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Inhibition of ribosome biogenesis in the epidermis is sufficient to trigger organism-wide growth quiescence independently of nutritional status in C. elegans [1]

['Qiuxia Zhao', 'Department Of Molecular Biosciences', 'University Of Texas At Austin', 'Austin', 'Texas', 'United States Of America', 'Rekha Rangan', 'Shinuo Weng', 'Cem Özdemir', 'Elif Sarinay Cenik']

Date: 2023-09

Interorgan communication is crucial for multicellular organismal growth, development, and homeostasis. Cell nonautonomous inhibitory cues, which limit tissue-specific growth alterations, are not well characterized due to cell ablation approach limitations. In this study, we employed the auxin-inducible degradation system in C. elegans to temporally and spatially modulate ribosome biogenesis, through depletion of essential factors (RPOA-2, GRWD-1, or TSR-2). Our findings reveal that embryo-wide inhibition of ribosome biogenesis induces a reversible early larval growth quiescence, distinguished by a unique gene expression signature that is different from starvation or dauer stages. When ribosome biogenesis is inhibited in volumetrically similar tissues, including body wall muscle, epidermis, pharynx, intestine, or germ line, it results in proportionally stunted growth across the organism to different degrees. We show that specifically inhibiting ribosome biogenesis in the epidermis is sufficient to trigger an organism-wide growth quiescence. Epidermis-specific ribosome depletion led to larval growth quiescence at the L3 stage, reduced organism-wide protein synthesis, and induced cell nonautonomous gene expression alterations. Further molecular analysis reveals overexpression of secreted proteins, suggesting an organism-wide regulatory mechanism. We find that UNC-31, a dense-core vesicle (DCV) pathway component, plays a significant role in epidermal ribosome biogenesis-mediated growth quiescence. Our tissue-specific knockdown experiments reveal that the organism-wide growth quiescence induced by epidermal-specific ribosome biogenesis inhibition is suppressed by reducing unc-31 expression in the epidermis, but not in neurons or body wall muscles. Similarly, IDA-1, a membrane-associated protein of the DCV, is overexpressed, and its knockdown in epidermis suppresses the organism-wide growth quiescence in response to epidermal ribosome biogenesis inhibition. Finally, we observe an overall increase in DCV puncta labeled by IDA-1 when epidermal ribosome biogenesis is inhibited, and these puncta are present in or near epidermal cells. In conclusion, these findings suggest a novel mechanism of nutrition-independent multicellular growth coordination initiated from the epidermis tissue upon ribosome biogenesis inhibition.

Funding: This work was supported by the National Institutes of Health (5R35GM138340-03 to ESC), the Welch Foundation (F-2133-20230405 to ESC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The RNA-seq libraries from this study can be accessed via the NCBI GEO database using the accession code GSE213367. The data is available at this link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE213367 Deseq2 analysis output tables for RNAseq, Mass-spec raw peptide counts and DEP output tables are available as supplementary tables, and uploaded to submission.

Copyright: © 2023 Zhao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The ida-1 gene, which exhibits epistasis to unc-31, encodes IA-2/IDA-1, a protein that genetically interacts with UNC-31/CAPS and affects neurosecretion in C. elegans [ 35 ]. UNC-31 is the C. elegans homolog of CAPS, a crucial factor in the priming step of Ca 2+ -dependent exocytosis of DCVs and the regulation of DCV cargo release [ 36 , 37 ]. Intriguingly, reducing the expression of epidermal ida-1 or unc-31 led to an increase in worm body length when epidermal ribosome biogenesis was inhibited. We also observed the presence of DCV puncta, indicative of the subcellular localization of IDA-1, in or near epidermal cells. Taken together, our findings highlight the significant role of DCV secretion in the vicinity of epidermal tissue in mediating the growth quiescence associated with epidermal ribosome biogenesis inhibition.

