T&T-seq Reveals Impaired Translation Regulation Abilities of POI-associated EIF4ENIF1 Mutations
Premature Ovarian Insufficiency (POI) refers to the decline and stagnation of ovarian function in women before the age of 40.POI-associated EIF4ENIF1 mutations and the distribution of functional domains in the EIF4ENIF1 protein have been separately described. However, not all the clinically observed EIF4ENIF1 mutations in POI cases fall in clearly defined functional domains of the EIF4ENIF1 protein. Herein, we introduce T&T seq as a new evaluation tool to sensitively measure the translation regulation capacities of EIF4ENIF1 proteins with clinically discovered mutations. The sequencing results showed that POI-associated EIF4ENIF1 mutations impaired its translation repression function to different degrees. Overall design: We employed T&T-seq, a translation-transcription dual-omic sequencing method in which actively translating mRNAs were captured by affinity beads, to examine the transcriptional and translational states in 293FT cell lines upon the overexpression of wild-type and mutated EIF4ENIF1 proteins. For the first experiment, Flag-tagged EIF4ENIF1 WT, Q842P, (1-684) truncation proteins were overexpressed in 293FT cells and the transcriptional-translational gene expression states were compared to that of 293FT cells transfected with empty vector plasmids. For the second experiment, EIF4ENIF1 WT, R208H and GFP proteins were simultaneously overexpressed in 293FT cells and the transcriptomes and translatomes were compared.