Figure 4. Non-cleavable FUBI-eS30 mutants have a dominant-negative effect on late cytoplasmic steps of 40S subunit biogenesis.

(A) Scheme of pre-40S maturation focusing on late nucleoplasmic to cytoplasmic maturation steps and highlighting the RBFs mentioned in the text. Nucleoplasmic pre-40S particles containing 18S-E pre-rRNA with its characteristic 5' ITS1 remnant and bound to various RBFs can be exported in an XPO1-dependent manner. In the cytoplasm, late-assembling RPS are incorporated, the 3' overhang of 18S rRNA is cleaved off by the endonuclease NOB1, and the RBFs are released and recycled. Together, these sequential maturation steps lead to the formation of a mature 40S subunit. (B) Immunofluorescence analysis of parental, WT or mutant (AA, GV) FUBI-eS30-StHA HeLa cell lines using the indicated antibodies against the constructs (HA) and the 40S RBFs PNO1, RIOK2, NOB1, and RIOK1. Note that HA and RIOK2 co-immunostaining was performed in parallel with Hoechst staining of DNA. Where indicated, cells were treated with leptomycin B (LMB; 20 nM, 90 min) to inhibit XPO1-mediated nuclear export prior to fixation of cells. Scale bar, 20 µm. (C) Quantification of RBF localization for selected conditions of the experiment shown in (B). Percentage of cells assigned to the respective phenotypic classes exemplified below was determined from three or in case of PNO1 -LMB four biological replicates for the indicated total number of cells per condition and cell line (n).

Figure 4—figure supplement 1. Expression of non-cleavable FUBI-eS30 mutants affects nucleolar but not early cytoplasmic 40S or cytoplasmic 60S biogenesis.

(A) The localizations of the 40S RBFs LTV1, ENP1, and RRP12 and of the 60S RBF NMD3 were analyzed in parental, WT or mutant (AA, GV) FUBI-eS30-StHA HeLa cell lines by immunostaining. Where indicated, cells were treated with 20 nM LMB for 90 min or for 4 hr in case of anti-NMD3 staining before fixation. Scale bar, 20 µm. (B) Anti-NMD3 immunofluorescence analysis of HeLa K cells treated with control or AAMP siRNA (10 nM, 72 hr). Scale bar, 20 µm. (C) Immunoblot analysis of (B) using the indicated antibodies showed efficient depletion of AAMP.

Figure 4—figure supplement 2. Non-cleavable FUBI-eS30 mutants induce a persistent 40S ribosomal subunit biogenesis defect.

(A) Flowchart of the timeline for the tetracycline (tet)-mediated induction of FUBI-eS30-StHA expression in HeLa cell lines without (left panel) or with (right panel) a chase period in tet-free medium. All cells were harvested at the same time for subsequent analysis by immunofluorescence or polysome profiling. (B) Parental, WT or mutant (GA, GV) FUBI-eS30-StHA HeLa cell lines were treated as in (A) and immunostained with antibodies against the constructs (HA) and the 40S RBF PNO1. Scale bar, 20 µm. (C) Extracts of WT or mutant (AA) FUBI-eS30-StHA HeLa cell lines matching the samples in (B) were separated on a linear 15–45% sucrose gradient by centrifugation. Gradient fractions were analyzed by immunoblotting using the indicated antibodies against the constructs (HA), 60S and 40S RBFs (NMD3 and NOB1, respectively) and ribosomal proteins of the small and large subunits (uS3 and uL23, respectively). Note the decrease of FUBI-eS30(AA)-StHA in the light fractions after the chase (respective fractions marked by red boxes). (D) Quantification of the soluble and 40S-bound anti-NOB1 signal in the respective sucrose gradient fractions of three biological replicates of the experiment shown in (C). Unpaired t-test, mean ± SD, N = 3, *p < 0.05, **p < 0.01. (E) Polysome profiles of parental (P, light orange), WT (dark orange), AA (dark blue), or GV (light blue) FUBI-eS30-StHA HeLa cell lines matching the samples in (B). The relative areas beneath the A₂₅₄ traces of the 40S, 60S, 80S, and the first three polysome peaks of three biological replicates were measured and quantified. Unpaired t-test, mean ± SD, N = 3, *p < 0.05, **p < 0.01.