Using the AID system, we examined the impact of RPOA-2, GRWD-1, and TSR-2 depletion on ribosome biogenesis. We found that depleting any of these proteins led to a deficiency in ribosome biogenesis, triggering a growth quiescence response across the organism at an early larval stage. Interestingly, this quiescence was resistant to rescue attempts by bypass mutations in the insulin signaling pathway (daf-16 and daf-18). The deficiency of ribosome biogenesis in tissues of equivalent volume resulted in a scaled, coordinated growth. We directed our attention towards the specific inhibition of ribosome biogenesis in the epidermis tissue, observing profound consequences for the entire organism. This led to a significant slowdown in organism-wide growth (quiescence) and induced gene expression changes in a diverse range of cell types in a cell nonautonomous manner. Overexpression of secreted proteins and dense-core vesicle (DCV) pathway proteins were observed, while both cytosolic and mitochondrial ribosomal proteins were significantly underexpressed throughout the organism. We also confirmed the overexpression of the DCV membrane-associated protein, IDA-1, in response to epidermal ribosome biogenesis inhibition.

In this study, we used an auxin-inducible degradation (AID) system [ 26 , 27 ] to specifically and reversibly modulate ribosome biogenesis at distinct stages in C. elegans. Ribosomes, consisting of 2 subunits, 60S and 40S, integrate different ribosomal proteins and ribosomal RNA. The transcription of 45S ribosomal DNA loci into rRNA is carried out by RNA Polymerase I (Pol I) [ 28 , 29 ]. Primarily, in the nucleolus, the newly translated ribosomal proteins are imported from the cytoplasm by dedicated chaperones [ 30 , 31 ]. For instance, Rrb1p chaperones uL3 to the nucleolus, and its depletion reduces the 60S ribosomal subunit levels, leaving the 40S subunit unaffected in yeast [ 32 ]. Similarly, Tsr2 chaperones the r-protein eS26 to the first assembling pre-ribosome, the 90S, and is necessary for the cytoplasmic processing of 20S pre-rRNA into mature 18S rRNA [ 33 ]. Tsr2 also regulates the release and reincorporation of eS26 from mature ribosomes, facilitating a reversible stress response [ 34 ]. Within C. elegans, rpoa-2 encodes the second largest subunit of RNA Pol I, while Y54H5A.1 (grwd-1) and Y51H4A.15 (tsr-2) encode the chaperone proteins required for the assembly of ribosomal proteins RPL-3 and RPS-26, respectively.

Our previous research revealed a ribosome biogenesis-mediated growth coordination in mosaic animals in C. elegans. Specifically, using unigametic inheritance [ 24 ], we generated embryos with an anterior–posterior (AB-P1) split of wild-type and ribosomal protein gene null cells (rpl-5(0)) at the two-cell cleavage step. These mosaic embryos, completing embryogenesis with maternal ribosomes, experienced L1 stage arrest. The growth of wild-type cells paralleled that of their rpl-5(0) neighbors, indicating an organism-wide checkpoint. This checkpoint persisted despite insulin signaling pathway bypass mutations (daf-16 and daf-18) and was associated with a stress response gene expression profile, suggesting that growth coordination between the 2 lineages can be independent of nutritional status [ 25 ].

Caenorhabditis elegans provides a suitable model for studying growth coordination due to its fast developmental cycle and available genetic and cytological tools. In contrast to insect clade development, which is centrally mediated by the ecdysone hormone, C. elegans developmental timing is dependent on an intricate network of heterochronic genes (reviewed in [ 11 ]). Furthermore, C. elegans can modulate their larval development according to external cues, such as nutrient availability, through dauer regulation and starvation-induced larval quiescence, primarily attributed to IIS and TGFβ signaling pathways (reviewed in [ 4 , 5 ]). Finally, numerous examples, such as starvation response, dietary restriction, and mitochondrial unfolded protein response-mediated longevity [ 12 – 23 ], demonstrate cell nonautonomous organism-wide communication within C. elegans.

