Functional impact of the eIF6 N106S mutation on ribosome biogenesis in wild type and Shwachman-Diamond syndrome cells.

backgroundEukaryotic initiation factor 6 (eIF6) is an essential regulator of ribosome biogenesis that prevents the premature association of the 60S and 40S ribosomal subunits. eIF6 eviction from nascent 60S particles, mediated by the ribosome biogenesis factors SBDS and EFL1, is required for the formation of translationally competent 80S ribosomes. SBDS and EFL1 deficiencies cause Shwachman-Diamond syndrome (SDS), a ribosomopathy mainly characterized by exocrine pancreatic insufficiency, bone marrow failure and a predisposition to hematological malignancies. Somatic EIF6 mutations that reduce the amount of eIF6 (e.g., nonsense mutation, deletion) or its ability to bind to the 60S subunit (e.g., N106S missense mutation) are frequently found in hematopoietic cells of SDS individuals. These somatic EIF6 mutations represent indirect somatic genetic rescue (SGR) in the context of SDS, as they increase cellular fitness to cause clonal expansion and are associated with clinical improvement in some cases. However, the functional consequences of these eIF6 mutations in human SDS cells have never been reported to date.

methodsAs fibroblasts from individuals with SDS carrying biallelic pathogenic variants in either SBDS or EFL1 exhibit impaired ribosome production and reduced translation, we employed these cells, in addition to fibroblasts from a healthy donor, as a cellular model to analyze the impact of the EIF6 N106S mutation, which was introduced by CRISPR/Cas9-mediated genome editing. The assessment of eIF6 localization, ribosome biogenesis, ribosomal RNA (rRNA) processing, global protein synthesis, cellular fitness, and p53 pathway activation was conducted in these cells.

resultsEndogenous expression of the eIF6 N106S mutant altered the subcellular distribution of eIF6 and induced pronounced defects, including impaired ribosomal RNA 3′-ETS processing, reduced availability of mature 60S subunits, and accumulation of halfmer polysomes. These alterations were associated with reduced cellular fitness in both healthy donor and SDS fibroblasts. In the context of SBDS deficiency, the N106S mutation partially alleviated aberrant retention of eIF6 on the 60S subunit but failed to restore global translation or confer a proliferative advantage.

conclusionsOur study reveals that the eIF6 N106S mutation uncouples partial correction of eIF6–60S dissociation from proper rRNA biogenesis and cellular fitness. These findings reveal intrinsic limits to altering eIF6–60S interactions without disrupting ribosome biogenesis. Furthermore, these results emphasize that therapeutic strategies targeting the eIF6–60S interaction must carefully balance rescue of ribosomal stress against disruption of ribosome biogenesis, particularly in disease contexts such as Shwachman-Diamond syndrome.