The transcription factor Xrp1 orchestrates both reduced translation and cell competition upon defective ribosome assembly or function
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Evaluation Summary:
A growing body of literature substantiates the physiological importance of the phenomenon of 'cell competition' induced by differences in the copy number of ribosomal proteins (Rp) in adjacent cells. Yet, the molecular players that effect cell competition in 'loser' cells have been elusive and poorly studied; the current study by Kiparaki et al makes significant headway in the field by demonstrating that a little known transcription factor, Xrp1, is the common effector of cell death in loser cells when competition is induced by Rp haplo-insufficiency. While differences in cellular translation levels were thought to be the main driver of cell death in loser cells, this work dissects this premise in detail to unequivocally show that in the absence of Xrp1, translation differences alone is not sufficient to induce loser cell death.
(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 agreed to share their name with the authors.)
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Abstract
Ribosomal Protein ( Rp ) gene haploinsufficiency affects translation rate, can lead to protein aggregation, and causes cell elimination by competition with wild type cells in mosaic tissues. We find that the modest changes in ribosomal subunit levels observed were insufficient for these effects, which all depended on the AT-hook, bZip domain protein Xrp1. Xrp1 reduced global translation through PERK-dependent phosphorylation of eIF2α. eIF2α phosphorylation was itself sufficient to enable cell competition of otherwise wild type cells, but through Xrp1 expression, not as the downstream effector of Xrp1. Unexpectedly, many other defects reducing ribosome biogenesis or function (depletion of TAF1B, eIF2, eIF4G, eIF6, eEF2, eEF1α1, or eIF5A), also increased eIF2α phosphorylation and enabled cell competition. This was also through the Xrp1 expression that was induced in these depletions. In the absence of Xrp1, translation differences between cells were not themselves sufficient to trigger cell competition. Xrp1 is shown here to be a sequence-specific transcription factor that regulates transposable elements as well as single-copy genes. Thus, Xrp1 is the master regulator that triggers multiple consequences of ribosomal stresses and is the key instigator of cell competition.
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Evaluation Summary:
A growing body of literature substantiates the physiological importance of the phenomenon of 'cell competition' induced by differences in the copy number of ribosomal proteins (Rp) in adjacent cells. Yet, the molecular players that effect cell competition in 'loser' cells have been elusive and poorly studied; the current study by Kiparaki et al makes significant headway in the field by demonstrating that a little known transcription factor, Xrp1, is the common effector of cell death in loser cells when competition is induced by Rp haplo-insufficiency. While differences in cellular translation levels were thought to be the main driver of cell death in loser cells, this work dissects this premise in detail to unequivocally show that in the absence of Xrp1, translation differences alone is not sufficient to induce loser …
Evaluation Summary:
A growing body of literature substantiates the physiological importance of the phenomenon of 'cell competition' induced by differences in the copy number of ribosomal proteins (Rp) in adjacent cells. Yet, the molecular players that effect cell competition in 'loser' cells have been elusive and poorly studied; the current study by Kiparaki et al makes significant headway in the field by demonstrating that a little known transcription factor, Xrp1, is the common effector of cell death in loser cells when competition is induced by Rp haplo-insufficiency. While differences in cellular translation levels were thought to be the main driver of cell death in loser cells, this work dissects this premise in detail to unequivocally show that in the absence of Xrp1, translation differences alone is not sufficient to induce loser cell death.