One of the best-studied examples of growth coordination comes from Drosophila studies, revealing that the growth of eye discs is coordinated upon knockdown of ribosomal protein genes, RpL7 or RpS3, specifically in the wing [ 7 ]. This finding suggests that system-wide growth coordination requires communication between different organs. In Drosophila, the coordination between wing and eye disc growth is regulated by the insect-specific Xrp1 and mediated by ecdysone inhibition through the secreted peptide hormone Dilp8. The JNK stress signaling pathway also plays a role in this process [ 7 , 10 ]. Since Xrp1 and Dilp8 are specific to the insect clade, it suggests the existence of evolutionarily divergent mechanisms. However, several key questions remain unanswered: (1) Do similarly divergent or conserved mechanisms operate in other species? (2) What role do specific tissues play in overall organism growth? (3) How is information relayed between body parts?

Organism-wide growth in metazoans is a complex process that is influenced by a combination of autonomous [ 1 – 3 ] and nonautonomous factors. These factors process information from nutritional cues via pathways including TORC1, TGFβ, and insulin/insulin-like growth factor signaling (IIS) (reviewed in [ 4 , 5 ]). Interestingly, growth coordination maintains proper body proportions, even if a specific organ’s growth is hindered. For example, when the left limb of a mouse has its cell cycle suppressed during development, the symmetry between the left and right limb remains unchanged [ 6 ]. In Drosophila, other compartments’ development slows down when one embryonic compartment’s growth is disturbed [ 7 – 9 ]. However, how growth regulation occurs in response to a specific organ’s growth impairment is not well understood, unlike the mechanisms governing nutrition-dependent organismal growth regulation.

Results

Modulation of ribosome biogenesis using the AID system To modulate ribosome biogenesis in an inducible fashion, we decided to use the AID system to target biogenesis factors [26,27]. In this approach, an auxin-inducible degron-tagged target protein can be depleted upon the expression of an auxin receptor F-box protein TIR1 and the small molecule auxin (indole-3-acetic acid (IAA)) [26]. We generated C. elegans strains with an AID degron::GFP cassette integrated into the genomic loci of an RNA Pol I subunit (rpoa-2), as well as the chaperones of RPL-3 and RPS-26 (grwd-1/Y54H5A.1 and tsr-2/Y51H4A.15, respectively) using CRISPR/Cas9-mediated editing [30,38]. These tagged proteins specifically function in ribosomal RNA transcription from repeated 45S ribosomal DNA loci, as well as nucleolar 40S and 60S ribosome subunit biogenesis, thus, specifically target ribosome biogenesis at 3 distinct steps (Figs 1A and S1A–S1F). PPT PowerPoint slide

