Coupled Caspase and N-End Rule Ligase Activities Allow Recognition and Degradation of Pluripotency Factor LIN-28 during Non-Apoptotic Development

Abstract

Recent findings suggest that components of the classical cell death machinery also have important non-cell-death (non-apoptotic) functions in flies, nematodes, and mammals. However, the mechanisms for non-canonical caspase substrate recognition and proteolysis, and the direct roles for caspases in gene expression regulation, remain largely unclear. Here we report that CED-3 caspase and the Arg/N-end rule pathway cooperate to inactivate the LIN-28 pluripotency factor in seam cells, a stem-like cell type in Caenorhabditis elegans, thereby ensuring proper temporal cell fate patterning. Importantly, the caspase and the E3 ligase execute this function in a non-additive manner. We show that CED-3 caspase and the E3 ubiquitin ligase UBR-1 form a complex that couples their in vivo activities, allowing for recognition and rapid degradation of LIN-28 and thus facilitating a switch in developmental programs. The interdependence of these proteolytic activities provides a paradigm for non-apoptotic caspase-mediated protein inactivation.

Keywords: Arg/N-end rule; Lin28; UBR; developmental timing; heterochronic; non-apoptotic; proteasome; proteostasis; stem cell.

Figure 1. CED-3 Caspase Initiates Proteolytic Processing of LIN-28 but Requires Other Factors for LIN-28 Inactivation

(A) Diagram of LIN-28 regulation by microRNAs and CED-3 caspase (Weaver et al., 2014). Other factors (Rougvie and Moss, 2013) omitted for simplicity. (B) Examination of endogenous CED-3 caspase for site-specific cleavage of LIN-28::GFP fusion (Figures S1A and S1B, experimental setup). Proteolysis reactions from whole worm extracts taken from wild-type animals (+) or ced-3(−) mutants (−) with either a LIN-28::GFP transgene (Tg) or a LIN-28(D31A)::GFP point mutant transgene (D31A). Arrow, full-length LIN-28::GFP fusion protein; arrowhead, ~30 kDa processed product (Figure S1C, independent replicates). (C) Quantitation of degradation experiments shown in (B) and Figure S1C. Mean values with standard deviations. *Significant, p values indicated below plot for degradation product at 6 hr, Unpaired, two-tailed t-test. (D) Diagram of lin-28 gene with DxxD CED-3 cleavage motif. The Δ32M-lin-28 mutation removes residues 2–31 (including DxxD motif). (E) Illustration of lin-28-dependent phenotypes (Rougvie and Moss, 2013). (F) Seam cell counts at fourth larval stage of lin-28(−) animals without or with the Δ32M-lin-28 transgene or endogenous lin-28 Δ32M mutation by Cas9 (*Significant, p<0.0001, Mann-Whitney). Number of animals scored and median values indicated. (G) Vulva phenotypes of lin-28(−) animals at adulthood without or with the Δ32M-lin-28 transgene or endogenous lin-28 Δ32M mutation by Cas9 (*Significant, p<0.0001, Fisher’s exact test). Number of animals scored indicated.

Figure 2. CED-3 Cleaved LIN-28 Is An Arg/N-End Rule Substrate and UBR-1 Ligase Works Non-Additively with CED-3 Caspase to Regulate LIN-28-Dependent Seam Cell Divisions

