Fig 2: Model of CLUH function in the liverCLUH forms granules containing target mRNAs and regulates their translation. In addition, CLUH granules recruit G3BP1, G3BP2, and mTOR, thereby enhancing mitophagy and inhibiting mitochondrial anabolic pathways. Together, these roles of CLUH are crucial in the liver to survive starvation. In the absence of CLUH, CLUH granules do not form resulting in reduced translation of Hmgcs2, Hadha, and Pcx mRNAs, in failure to suppress mTORC1-dependent anabolic pathways, and in a block in mitophagy.

Fig 3: Astrin loss promotes anabolic pathways.(A) Volcano plot of label-free proteomics of WT and SPAG5 ind KO HeLa cells (n = 4 independent replicates). CLUH targets are marked in blue. KEGG pathways of downregulated (B) or upregulated (C) proteins (with a cutoff of p = 0.05; q = 0.15) detected in proteomics analysis of SPAG5 ind-KO cells (A and Supplementary file 3) using the EnrichR webtool. Targeted metabolomics of WT, SPAG5, and KNSTRN ind-KO cells after 8 hr Hanks' Balanced Salt Solution (HBSS) starvation showing glycolytic intermediates and lactic acid (D), sedoheptulose-7-P (E), pyruvic acid and TCA cycle intermediates (F), nucleotide levels (G), and ornithine and polyamine levels (H). Bars show mean ± standard error of the mean (SEM) and dots represent values of individual replicates. One-way analysis of variance (ANOVA) with post hoc Tukey’s multiple comparison tests on log converted fold changes were performed with *p = 0.05; **p = 0.01; ***p = 0.001.

Fig 4: CLUH interacts with astrin-1 and kinastrin.(A, B) Western blots of reciprocal co-IPs of endogenous CLUH, astrin, and kinastrin in HeLa cells. Two different antibodies have been used to pull down CLUH (1, 2). Asterisk marks IgG light chain. (C) Scheme of human SPAG5 cDNA with indicated UTRs and ORFs. Closeup shows positions of the uATG and ATG1–6 with surrounding Kozak sequences. (D, E) Western blots of HeLa cells overexpressing FLAG-tagged astrin constructs. Pan-actin was used as loading control. (F) Western blots of reciprocal co-IPs of endogenous CLUH and overexpressed FLAG-tagged astrin full length (ATG1-SPAG5) or a N-terminal deleted variant (?151-SPAG5) in WT and CLUH KO HeLa cells. Pan-actin was used as loading control for input samples. Asterisks indicate additional astrin bands appearing upon overexpression of the N-terminal deleted variant. (G) Confocal immunofluorescence pictures of HeLa cells overexpressing FLAG-tagged astrin-1 and astrin-2 (SPAG5), astrin-1 (ATG3 + 4 + 5GGG-SPAG5) or astrin-2 (ATG1GGG-SPAG5) alone stained for FLAG (green) and CLUH (red). DAPI was used to stain nuclei (blue). Small boxes in left corners show a ×4 magnified area of boxed regions. Scale bar, 10 µm. (H) Polysome profiling of HeLa cells chemically crosslinked with dithiobis succinimidyl propionate (DSP). At the top, absorbance profile at 254 nm of the fractions is shown with indicated peaks of 40S and 60S ribosomal subunits, 80S monosome and polysomes; at the bottom the corresponding western blots of the fractions are shown. RPL7 was used as a marker for ribosomes. Figure 1—source data 1.Uncropped blots for Figure 1A,B,D,E, F. Figure 1—source data 2.Uncropped blots for Figure 1H. Figure 1—source data 3.Unedited blots for Figure 1A,B,D,E, F,H.

Fig 5: CLUH forms granules upon starvation AConfocal images of liver cryosections and primary hepatocytes of Li-Cluh WT and Li-Cluh KO mice stained with anti-CLUH antibody. Scale bar, 10 µm.BConfocal images of liver cryosections of fed and starved Li-Cluh WT and Li-Cluh KO mice stained with anti-CLUH antibody. Right panels show 6.5× magnified boxed areas. Scale bar, 10 µm.CConfocal images of primary hepatocytes cultured in indicated media and stained with anti-CLUH antibody. Right panels show 4.5× magnified boxed areas. Scale bar, 10 µm.D, EConfocal images of primary hepatocytes cultured in indicated media and stained with (D) anti-G3BP1 or (E) anti-DCP1A and anti-CLUH antibodies. Right panel shows 5× enlargement of indicated area. Scale bar, 10 µm.