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TIFF original image Download: Fig 1. AID system facilitates degradation of ribosome biogenesis factors. (A) The scheme illustrates ribosome biogenesis factors investigated in this study and is created with BioRender.com. rpoa-2 encodes the second-largest subunit of RNA Pol I, while grwd-1 and tsr-2 encode chaperone proteins that assist RPL-3 and RPS-26 in nuclear large and small ribosomal subunit assembly, respectively. (B) Localization of endogenous RPOA-2, GRWD-1, and TSR-2 in live animals. A degron::GFP cassette was integrated to the N terminus of the endogenous rpoa-2 gene or C terminus of grwd-1 and tsr-2 genes. The L4 stage animals were imaged using DIC and fluorescence. RPOA-2 is localized in the nucleolus, while GRWD-1 and TSR-2 are primarily localized in the nucleus. (C) The AID system enables the degradation of RPOA-2, GRWD-1, and TSR-2. L3 stage animals were incubated with 1 mM IAA and imaged after 24 hours. For quantification, each 20× image was analyzed using Fiji software. Data represent GFP intensity (corresponding to RPOA-2, GRWD-1 or TSR-2) normalized by mRuby intensity (TIR1) from 25 animals. Animals were immobilized on slides using 1 mM levamisole. Statistical significance was determined via an independent t test. Scale bar, 50 μm. The underlying data for (C) can be found in the Tab A in S1 Data. AID, auxin-inducible degradation; IAA, indole-3-acetic acid; RNA Pol I, RNA Polymerase I. https://doi.org/10.1371/journal.pbio.3002276.g001 To further validate RPOA-2, GRWD-1, and TSR-2 have analogous roles in C. elegans ribosome biogenesis as described for their homologs, we conducted polysome profiling experiments. Our data indicated that depleting RPOA-2 reduced the amount of ribosomal subunits, monosome, and polysome peaks, without preferential depletion of a specific subunit (S2C Fig). The depletion of GRWD-1 significantly reduced the large subunit (60S), monosome and polysome peaks, with an accumulation of the small subunit (40S) (S2D Fig). This observation is in line with the previous studies on the yeast ortholog encoded by RRB1 [32]. TSR-2 depletion led to a decrease in mature ribosomes and an overall increase in 60S levels (S2E Fig), in agreement with the earlier studies on the yeast ortholog encoded by TSR2 [33]. Therefore, our results suggest that depleting RPOA-2, GRWD-1, or TSR-2 significantly reduces translating ribosome populations, a finding that corroborates previous studies on yeast orthologs. Strains expressing degron::GFP-integrated RPOA-2, GRWD-1, or TSR-2 were found to be homozygous viable and phenotypically identical to the wild type. These exhibited nucleolar RPOA-2 [39], nuclear GRWD-1, and nuclear TSR-2 localization patterns (Figs 1B and S2A), indicating that the degron::GFP tags are consistent with normal organism growth. To evaluate the AID system, we crossed strains expressing degron::GFP integrated the ribosome biogenesis factor (RPOA-2, GRWD-1, or TSR-2) with strains ubiquitously expressing TIR1 under the eft-3 promoter. L3 stage animals expressing both the degron::GFP tag and TIR1 showed complete depletion of GFP signals when exposed to 1mM IAA overnight (Fig 1C). Similarly, in the presence of IAA, RPOA-2 tagged with degron::GFP was undetectable by western blot within 3 hours (S2B Fig). This suggests that the AID system successfully degraded the ribosome biogenesis factors (RPOA-2, GRWD-1, and TSR-2).

Embryonic inhibition of ribosome biogenesis results in a reversible quiescence To assess the effect of IAA-mediated depletion of RPOA-2, GRWD-1, or TSR-2 on embryonic development, we treated stage-synchronized embryos expressing RPOA-2, GRWD-1, or TSR-2 tagged with a degron::GFP, in the presence of ubiquitous TIR1, with 1 mM IAA for 24 hours. As anticipated, given the sufficiency of maternal ribosomes for C. elegans embryonic development [25], all embryos completed embryogenesis and hatched, despite the depletion of ribosome biogenesis factors with IAA treatment. To evaluate postembryonic development without new ribosome biogenesis, we measured larval body length following a 3-day incubation (with or without IAA) starting from stage-synchronized embryos (Fig 2A, top). All 3 strains (rpoa-2, grwd-1, or tsr-2 degron::GFP integrated in the presence of eft-3p::TIR1) exhibited an overall stall in growth and development when exposed to IAA (Figs 2A and S3A). PPT PowerPoint slide

