INTEGRATED TRANSCRIPTOMIC AND TRANSLATIONAL PROFILING OF EIF6-SBDS IN SHWACHMAN-DIAMOND SYNDROM REVEALS DIFFERENT CELLULAR STATES SHAPED BY RIBOSOME COMPOSITION AND FIDELITY

Ribosome biogenesis is an essential and tightly regulated process that underpins cellular growth, proteostasis, and metabolic homeostasis. Disruptions in its final cytoplasmic maturation steps can profoundly alter translational output and lead to human disease. Shwachman–Diamond syndrome (SDS) exemplifies this class of disorders. Caused by biallelic loss-of-function mutations in SBDS, SDS is characterised by exocrine pancreatic dysfunction, growth impairment, bone-marrow failure, and a markedly increased risk of myelodysplastic syndrome and acute myeloid leukaemia. SBDS is required for release of the anti-association factor eIF6 from nascent 60S subunits, a prerequisite for productive 80S assembly and translational initiation. Consequently, SBDS deficiency limits the pool of functional ribosomes and constrains global protein synthesis. Recent sequencing of SDS bone marrow has revealed a striking and recurrent event: the acquisition of somatic mutations in eIF6, most frequently the missense variant N106S, arising exclusively in individuals with germline SBDS mutations. Their selective occurrence in SDS haematopoiesis has led to the hypothesis that these mutations act as a rescue event, although the underlying mechanism has remained unclear. Here, we investigate how the eIF6 N106S mutation modifies ribosome function and interacts with SBDS depletion. Using Prime Editing, we generated isogenic HEK293T lines carrying a N106S allele in heterozygosis and established matched SBDS-depleted models via shRNAi. Polysome profiling, RNA-seq, and Ribo-seq were used to assess how each perturbation influences translational output, decoding fidelity, and stress-response pathways. Additional analyses of rRNA fragments, snoRNA dynamics, and inferred ribosomal-protein association provided complementary insight into consequences on ribosome assembly. Translation assays showed that both SBDS depletion and the N106S mutation reduce global protein synthesis. Unexpectedly, their combination further repressed translation rather than restoring it, contradicting the view that N106S simply alleviates the SBDS-dependent anti-association defect. Transcriptome profiling revealed that the double-mutant state does not recapitulate either single perturbation but instead produces a coordinated shift in stress signalling and cellular metabolism. Rather than approximating wild-type behaviour, this condition establishes a distinct regulatory environment characterised by reduced inflammatory signalling and dampened anabolic activity. Taken together, these findings indicate that the N106S mutation does not restore the translational output impaired by SBDS deficiency but instead gives rise to a modified cellular state in which ribosome function is altered: decoding fidelity appears improved, proteotoxic stress is diminished, and transcriptional and metabolic programs diverge from both wild-type and single-mutant cells. These observations offer a plausible explanation for the selective retention of EIF6 mutations in SDS haematopoiesis and provide a foundation for future studies evaluating how modulation of the eIF6–60Scould be exploited in therapeutic treatment for SDS patients.