Fig 1: Structural analysis of ribosome-bound EDF1 and Mbf1.(A) Overview of EM map and models of the 40S subunit of human non-rotated EDF1-bound ribosome. Selected r-proteins and EDF1 (orange) are shown as models in the EDF1•80S map (PDB: 6ZVH). (B) Overview of Mbf1 (violet red) bound to the yeast rotated ribosome with hybrid tRNAs. (PDB: 6ZVI). (C) Alignment of EDF1 and Mbf1 sequences colored by conservation and domain architecture of EDF1. (D) Overall structure of ribosome-bound EDF1 and Mbf1 showing a highly similar fold and binding mode with the C-terminus sandwiched between helix 16 (h16) and helix 33 (h33) of the 18S rRNA and the r-protein uS3 close to the mRNA entry channel, and the N-terminus forming a helix at the base of helix 16. (E) EDF1 and Mbf1 interact with rRNA helix 18 (h18), displacing the C-terminus of eS30. Binding of EDF1 and Mbf1 shifts helix 16 towards the ribosome, resulting in a clash of the canonical eS30 position with the new position of helix 16. See also Figure 3—figure supplement 1, Figure 3—figure supplement 2 and Figure 3—source data 1.Figure 3—source data 1.Cryo-EM data collection, refinement and validation statistics.
Fig 2: Interaction analyses of EDF1 under basal growth and ribotoxic-stress conditions.(A) Immunoaffinity purification of endogenous EDF1 from untreated (UT) or low dose emetine treated (EL; 1.8 µM, 15 min) HEK293T cells using Protein A-coupled EDF1-antibody. Scatter plot showing log2(LFQ) intensity of proteins identified under EL (y-axis) and UT (x-axis) conditions. (B) BioID analyses of BirA*-EDF1 with and without doxycycline induction. Volcano plot of fold change in protein LFQ intensity with or without BirA*-EDF1 expression induction by doxycycline (dox). Selected candidates highlighted in red. A cutoff of (+dox/-dox) =16 fold and p-value = 0.01 was set to eliminate known BioID contaminants. See also Figure 5—figure supplement 1, Figure 5—source data 1, and Figure 5—source data 2.Figure 5—source data 1.Related to Figure 5A; Immunoaffinity purification of endogenous EDF1 from untreated (UT) and 1.8 µM emetine treated (EL) HEK293T cells for 15 min.Figure 5—source data 2.Related to Figure 5B and Figure 5—figure supplement 1B–1C; BioID analyses of BirA*-EDF1 with or without doxycycline induction for 16 hr.
Fig 3: Interactions and functional implications of EDF1 and Mbf1.(A) EDF1 (orange) and Mbf1 (violet red) interact with ribosomal protein uS3 via a helix-helix interaction. In the human structure, Y107 of uS3 is stacks with H70 of EDF1. Conserved residues required for frameshift inhibition in yeast are colored in steel blue. (B) Overview of Mbf1’s position with respect to the mRNA path on the 40S ribosomal subunit. (C) Mbf1 clamps the mRNA into a headlock, with the aromatic amino acid Y48 exposed to facilitate interaction with the mRNA. The KKY-motif is well conserved between Mbf1 and EDF1 (KKW). (D) Comparison of the mRNA path of a Mbf1-bound colliding ribosome with that of a canonical colliding ribosome (PDB: 6I7O). The mRNA and helix 16 are shifted in Mbf1-bound ribosomes. (E) Overview of the Mbf1-ribosome interaction in collided polysomes. Mbf1 binds the second and third ribosomes of the trisome unit.
Fig 4: Functional relationship among mbf1, E(z) and pcm. (A) pcm is downregulated by Psc and Pc. Expression of the indicated genes in third instar male larvae was analyzed by RT-qPCR in the wild-type or Polycomb group mutant background. pcm does not appear to be a direct target of Polycomb silencing (Zeng et al., 2012). Data are mean±s.d. relative to the wild-type mRNA level; *P<0.01 (Student's t-test). (B) Western blot analysis of Pcm in wing discs from the indicated lines. Numbers indicate relative Pcm levels normalized to Tubulin levels. (C) pcm mutation suppresses the extra sex comb phenotype. *P<0.01 (Fisher's exact test). (D) pcm mutation restores the E(z) mRNA level in Psc1/+ or Psc1/+; mbf12/+. The E(z) mRNA levels in third instar male larvae of the indicated lines were analyzed by RT-qPCR. Data are mean±s.d. relative to the wild-type mRNA level. NS, not significant; *P<0.01 (Student's t-test). (E) (Top) Misexpression of Ubx in the wing disc of Psc1/+; mbf12/+ and its suppression by pcmΔ1. Arrows indicate Ubx-positive spots. (Bottom) Immunostaining of E(z) protein in the wing discs shown above. (Right) Adult wing defect (arrowheads) in Psc1/+; mbf12/+ and its suppression by pcmΔ1. The number of wings with the defect among the total number of wings examined is indicated. **P<0.05 (Fisher's exact test).
Fig 5: EDF1 is critical for JUN-centric transcriptional response to ribosomal collisions.(A) Immunoblots of HEK293-Flp-In TREx WT cell extracts showing phosphorylation of JUN at serine 73, and ubiquitylation of eS10 in response to emetine treatment at the indicated concentrations for 15 min. (n = 2) (B) Schematic for RNA sequencing analyses of HEK293-Flp-In TREx WT and ?EDF1 treated with 0 µM (UT) and 1.8 µM emetine for 30 (EL30) and 120 min (EL120). (C) Volcano plots of fold change of normalized transcript reads in emetine-treated compared to untreated samples for HEK293-Flp-In TREx WT cells at 30 (left) and 120 (right) minutes. (D) Scatter plot of log2(EL/UT) fold change of normalized transcript reads from emetine-treated HEK293-Flp-In TREx WT cells after 30 min (x-axis) or 120 min (y-axis). (E) Volcano plots of the ratio of fold change in normalized transcript abundance in response to emetine treatment between ?EDF1 and WT cells at 30 (left) and 120 (right) minutes. (F) Normalized transcript reads of selected genes for untreated (UT) and 1.8 µM emetine treated samples at 30 (EL30) and 120 min (EL120) in WT (filled circle) and ?EDF1 (open circle) cell lines. (G) Cartoon showing the multifaceted roles of EDF1 in coordinating different arms of the ribosome-mediated QC pathway and promoting a JUN-centric transcriptional program in response to ribosome collisions. See also Figure 7—figure supplement 1.
Supplier Page from Abcam for Anti-EDF1 antibody