(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 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Kiparaki et al. extend the Baker lab's prior work, which showed induction Xrp1 in sub-optimal cells that are heterozygous for mutation in a ribosomal protein (Rp+/-). Rp+/- cells are eliminated by wild-type neighbors in the Drosophila wing imaginal disc epithelium. The Baker lab previously identified a mutation in rpS12 that renders cells resistant to Rp-dependent cell competition. In the current work, they show that Xrp1 mediates the reduced translation in and cell competition of Rp+/- cells. They demonstrate that Rp+/- have defects in ribosome biogenesis and have protein aggregation. A key advance to the field is their demonstration that Rp+/- cells display cell-autonomous phospho-eiF2aplha via PERK (but not Gcn2) and that eiF2alpha phosphorylation is downstream of induction of Xrp1. However, there appears …
Reviewer #1 (Public Review):
Kiparaki et al. extend the Baker lab's prior work, which showed induction Xrp1 in sub-optimal cells that are heterozygous for mutation in a ribosomal protein (Rp+/-). Rp+/- cells are eliminated by wild-type neighbors in the Drosophila wing imaginal disc epithelium. The Baker lab previously identified a mutation in rpS12 that renders cells resistant to Rp-dependent cell competition. In the current work, they show that Xrp1 mediates the reduced translation in and cell competition of Rp+/- cells. They demonstrate that Rp+/- have defects in ribosome biogenesis and have protein aggregation. A key advance to the field is their demonstration that Rp+/- cells display cell-autonomous phospho-eiF2aplha via PERK (but not Gcn2) and that eiF2alpha phosphorylation is downstream of induction of Xrp1. However, there appears to be a possible loop or circuit between phospho-eiF2alpha and Xrp1 because depletion of the phosphatase that dephosphorylates eiF2alpha causes cell autonomous induction of Xrp1. Another advance to the field is the observation that knockdown of numerous translation factors also leads Xrp1 induction, eiF2alpha phosphorylation, reduced translation and cell competition. They use genetics to try to separate reduced translation downstream of Xrp1 from cell competition translation downstream of Xrp1, but some additional experiments are needed to support these conclusions. The upregulation of Xrp1 when translation components were depleted dependent could be reduced in the competition resistant background rpS12G97D. They use molecular biology and cell culture to show that Xrp1 is a sequence specific transcription factor. They mutate three Xrp1 sequences in the commonly used anti-oxidant reporter GstD1-GFP and show that expression of this reporter is now greatly diminished. The latter result suggests that the anti-oxidant response observed in Rp/+ cells may result from Xrp1. The conclusions of this paper are moderately supported by data, and these results could be valuable to the field of cell competition.
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Reviewer #2 (Public Review):
The authors make excellent use of Drosophila genetics tools in combination with molecular biology techniques to broadly implicate the transcription factor Xrp1 as the effector of loser cell status in cell competition. The authors rigorously demonstrate in multiple Rp (also known as minute in Drosophila) haplo-insufficiencies, that there are very little differences in number of ribosomal subunits large or small. They also painstakingly deplete a variety of different translation factors in mitotic clones to reduce new protein synthesis to show that low translation levels lead to Xrp1 induction. They also corroborate recently published results (Baumgartner et al NCB 2021, Recasens-Alvarez et al NCB 2021) showing that proteotoxic stress, as marked by phosphorylation of eIF2⍺ is elevated in loser cell populations …
Reviewer #2 (Public Review):
The authors make excellent use of Drosophila genetics tools in combination with molecular biology techniques to broadly implicate the transcription factor Xrp1 as the effector of loser cell status in cell competition. The authors rigorously demonstrate in multiple Rp (also known as minute in Drosophila) haplo-insufficiencies, that there are very little differences in number of ribosomal subunits large or small. They also painstakingly deplete a variety of different translation factors in mitotic clones to reduce new protein synthesis to show that low translation levels lead to Xrp1 induction. They also corroborate recently published results (Baumgartner et al NCB 2021, Recasens-Alvarez et al NCB 2021) showing that proteotoxic stress, as marked by phosphorylation of eIF2⍺ is elevated in loser cell populations that are heterozygous for Rp. The authors further examine the role of P- eIF2⍺ and convincingly show that while restoring P-eIF2⍺ levels does not eliminate cell death in loser cells, depletion of Xrp1 is sufficient to do so- this data and other support the author's central conclusion that Xrp1 is a common effector of loser status during cell competition.
Overall the study is solid and the data are strong but there remain some technical gaps that would need to be clarified:
1. The primary strength of their paper is in establishing a common 'loser' cell mechanism and but this is dampened by their incomplete analysis of Xrp1 in all the Rp mutants they test in Figure 1, leaving open the possibility that some minutes (such as RpL27A, which has slightly different effects from RpL14) might yet induce competition in a Xrp1-independent way.
2. The examination of whether Xrp1 localizes to the nucleolus by co-staining with Fibrillarin (Figure 2 supplement 2) is not done at sufficiently high magnification or resolution to support their conclusion that they do not observe nucleolar localization or displacement of Xrp1.
