Fig 1: NuMA interacts with proteins and nucleic acid components of ribosomal biogenesis. S1 cells were cultured for 8 days with complete medium followed by 2 days without EGF to induce proliferation arrest; T4–2 cells were cultured for 6 days. (A) Immunoprecipitation (IP) of nuclear extracts from S1 and T4–2 cells with NuMA antibodies (NuMA) or with non-specific immunoglobulins (IgG), followed by western blot analysis of the input and immunoprecipitated samples using NuMA and NM1 antibodies. (B) Immunoprecipitation of nuclear extracts from S1 cells followed by western blot analysis for NuMA and RNA polymerase I (RNA pol I). (C) ChIP from T4–2 cells with NuMA antibody (n = 4) or RNA Polymerase I antibody (n = 2), followed by RT-qPCR for coding (pro-1, H4, H8, H13) and non-coding (H18) regions of rDNA. The drawing shows the organization of the rDNA gene (IGS = intergenic sequence). Data are normalized to those obtained with IgG control (see ‘Materials and Methods’ section). (D) RNA immunoprecipitation fromT4–2 cells with NuMA antibody or with non-specific IgG of nuclear extracts and soluble extracts (the latter were treated with 50 µg/ml cycloheximide [CH]). Both the DNAse treated non-reverse transcribed RNA (nonRT RNA) and the DNAse treated reverse-transcribed cDNA (cDNA) samples were subjected to PCR using primers specific for human 18S and 28S rRNAs (n = 2). (E and F) Immunoprecipitation of nuclear extracts from S1 cells followed by western blot analysis for NuMA, RPL26 and RPL24.
Fig 2: Allelic variants of SPATA5 exhibit ribosome assembly defects(A) Schematic of SPATA5 domain structure. The two allelic variants indicated in red have been linked with microcephaly, hearing loss, and intellectual disability in humans.(B) Schematic describing how the labeling of SNAP-tagged RPL28 was conducted.(C) HEK293T cells were transduced with an sgRNA targeting SPATA5, transfected with plasmids carrying transgenes corresponding to wild-type SPATA5 or the disease-linked (Thr330del and D628G) allelic variants, and old ribosomes were pulse labeled with TMR-Star (red) and new RPL28 proteins were labeled with Oregon green (green), as indicated. The wild-type transgene rescued the RPL28 nucleolar retention phenotype caused by loss of SPATA5, whereas cells expressing the two allelic variants continued to display low levels of newly labeled nucleolar RPL28 after a 4-h chase period (yellow arrows).(D) Staining cells for RSL24D1 (green) and DNA (blue) showed that, in contrast to wild-type transgenic controls, expression of the Thr330del and D628G variants did not fully rescue the localization defects of RSL24D1 in SPATA5 mutant cells.(E) Quantification of Oregon green (OG)-labeled new SNAP-tagged RPL28 within nuclei in the indicated genetic backgrounds. n = 3 biological replicates.(F) Quantification of nuclear RSL24D1 staining in the indicated genetic backgrounds. n = 3 biological replicates. Statistical comparisons in (E and F) are between wild-type cells and cells with each of the indicated rescuing transgenes. Mean ± SEM is shown. p values were calculated by nested one-way ANOVA with Dunnett’s multiple comparisons test.(G) Western blot analysis of cytoplasmic extracts and Ribo-SNAP pull-down samples probed with RSL24D1 and RPL24 antibodies. Wild-type transgenes could partially rescue the increase of cytoplasmic RSL24D1 and its aberrant prolonged association with the 60S subunit in SPATA5 mutant cells. By contrast, the amount of cytoplasmic and ribosome bound RSL24D1 were modestly elevated upon expression of the Thr330del and D628G allelic variants relative to the wild-type control. Scale bars, 10 µm.
Fig 3: C1orf109 and SPATA5 mutant cells exhibit defects in the recycling of RSL24D1(A) Wild-type (WT) controls and HEK293T cells transduced with sgRNAs targeting C1orf109 and SPATA5 stained for RSL24D1 (green) and DNA (blue). While most RSL24D1 localizes to the nucleoli of control cells, disruption of C1orf109 and SPATA5 results in ectopic localization of RSL24D1 to the cytoplasm. Scale bars, 20 µm.(B) Overview of SNAP-tagged ribosome pull-down.(C) Western blot analysis of cytoplasmic extracts and Ribo-SNAP pull-down samples probed with either RSL24D1 or RPL24 antibodies. Loss of C1orf109 and SPATA5 results in a dramatic increase in cytoplasmic localization and ribosome associated RSL24D1.(D) Western blot of sucrose gradient fractions from WT cells probed with antibodies against RPL28 and RSL24D1. Most RSL24D1 was observed in the 60S/80S fraction, with a sharp drop-off in the 80S/light polysome (LP) fraction.(E) Western blot of sucrose gradient fractions from C1orf109KO cells probed with antibodies against RPL28 and RSL24D1.(F) Western blot of sucrose gradient fractions from SPATA5KO cells probed with antibodies against RPL28 and RSL24D1.
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