Fig 2: Endogenous EIF5B clustering is stress inducible and is sufficient to promote HCV, Insr, and Igf1r IRES activity(A) Diagram of GFP-Frankenbody and tagged endogenous EIF5B proteins.(B) Images of HCT116 10B cells transfected with GFP-Frankenbody construct, visualized with live-cell fluorescent microscopy.(C) 375 μM sorbitol induces clustering of endogenous EIF5B.(D) 500 μM NaAsO2 induces clustering of endogenous EIF5B.(E) Percentage of cells with puncta. n = 1,000 cells for all conditions.(F) Distribution of the number of puncta per cell.(G) Addition of sorbitol or NaAsO2 increases the F/R ratio of bicistronic reporters in HCT116 cells.(H) Diagram of Frankenberry protein predicted effect on tagged endogenous EIF5B proteins.(I) Parental HCT116 cells transfected with the Frankenberry construct.(J) HCT116 10B cells transfected with Frankenberry.(K) HCT116 10B cells transfected with Frankenberry and treated with 1 μM dTagV-1. Scale bars: 100 μm.(L) Co-expression of Frankenberry and bicistronic reporters in parental HCT116 cell line, where endogenous EIF5B is untagged.(M) Co-expression of Frankenberry in HCT116 10B cell line.(N) Immunoblot of eIF2α and phospho-eIF2α. Each antibody was imaged in a different channel.For all experiments, at least three biological replicates were assayed in triplicate, and the mean of values is plotted with ±SEM. Statistical significance was determined via unpaired, two-tailed t test with no assumption of equal variance, and the alpha value was set at 0.05; ****p < 0.00001, ns denotes conditions that are not statistically significant. Circles indicate individual measurements.

Fig 3: The EIF5B granules form by self-association(A) Live-cell wide-field fluorescent microscopy imaging of mNeongreen, mNeongreen-NTR, mNeongreen-CTR, mRuby, and mRuby-EIF5B fluorescent proteins expressed in HCT116 cells. Scale bars: 100 μm.(B) Percentage of HCT116 cells expressing the indicated protein with puncta. n = 1,000 cells for all conditions.(C) Distribution of the number of puncta per cell in HCT116 cells expressing the indicated protein. n = 1,000 cells for all conditions.(D) mNeongreen fusion protein is indicated. mRuby-EIF5B is co-expressed in all conditions. Left column is an image of the green channel. Middle column is an image of the red channel. Right column is an image of the merge. Scale bars: 100 μm.

Fig 4: HCV, Insr, and Igf1r IRES activity is specifically stimulated by EIF5B overexpression(A) Diagram of bicistronic reporter plasmids and expression vectors used in the assays.(B) Diagram of the reporter RNAs produced by transcription of the transfected plasmids. Black circle indicates cap.(C) Dual-luciferase assay demonstrating the effect of overexpressing each protein candidate via transient transfection. Translation is represented as a ratio of IRES-mediated firefly luciferase signal over cap-dependent Renilla luciferase (F/R).(D) Raw Renilla luminescence from bicistronic assay shown in (C).(E) Raw firefly luminescence from the bicistroinc assay shown in (C).(F) Effect of overexpression of EIF5B on the bicistronic reporter in HCT116 cells.(G) Effect of overexpression of EIF5B on the activity of the HCV, Insr, and Igf1r monocistronic IRES reporter HCT116 cells.(H) EIF5B promotes HCV, Insr, and Igf1r IRES activity in vitro.For all experiments, at least three biological replicates were assayed in triplicate, and the mean of values is plotted with ± standard error of the mean (SEM). Statistical significance was determined via unpaired, two-tailed t test with no assumption of equal variance, and the alpha value was set at 0.05; ****p < 0.00001, ns denotes conditions that are not statistically significant. Circles indicate individual measurements.

Fig 5: EIF5B granules contain RNA and do not co-localize with G3BP1(A–D) Co-transfection of Frankenberry and mNeongreen-G3BP1 in HCT116 10B cells. Scale bars: 100 μm. (A) Frankenberry imaging of endogenous EIF5B, (B) mNeongreen-labeled G3BP1, (C) merge, and (D) Merge with individual cells outlined.(E) Pearson’s correlation coefficient for Frankenberry and mNeongreen-G3BP1 is −0.187 for n = 1,000 cells.(F) Pearson’s correlation coefficient for endogenous EIF5B, labeled with GFP-Frankenbody, and mRuby_G3BP1, both transiently expressed in HCT116 10B under unstressed (−) conditions or with treatment of 375 μM sorbitol or 500 μM NaAsO2. The Pearson’s correlation coefficients are −0.238, −0.072, and −0.056, respectively.(G) The expression of Insr_3xSpinach2 alone.(H) Co-expression of Insr_3xSpinach2 with EIF5B NTR.(I) The expression of the BG_3xSpinach2 alone.(J) Co-expression of BG_3xSpinach2 with EIF5B NTR.(K–M) The co-expression of the Insr_3xSpinach2 and Frankenberry (K) green channel to image Insr RNA, (L) red channel to image Frankenberry, and (M) merge.(N–P) The co-expression of the BG_3xSpinach2 and Frankenberry (N) green channel to image BG RNA, (O) red channel to image Frankenberry, and (P) merge. All constructs were imaged in unfixed HCT116 10B cells. Scale bars: 100 μm.