(A) Illustration of Arg/N-end rule substrates. (B) Diagram of ubiquitin fusion technique. X indicates the N-terminal residue following cleavage by deubiquitinase. (C) Phosphorscreen image of ³⁵S-labeled proteins following in vitro N-end rule assays. (D) Log scale quantitation of three independent in vitro N-end rule assays (Figure S1D, independent replicates and Figure S1E using CED-3 cleaved LIN-28 directly as input). Means, dots. Standard deviations, brackets (E–F) Analysis of ubr-1(−) on seam cell divisions in L4 stage animals (Figures S2A–S2E, ubr-1 homology and mutation). Pseudo-colored images in (E) show seam cell nuclei. Scale bars in (E), 50 μm. Total numbers of animals scored indicated. Orange bars, median values (given below each dot plot). Hatched line, 16 seam cells typically found in wild-type animals (same throughout). **Significant compared to single mutants, p<0.001, Mann-Whitney test. (Figures S3A–S3C, additional data on seam cell specification). (G) Analysis of ate-1(RNAi) on seam cell divisions in L4 stage animals. *Significant, p <0.0001, ain-1(−) (mock RNAi) compared to wt (mock RNAi); **Significant, p <0.0001, ain-1(−); ate-1(RNAi) compared to ain-1(−) (mock RNAi) and wt treated with ate-1(RNAi)(Mann-Whitney). Number of animals scored indicated. (H) Analysis of ubr-1(−) and ced-3(−) to determine impact on seam cell number [**Significant compared to single mutants, p<0.0001, ‡Also significant compared to ubr-1(−);ced-3(−), p = 0.0003, but not significant compared to ain-1(−);ubr-1(−) or ain-1(−);ced-3(−), Mann-Whitney test]. Data independent of panel (F) (Figure S3D, genetic models). (I) Analysis of ubr-1(−) and ced-3(−) to determine impact on adult alae formation. Percentages of gapped and low quality alae indicated [**Significant compared to single mutants, p<0.05, ‡Also significant compared to ubr-1(−);ced-3(−) but not significant compared to ain-1(−);ubr-1(−), Chi-square test].

Figure 3. Inter-Dependent UBR-1 and CED-3 Activities Limit LIN-28 Expression

(A) Western blot of endogenous LIN-28. Arrows, A and B isoforms. (B) Left panel: quantitation of full-length LIN-28 at 30h (Figure S3E, independent replicates) with mean and standard deviation (wt mean set to 1.0) (*Significant, p = 0.0476, unpaired t test with Welch’s correction). Right panel: quantitation of lin-28 mRNA levels by qRT-PCR (p=0.4899, unpaired t test with Welch’s correction). (C–E) Analysis of LIN-28::GFP fusion protein expression during third larval stage. Box-whisker plot in (C) of gonad lengths to confirm stage-matching of input animals (n = 20 for each genotype). Images in (D) are differential interference contrast (DIC) and pseudo-colored LIN-28::GFP. Scale bars in (D), 50 μm. Scatter plot in (E) shows LIN-28::GFP intensity as a function of gonad length [data from (C)]. (F) Comparisons of p values for results given in (E). *Significant, p <0.05, wt compared to either single mutant or double mutant, Mann-Whitney test. (G) Proteolysis reactions of whole worm extracts taken from either ubr-1(−) mutant (−) or ubr-1 wild-type (+) animals with an integrated LIN-28::GFP transgene (Tg) (Figure S3F, independent replicates). Arrow in (G), full-length LIN-28::GFP fusion protein; arrowhead, ~30 kDa processed product. (H) Quantitation of three independent degradation experiments shown in (G) and Figure S3F. Mean values with standard deviations shown (*Significant, p values indicated, wt compared to ubr-1(−) at the indicated times, Unpaired, two-tailed t-test).

Figure 4. Caspases Likely Form Complexes with Arg/N-end rule Components

(A–B) Co-immunoprecipitation (Co-IP) analyses for UBR-1 and CED-3 caspase. The CED-3(C358S) mutation prevents cell death (Figure S4A). Tag sequences: see STAR Methods. WB: Western blot. Two tags were used with CED-3 to demonstrate the specificity of this interaction since UBR-1 tags are sterically inaccessible for immunoprecipitation (explanation in STAR Methods). (C) Diagram of CED-3 auto-processing (Xue et al., 1996) indicated (black arrowheads). The p15 domain can be further processed to a p13 subunit. Red asterisk marks Cys residue in active site. (D) Co-IP analyses with Western blot (WB) showing that the p17 subunit specifically associates with UBR-1. The auto-processed domains and subunits were cloned individually and tested for interaction with UBR-1 (Figures S4B–S4D, independent replicates). (E) Co-IP analyses to show the interaction between full-length ATE-1 and CED-3 caspase. (F) Co-IP analyses of human caspases and human UBR2 (Figure S4E, independent replicate and Figure S4F, caspase homology). (G) Model for a complex containing CED-3 caspase and Arg/N-end rule components.