1. Coupled Caspase and N-End Rule Ligase Activities Allow Recognition and Degradation of Pluripotency Factor LIN-28 during Non-Apoptotic Development 
Benjamin P. Weaver, Yi M. Weaver, Shohei Mitani, Min Han 
Developmental Cell 
Volume 41, Issue 6, Pages e6 (June 2017)
DOI: /j.devcel
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2. Developmental Cell 2017 41, 665-673. e6DOI: (10. 1016/j. devcel. 2017
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3. 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) are 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 SDs. ∗Significant, p values indicated below plot for degradation product at 6 hr, unpaired two-tailed t test. WT, wild-type.
(D) Diagram of lin-28 gene with DxxD CED-3 cleavage motif (red arrowhead, cleavage site). The Δ32M-lin-28 mutation removes residues 2–31 (including DxxD motif) (pink box, modified first exon).
(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 (∗p < , 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 (∗p < , Fisher's exact test). Number of animals scored indicated.
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4. 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). Dots, mean; error bars, SD.
(E and 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). ∗∗p < compared with single mutants, Mann-Whitney test (see Figures S3A–S3C for additional data on seam cell specification).
(G) Analysis of ate-1(RNAi) on seam cell divisions in L4 stage animals. ∗p < , ain-1(−) (mock RNAi) compared with wild-type (WT; mock RNAi); ∗∗p < , ain-1(−); ate-1(RNAi) compared with 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 (∗∗p < , significant compared with single mutants; ‡p = , also significant compared with ubr-1(−);ced-3(−), but not significant compared with ain-1(−);ubr-1(−) or ain-1(−);ced-3(−), Mann-Whitney test). Data independent of (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 (∗∗p < 0.05, significant compared with single mutants; ‡also significant compared with ubr-1(−);ced-3(−), but not significant compared with ain-1(−);ubr-1(−), chi-square test).
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5. Figure 3
Interdependent UBR-1 and CED-3 Activities Limit LIN-28 Expression
(A) Western blot of endogenous LIN-28. Arrows indicate A and B isoforms.
(B) Left panel: quantitation of full-length LIN-28 at 30 hr (Figure S3E, independent replicates) with mean and SD (WT mean set to 1.0). ∗p = , unpaired t test with Welch's correction. Right panel: quantitation of lin-28 mRNA levels by qRT-PCR with mean and SD shown (p = , unpaired t test with Welch's correction).
(C–E) Analysis of LIN-28::GFP fusion protein expression during third larval stage. Box-and-whisker plot in (C) of gonad lengths to confirm stage-matching of input animals (n = 20 for each genotype). Images in (D) are DIC and pseudo-colored LIN-28::GFP. Scale bars in (D), 50 μm. Scatterplot 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). ∗p < 0.05, WT compared with 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 points to full-length LIN-28::GFP fusion protein and arrowhead to ∼30-kDa processed product.
(H) Quantitation of three independent degradation experiments shown in (G) and Figure S3F. Mean values with SDs are shown (Significant, p values indicated, WT compared with ubr-1(−) at the indicated times, unpaired two-tailed t test).
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6. Figure 4 Caspases Likely Form Complexes with Arg/N-End Rule Components
(A and B) Co-immunoprecipitation (CoIP) 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 (for explanation see STAR Methods). NS, non-specific band.
(C) Diagram of CED-3 autoprocessing (Xue et al., 1996) indicated by black arrowheads. The p15 domain can be further processed to a p13 subunit. Red asterisk marks Cys residue in active site.
(D) CoIP analyses with western blot (WB) showing that the p17 subunit specifically associates with UBR-1. The autoprocessed domains and subunits were cloned individually and tested for interaction with UBR-1 (Figures S4B–S4D, independent replicates). NS, non-specific band.
(E) CoIP analyses to show the interaction between full-length ATE-1 and CED-3 caspase. NS, non-specific band.
(F) CoIP 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. Red asterisk, active site. Dotted curved lines, physical interactions found in this study.
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