriad of late (but not early) biogenesis factors being mislocalised due to impaired recycling. The authors then use an unbiased interaction analysis to identify candidate processing enzymes. The two top candidates are analysed by siRNA, with only one, USP36, shown to impact processing of endogenous FAU. Purified USP36 was shown to cleave purified FUBI-fusions in vitro. Together with the nucleolar localisation of USP36, the findings paint a compelling and consistent picture of USP36 being a key enzyme in the processing of FUBI, a reaction of clear biological importance with consequences for organism viability and homeostasis.

    The study is systematic, well controlled, logically organised, and convincing. In areas where there remain further work, the authors are suitably cautious and present alternative explanations. The writing is exceptionally clear, and the quality of the data are uniformly high. The study will provide an important foundation for future work on FAU, its processing, and on the potential functions of post-processed FUBI.

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