Fig 2: HMGN3 is a direct target of IRES-dependent DAP5-mediated translation. (A) Schematic of in vitro and in vivo IRES activity assays. (B) In vitro IRES activity screen. RNA hairpin (hp) FL vectors containing the 5′ UTRs of the indicated genes were translated in RRLs. Luciferase activity was measured and compared with background luciferase activity from the empty hairpin vector, which was set as 1. (*) P < 0.02; (**) P < 0.005. (C) IRES activity in vivo. HMGN3 5′ UTR and empty hairpin vectors were transfected into NT and DAP5 knockdown hESCs, and relative luciferase activity was measured. (D) DAP5 was immunoprecipitated from H9 pluripotent cells followed by purification of associated RNA. mRNA levels of the indicated genes were determined by real-time PCR and normalized to corresponding levels within total mRNA. Levels of DAP5 self-immunoprecipitation, used as a positive control, were set as 1. (E, top) Radiolabeled RNAs corresponding to the HMGN3 5′ UTR and the first or second half of the 5′ UTR were incubated with or without DAP5 protein and resolved by gel electrophoresis. (Bottom) The radiolabeled HMGN3 5′ UTR was incubated with DAP5 protein in the presence of increasing concentrations of competing unlabeled full-length 5′ UTR or either half. In all graphs, data are presented as mean ± SD of three (B,C) or four (D) biological repeats. Significance was determined by t-test.

Fig 3: Translation of endogenous DAP5 (eIF4G2/p97) is resistant to CHX. (A) HeLa cells were transfected with the GUG-nLuc-3XFlag reporter. After 24 h, cells were treated with vehicle (Veh., 0.1% DMSO) or 100 µg/mL CHX and then incubated for 15 min or 24 h before being subjected to ribosome profiling and RNA-seq. (B) A volcano plot showing changes in TE of endogenous mRNAs (24 h of CHX vs. 15 min of CHX) compared with the associated false discovery rates (FDRs). FDRs for each mRNA are listed in Supplemental Table S1. (C,D) Ribosome footprints (C) and RNA-seq fragments (D) that mapped to the GUG-encoded DAP5 locus in cells that had been treated with CHX for 15 min (blue) or 24 h (red). Raw counts are depicted, and the ribosome footprint and RNA-seq RPKMs (which account for library size and ORF length) of the DAP5-coding sequence are also given for each replicate and time point. (E) Western blotting was used to assess endogenous DAP5 protein levels in HeLa cells before (control) and after 24 h of treatment with 1, 10, or 100 µg/mL CHX. Tubulin was used as a loading control. (F) RT-qPCR was used to assess endogenous DAP5 mRNA levels before (control) and after 24 h of 1 µg/mL CHX treatment. FH and NPTX1 mRNAs (whose levels did not change during CHX treatment as determined by RNA-seq) were used as dual internal references. Data are shown as mean ± SD. n = 3. A two-tailed unpaired t-test with Welch's correction was used. (G) After 1 or 24 h of treatment with 100 or 1 µg/mL CHX, HeLa cells were subjected to PURO labeling and Western blotting to confirm global translation inhibition. Tubulin was used as a loading control. (H) Western blotting was used to examine expression of the GUG- and AUG-encoded DAP5-3XFlag reporters before and after 24 h of 100 µg/mL CHX treatment. Tubulin was used as a loading control. AUG* denotes that one-fourth of the AUG-DAP5-3XFlag plasmid was transfected to avoid overexposure during film development.

Fig 4: RBP groups inferred from eCLIP data and co-IP validation. a We show the summary of datasets for different CLIP technologies and cell lines used in this study. The Venn diagram shows only a few number of shared RBPs among three cell lines. b Example RBP groups having interactions supported by other data/evidence in HepG2 and K562 cell lines. c Co-IP of FKBP4 and SRSF1 (group 8) in HepG2 cell line. d Co-IP of EIF4G2 and IGF2BP1 (group 3) in K562 cell line

Fig 5: Phenotypic and molecular characterization of DAP5 knockdown hESCs. (A) Light microscopic images of NT and DAP5 shRNA-expressing hESCs grown on mouse embryonic fibroblast (MEF) feeder cells. (B) Quantitative real-time PCR was performed on nondifferentiated NT shRNA-expressing or DAP5 shRNA-expressing hESCs. For each gene, the mRNA level of the NT knockdown cells was set at 1. Shown are means ± SD of independent biological duplicates. Statistical significance between pairs was determined by t-test. (*) P < 0.005; others are not significantly different. (C) NT and DAP5 knockdown hESC colonies were immunostained for plutipotent markers Nanog and Oct3/4. DAPI was used to stain nuclei. (D) Relative mRNA levels of NANOG, HOXA1, and RARB in NT or DAP5 knockdown hESCs treated with RA at the indicated concentrations for 48 h. (*) P < 0.05; (**) P < 0.01; (***) P < 0.005; (****) P < 0.0005. (E) Western blot of wild-type H9 (−), control (NT), and DAP5 shRNA-expressing cells undifferentiated or treated with 100 nM RA for 48 h, showing expression of DAP5 and pluripotent markers Nanog and Oct4. GAPDH was used as loading control. (F) NT and DAP5 shRNA hESCs were transfected with control mCherry or DAP5 plasmids and then treated with RA for 24 h. RT–PCR was performed to quantify mRNA levels of HOXA1 and RARB, and protein levels of Nanog were monitored by Western blotting. Densitometric analysis of Nanog protein levels, normalized to GAPDH protein levels, is presented below the blots. In the graphs in D and F, data are presented as mean ± SD of triplicates, with statistical significance determined by t-test.