Coordinate Regulation of Ribosome and tRNA Biogenesis Controls Hypoxic Injury and Translation

Abstract

The translation machinery is composed of a myriad of proteins and RNAs whose levels must be coordinated to efficiently produce proteins without wasting energy or substrate. However, protein synthesis is clearly not always perfectly tuned to its environment, as disruption of translation machinery components can lengthen lifespan and stress survival. While much has been learned from bacteria and yeast about translational regulation, much less is known in metazoans. In a screen for mutations protecting C. elegans from hypoxic stress, we isolated multiple genes impacting protein synthesis: a ribosomal RNA helicase gene, tRNA biosynthesis genes, and a gene controlling amino acid availability. To define better the mechanisms by which these genes impact protein synthesis, we performed a second screen for suppressors of the conditional developmental arrest phenotype of the RNA helicase mutant and identified genes involved in ribosome biogenesis. Surprisingly, these suppressor mutations restored normal hypoxic sensitivity and protein synthesis to the tRNA biogenesis mutants, but not to the mutant reducing amino acid uptake. Proteomic analysis demonstrated that reduced tRNA biosynthetic activity produces a selective homeostatic reduction in ribosomal subunits, thereby offering a mechanism for the suppression results. Our study uncovers an unrecognized higher-order-translation regulatory mechanism in a metazoan whereby ribosome biogenesis genes communicate with genes controlling tRNA abundance matching the global rate of protein synthesis with available resources.

Keywords: aminoacyl tRNA synthetase; hypoxic cell death; protein synthesis; proteomics; ribosomal; ribosomal RNA helicase; tRNA ligase.

Figure 1.. DDX-52 is an RNA helicase required for hypoxic death and for development at high temperature.

Different hypoxic incubation times are used based on the level of killing of the wild type strain N2 and the degree of resistance of the strains being tested. (A) ddx-52(gc51) causes hypoxia resistance. Fraction dead after a 24 hour recovery from a 24 hour hypoxic incubation. All data are mean +/− s.e.m. N=40 independent trials for both N2 and ddx-52(gc51). ). * p < 0.01 versus N2, 2-sided unpaired t-test. (B) Complementation testing with two ddx-52 alleles. % dead after 24 hour recovery from a 20 or 22 hour hypoxic incubation. Data are mean % dead +/− s.e.m N=3 for all strains. * p < 0.01 versus N2, 2-sided unpaired t-test. (C) Transformation rescue of gc51 hypoxia resistance by expression of wild type ddx-52. Death after 24 hour recovery from 24 hours hypoxia for strains with and without extrachromosomal copy of wild type ddx-52 or the transformation marker rol-6. N=3. * p<0.01, 2-sided unpaired t-test. (C) DDX-52 protein domains. Protein domain prediction (InterPro). Image drawn to scale. Alignment of the ATP binding motif of DDX-52 with the closest ortholog of the indicated species (BLAST). Numbers indicate amino acid positions. (E) DDX-52::GFP is ubiquitously expressed in a nuclear pattern in embryos (above – scale bar=50 μm) and adults (below - scale bar=100 μm). (F) DDX-52::GFP nucleolar expression. Merged DIC, fluorescent channel image. Nucleus indicated by bracket; nucleolus by arrow. DDX-52::GFP expression in the nucleolus. Scale bar=20 μm (G, H, I) ddx-52(gc51) causes developmental arrest at 26°C. Newly laid eggs were incubated at 26°C for 72 hours. N2 (G) and ddx-52(gc51) (H) animals were then visually scored for the fraction of animals normally developing to the L4 larval or adult stage (I). See Table S1. Scale bars = 1mm. Data are mean % adults/L4 +/− s.e.m (N=22 trials, >100 animals/trial). * p<0.01, 2-sided unpaired t-test

Figure 2.. Isolation of genetic suppressors of ddx-52(gc51).