3. The activation of Ire1 measured by Xbp1-GFP (Figure 4 supplement 1) is not tested in clones, but rather in whole heterozygous discs, which leaves open the possibility that other UPR pathways do respond to minute-mediated competition. This is particularly relevant since the premise of cell competition is based on elimination due to differences in neighbors but survival in otherwise similar conditions.
4. The authors claim that depletion of Xrp1 blocks cell death in competition induced by all translation factors does not consider their data in Figure 7 supplement 1G which shows substantial Dcp1 staining in clonal populations where eIF5A and Xrp1 are both depleted.
5. Data in Figure 9F-N showing induction of Gstd1-GFP in unconvincing for reasons similar to point 3 above, in that they do not represent the context of cell competition using clonal analysis. Thus the authors' conclusion that Gstd1-GFP, which was found by Baumgartner et al, NCB 2021 to be elevated in Rp+/- cells, is a target of Xrp1 is unsupported.
6. This study shares a common weakness in many that examine phospho-eIF2⍺ outside the context of stress : the treatment of P-eIF2⍺ as a constant entity whereas others have demonstrated that it can vary with the circadian rhythm (Karki et al PNAS 2020), amongst other factors. While this is a technical limitation that is difficult to overcome, its acknowledgement is nonetheless warranted in the discussion. -
Reviewer #3 (Public Review):
The authors begin by investigating the mechanism by which cells that are heterozygous for mutations in specific ribosomal proteins (rp) are eliminated by cell competition. They first show that ribosome subunit concentrations show modest changes and even these changes are different depending on which rp is mutated. There are hints that mutations in subunits of the large subunit may have different consequences to mutations that affect the small subunit. In these mutants, there is an increase in the levels of intermediates in the ribosomal assembly pathway. What seems to be common in all these cases is that the translation factor eIF2alpha is phosphorylated and this phosphorylation is dependent upon the transcription factor Xrp1 and the PERK kinase which is usually activated by the unfolded protein response. …
Reviewer #3 (Public Review):
The authors begin by investigating the mechanism by which cells that are heterozygous for mutations in specific ribosomal proteins (rp) are eliminated by cell competition. They first show that ribosome subunit concentrations show modest changes and even these changes are different depending on which rp is mutated. There are hints that mutations in subunits of the large subunit may have different consequences to mutations that affect the small subunit. In these mutants, there is an increase in the levels of intermediates in the ribosomal assembly pathway. What seems to be common in all these cases is that the translation factor eIF2alpha is phosphorylated and this phosphorylation is dependent upon the transcription factor Xrp1 and the PERK kinase which is usually activated by the unfolded protein response. This also seems to result in the accumulation of cytoplasmic aggregates.
The authors then go on to show that eIF2alpha phosphorylation in turn induces Xrp1 and cell competition and that many different disruption of translation all result eventually in increased Xrp1 and cell competition. However, the key element seems to be the upregulation of Xrp1 because reducing eIF2alpha phosphorylation can still cause cell competition provided Xrp1 is upregulated.
The authors also show that Xrp1 functions as a positive regulator of gene expression by binding to a specific motif. They provide good evidence that a key reporter used in previous work that attributed cell competition to oxidative stress can be activated by Xrp1.
This paper significantly advances our knowledge of cell competition. The manuscript has a lot of information and is at times difficult to digest. However, the work is very thorough and all the appropriate controls have been included. It provides important insights into cell competition and the central role of Xrp1.
At one point, the authors interpret their experiments to conclude that "Interrupting the translation cycle activates Xrp1-dependent cell competition independently of diminished translation". I wonder if the experiments really show this. In every instance where competition is shown to occur, there is some degree of reduced translation and Xrp1 is elevated. So it is quite possible that both conditions need to be fulfilled for competition to occur. In some instances, when Xrp1 is reduced (and therefore competition does not occur), then translation levels are no longer diminished with the same perturbation. However, this does not tell us that diminished translation is not necessary when competition does occur (or am I misunderstanding something?). To support the conclusion they make, the authors would need to show a condition where Xrp1 is expressed, translation is not diminished and competition still occurs. (Maybe they showed this and I missed it). If they have not, they need to temper their conclusion.
Despite this issue, I think this is an excellent body of work that enhances our understanding of cell competition.
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