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TIFF original image Download: Fig 2. AID-mediated organism-wide ribosome biogenesis inhibition leads to developmental quiescence at the L2 stage. (A) Synchronized embryos of degron::GFP::rpoa-2, grwd-1::degron::GFP, or tsr-2::degron::GFP strains in the presence of eft-3p::TIR1 were treated either with (+) or without (−) 1 mM IAA for 3 days. Animals were imaged using DIC. Scale bar, 50 μm. (B) The overall body length of animals (from A) was analyzed using Fiji software. Data were obtained from 9 animals without IAA treatment and 21 animals with IAA treatment from each strain. Statistical significance was determined using an independent t test. (C) Mesoblast precursor (M) cell division was observed over a span of 4 days following embryo synchronization. Up to 18 M cells were observed in both degron::GFP::rpoa-2; eft-3p::TIR1 animals treated with 1 mM IAA (top) and homozygous arrested rpoa-2(ok1970) animals (bottom). Scale bar, 10 μm. Animals were immobilized on slides using 1 mM levamisole in (A and C). (D) Synchronized embryos expressing TIR1 globally and harboring degron::GFP-integrated ribosome biogenesis factors (RPOA-2, GRWD-1, and TSR-2) were incubated with 10 μM IAA for 3 days, followed by 6 days after removal of IAA. The percentage of gravid adults was assessed from at least 40 animals. The underlying data for (B and D) can be found in Tab B in S1 Data. AID, auxin-inducible degradation; IAA, indole-3-acetic acid. https://doi.org/10.1371/journal.pbio.3002276.g002 It is important to note that, in the absence of IAA, the global expression of TIR1 induces a modest background degradation of degron::GFP (S3B Fig) [40,41], with a higher basal degradation in tsr-2::degron::GFP strains compared to rpoa-2 and grwd-1 degron::GFP strains (S3B Fig). Thus, animals ubiquitously expressing TIR1 and degron::GFP-integrated TSR-2 developed significantly more slowly even in the absence of IAA, suggesting that basal degradation of TSR-2 affects postembryonic development (Fig 2A and 2B). To accurately stage animals upon the universal embryonic depletion of RPOA-2, we examined 2 distinct postembryonic lineages: the mesoblast precursor cell (M cell) and vulval precursor cells hlh-8p::GFP and egl-17p::mCherry reporters [42,43]. During the L1 stage, the M cell undergoes mitosis to generate 18 cells, 2 of which migrate during the L2 stage, subsequently dividing and differentiating into sex muscle cells at later larval stages [44]. With global depletion of RPOA-2, we observed 18 M cells, indicating that the quiescent larvae progressed at least to the late L1 stage (Fig 2C, top). Comparable M cell division patterns in rpoa-2(ok1970) null animals (Fig 2C, bottom) suggest that the ubiquitous depletion of RPOA-2 by the AID system can mimic the genetic deletion of rpoa-2. At the L3 larval stage, vulval precursor cells P(5–7).p adopt primary or secondary cell fates and undergo invariant cell divisions [45]. An inspection of these cells suggests that rpoa-2(ok1970) null animals halt development at the L2 stage (S3C Fig). In conclusion, the universal depletion of these ribosome biogenesis factors and the genetic loss of rpoa-2 lead to a growth standstill at the L2 stage. Contrary to the developmental quiescence observed at the early larval stage, when ribosome biogenesis was inhibited from the L4 stage onward, the animals matured into gravid adults (S4A Fig). This implies that the developmental quiescence is specific to the early larval stage. During the quiescent larval stage characterized by the depletion of a ribosome biogenesis factor (RPOA-2, GRWD-1, or TSR-2), animals relied on preexisting ribosomes for survival. We then investigated whether these remaining ribosomes could facilitate the recovery of these animals to gravid adulthood by enabling the synthesis of new ribosomes when IAA was removed. AID-mediated protein degradation can be reversed in the presence of low IAA concentrations (10 μM, 25 μM), with a potential for complete protein recovery post IAA removal [26]. To examine this reversibility, embryos were exposed to 10 μM IAA for 3 days and then transferred to IAA-free plates for 6 days. The recovery rates post IAA removal were notably less than 100% but significantly higher for GRWD-1 and TSR-2 global depletion compared to RPOA-2 (12.2%, 47.6%, and 61%, respectively, after a 3-day depletion of RPOA-2, GRWD-1, or TSR-2) (Fig 2D). Additionally, postembryonic growth reversibility for globally depleted RPOA-2 was observed to be both time and IAA concentration dependent, with gravid adults noted after up to 5 days of incubation with 10 μM IAA (S4B Fig). These findings suggest that restarting new ribosome biogenesis can alleviate growth quiescence in a fraction of animals.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002276

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