(A) Suppressor screen strategy. (B) Isolated suppressor strains. Animals with the noted genotypes were grown at 26°C for 72 hours and number of animals developing into adults/L4 larvae was scored. Data are mean +/− s.e.m (N≥3). *p<0.01 versus ddx-52(gc51), unpaired 2 sided t-test. (C) Model for predicted effect of DDX-52 and suppressor gene products, NCL-1, LARP-1, ULP-4, AATF-1 and NOL-10 on ribosome biogenesis. (D-F) Effect of suppressors on ddx-52(gc51) hypoxia resistance. % death after recovery from 20 or 24 hours of hypoxia (E,F 20 hours hypoxia). Data are mean +/− s.e.m (N≥3). *p<0.05 vs ddx-52(gc51) at same incubation time, 2-sided unpaired t-test. See Figure S1, Table S2.

Figure 3.. Genetic interaction of hypoxia resistant mutants with suppressors and each other.

% death after 24-hour recovery from the noted hypoxic incubation times. Data are mean +/− s.e.m (N≥3). * p<0.05 versus single hypoxia resistant mutant, 2-sided unpaired t-test (A) effect of larp-1(lf), ncl-1(lf) and the insulin receptor suppressor daf-16(lf) on hypoxia resistance of tars-1(rf). (B) effect of ulp-4(rf), nol-10(gf) and aatf-1(gf) on tars-1(rf). (C) effect of larp-1(lf) ncl-1(lf) and ulp-4(rf) on xpo-3(lf). (D) effect of larp-1(lf) ncl-1(lf) on rtcb-1(rf). (E) effect of larp-1(lf) ncl-1(lf) on pept-1(lf). (F) lack of additivity of the hypoxia resistance of tars-1(rf) and ddx-52(lf) (G) lack of additivity of tars-1(rf) and xpo-3(lf) (H) additivity of tars-1(rf) and pept-1(lf). See Figure S2, S3, S4.

Figure 4.. Restoration of translation in tars-1(rf) but not in pept-1(lf) by larp-1(lf) ncl-1(lf).

(A) Polysome profiles of N2, tars-1(gc52), and tars-1(gc52); larp-1(q783) ncl-1(gc53). Representative traces are shown (N=4). (B) larp-1(lf) ncl-1(lf) restore wild type levels of incorporation of ³⁵S methionine in tars-1(rf) but not in pept-1(lf) Wild type (N2), tars-1(gc52), larp-1(q783) ncl-1(gc53), tars-1(gc52); larp-1(q783) ncl-1(gc53), pept-1(Ig601) and larp-1(q783) ncl-1(gc53); pept-1(Ig601) animals were fed ³⁵S labeled OP50 bacteria for 24 hours. Counts per μg proteins extracted from the different strains were plotted. Data represent mean +/− SD (N>=4). * - p<0.0001, 2-tailed unpaired t-test

Figure 5.. Proteomic analysis of tars-1(lf), larp-1(lf) ncl-1(lf) and tars-1(lf); larp-1(lf) ncl-1(lf) triple mutant combination.

(A,B) SILAC incorporation rate (half-life) plotted against threonine frequency for tars-1(gc52) (A) and tars-1(gc52); larp-1(q783) ncl-1(gc53) (B). (C) log2[gc52/N2] ratio of abundance of ribosomal-related proteins in tars-1(gc52) versus N2 quantitated by TMT and LFQ methods. (D) log tars-1(gc52)/N2 ratio of abundance of proteins in entire proteome versus ribosomal-related proteins in by TMT and LFQ methods. See Data S1, Data S2, Figures S5. *** - p<0.001, unpaired 2-tailed t-test

Figure 6.. Translational machinery components regulating hypoxia resistance.

Proteins with hypoxia resistant mutants are in purple. Proteins with suppressor mutants are in red. The normal function of the protein is indicated by an arrow (promotes) or by a T-like symbol (inhibition).