POI-associated EIF4ENIF1 mutations exhibit impaired translation regulation abilities

•A novel EIF4ENIF1 variant c.623G > A;p.R208H was identified in a sporadic POI case.•EIF4ENIF1 208H and Q842P exhibited reduced translation inhibition ability in vitro.•T&T-seq as a sensitive method to evaluate variants of translation regulator genes. Various genetic variants have been found to be associated with the clinical onset of premature ovarian insufficiency (POI). However, when measured in vitro, the functional influence of the variants can be difficult to determine. By whole-exome sequencing (WES) of 93 patients with sporadic POI, we found a missense variant c.623G > A;p.R208H in the EIF4ENIF1 gene. In silico prediction of the variant using different algorithms suggested it might be a damaging variant. We compared the property of EIF4ENIF1 R208H and Q842P, a POI-related mutant that we reported previously, with wildtype (WT) protein using 293FT cells in vitro. Surprisingly, a change in subcellular distribution and granule forming ability (Q842P) and nuclear import capacity (R208H) was not observed, despite domain prediction evidences. Since EIF4ENIF1 was reported to inhibit translation, we employed T&T-seq, a translation-transcription dual-omics sequencing method, to profile gene expression upon overexpression of EIF4ENIF1 WT and mutants. EIF4ENIF1 WT overexpression group exhibited significantly (P