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  1. Evaluation Summary:

    Ribosomal proteins are prone to aggregation. This work provides strong experimental support for a novel mechanism by which the level of the mRNA encoding two different ribosomal proteins is fine-tuned by variation in the abundance or availability of their chaperones. This regulation is proposed to operate through mRNA degradation at the translating ribosome. The data are extensive - from genetic observations that hint at the existence of a feedback loop, through interaction mechanisms, to biological significance. The logical links between the different steps in the analysis are clear, sound and well set out. The conclusions are unanticipated, but convincingly supported and very likely relevant and important for other systems beyond yeast.

    (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. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

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  2. Reviewer #1 (Public Review):

    Pillet et al. describe a novel feedback mechanism that coordinates the expression of ribosomal proteins RPL3 and RPL4 with the pace of ribosome assembly through the co-translational regulation of their mRNA stability mediated by the Ccr4-Not deadenylase complex. The authors present a wealth of compelling data in support of a novel regulatory mechanism regulating the production of ribosomal proteins in yeast. Their conclusions are justified based on corroborating evidence generated from appropriately controlled experiments.

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  3. Reviewer #2 (Public Review):

    Ribosomal proteins are prone to aggregation, and 9 or the 79 r-proteins in yeast are known to associate with a dedicated chaperone protein required for their function. The authors report here on a continuation of their study of two r-proteins, Rpl3 and Rpl4, whose chaperones are Rrb1 and Acl4, respectively. The present study was motivated by the observation that acl4Δ strains, which grow extremely slowly, give rise to spontaneous suppressor mutations, 48 of which were identified by whole-genome sequencing. One suppressor is a point mutation in the RPL4 gene, whereas all the others are in genes linked to the well-studied Ccr4-Not complex (CAF130 and NOT1), known to be involved, among other things, in cytoplasmic mRNA degradation. They also identified suppressor mutations in YJR011C, which they show interacts with Caf130, and thus dub CAL4 (Caf130-associated regulator of Rpl4). Using RNA-seq, they show that all of caf130Δ and cal4Δ Interestingly, they point out that both Caf130 and Cal4 have been physically connected with nascent polypeptide-associated complex (NAC) proteins Btt1 and Egd1/2, and they go on to show that ablation of the genes encoding Btt1 and Egd2 also leads to some level of acl4Δ suppression. The authors then present evidence indicating that Caf130 connects both Cal4 and Btt1 to Ccr4-Not through an N-terminal domain of Not1 that is not required for its essential function(s). They thus identify a previously unknown function of the Not1 N-terminus which they show, Interestingly, is expressed only in a minor Not1 isoform, with the majority isoform initiating from a downstream ATG. The working model that emerges at this point is that RPL3 and RPL4 mRNA levels are regulated by targeted Ccr4-Not degradation at the ribosome, directed by an interaction with the emerging r-protein and some as-yet-unidentified component of the system. In what follows, the authors map regions of both Rpl3/4 that are required for their mRNA regulation and show that they are adjacent to but distinct from their chaperone binding sites. They then show that overexpression of either chaperone gene increases mRNA levels of the corresponding r-protein gene, and that deregulated expression of the RPL3 and 4 genes induces aggregation of their encoded proteins and lethality in the absence of the Tom1 E3 enzyme, known to be involved in r-protein degradation.

    The experiments reported here are well designed and the data are consistent with the authors' molecular model for regulation of RPL3/4 mRNA levels. The strength of this work lies in (1) its molecular (Y2H) and biochemical (co-IP) characterization of a network of protein-protein interactions linking parts of the Ccr4-Not complex to the nascent peptide associated complex (NAC), (2) its detailed mapping of sequences within both Rpl3 and Rpl4 that are required for regulation, and (3) its demonstration that these sequences can confer mRNA regulation to an otherwise heterologous transcript.

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  4. Reviewer #3 (Public Review):

    This paper follows from previous analyses of the regulation of Rpl3 and Rpl4 expression and pre-ribosome incorporation. Starting from the observation that suppressors for loss of the Rpl4 chaperone Acl4 are relatively common, the authors discovered that RP abundance is linked to mRNA stability via the ribosome-associated nascent polypeptide-associated complex (NAC). In the absence of specific Rlp4 or Rpl3 chaperones, NAC-beta components Edg1 and Btt1 recruit the NOT-complex via the non-essential, sub-stoichiometric, associated proteins Caf130 and Yjr011c (now Cal4). Caf130 binds the N-terminus of Not1 and Cal4 binds Caf130. Crr4-NOT then promotes mRNA degradation - presumably cotranslationally, although this is not shown. Finally, they show that tight regulation of Rpl3 and Rpl4 is important in avoiding toxic protein aggregation.

    Overall, the work reports a quite complete body of research - from initial genetic observations, through interaction mechanisms, to biological significance. The logical links between the different steps in the analysis are clear, sound and well set out. The findings are unexpected but convincingly supported and seem very likely to be relevant and important for other regulatory systems and conserved in eukaryotes.

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