Fig 2: SPATA5 and C1ORF109 interact with CINP and SPATA5L1(A) Immunoprecipitation of FLAG-tagged C1ORF109 from HEK293T cell extracts crosslinked with DSP reveals that C1ORF109 physically interacts with SPATA5, SPATA5L1, and CINP, but not with RPL28.(B) Pull-down of SNAP-tagged RPL28 shows that SPATA5, SPATA5L1, and CINP interact with ribosomes, but C1ORF109 does not.(C) Knockout of SPATA5L1 or CINP leads to defects in the trafficking of newly labeled RPL28 from the nucleolus to the cytoplasm.(D) Control, SPATA5L1KO, and C1orf109KO cells stained for RSL24D1 (green) and DNA (blue).(E) Matchmaker alignment of CINP and C1ORF109 AlphaFold predicted structures.(F) Control, CINPKO, and C1orf109KO cells transfected with a control or C1orf109 rescue construct stained for old and new ribosomes.(G) Cytoplasmic lysates from C1ORF109–3xFLAG expressing cells subjected to sucrose gradient fractionation and probed for SPATA5, SPATA5L1, C1ORF109, and CINP.

Fig 3: Loss-of-function screen identifies genes that contribute to ribosome biogenesis and turnover(A) Overview of CRISPR-Cas9 screen design.(B) MAGeCK analysis identified positive hits that result in lower levels of newly labeled RPL28 and increased levels of old ribosomes.(C) Functional categorization of genes identified in the screen.(D) Validation of representative hits from each of the functional categories using HEK293T cells. Strong retention of newly labeled nucleolar RPL28 after a 4-h chase period distinguishes C1orf109 and SPATA5 from other positive hits. Scale bars, 20 µm.

Fig 4: 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 5: 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.