Highlights d MIWI/piRNA activates mRNA translation via imperfect basepairing interactions d HuR and eIF3f are required for MIWI/piRNA-mediated target mRNA activation d piRNA system controls the translation of a subset of mRNAs in mouse spermatids d piRNA system plays a central role in acrosome formation during spermiogenesis

Postmeiotic spermatids use a unique strategy to coordinate gene expression with morphological transformation, in which transcription and translation take place at separate developmental stages, but how mRNAs stored as translationally inert messenger ribonucleoproteins in developing spermatids become activated remains largely unknown. Here, we report that the RNA binding protein FXR1, a member of the fragile X–related (FXR) family, is highly expressed in late spermatids and undergoes liquid-liquid phase separation (LLPS) to merge messenger ribonucleoprotein granules with the translation machinery to convert stored mRNAs into a translationally activated state. Germline-specific Fxr1 ablation in mice impaired the translation of target mRNAs and caused defective spermatid development and male infertility, and a phase separation–deficient FXR1 L351P mutation in Fxr1 knock-in mice produced the same developmental defect. These findings uncover a mechanism for translational reprogramming with LLPS as a key driver in spermiogenesis.

Fast and accurate profiling of exogenous gene expression in host cells is crucial for studying gene function in cellular and molecular biology, but still faces the challenge of incomplete co‐expression of reporter genes and target genes. Here, a single‐cell transfection analysis chip (scTAC) is presented, which is based on the in situ microchip immunoblotting method, for rapid and accurate analysis of exogenous gene expression in thousands of individual host cells. scTAC not only can assign information of exogenous gene activity to specific transfected cells, but enables the acquisition of continuous protein expression even in low co‐expression scenarios. It is demonstrated that scTAC can reveal the relationship of expression level between reporter genes and target genes, which is helpful for evaluating transient transfection strategy efficiency. The advantages of this method for the study of fusion protein expression and downstream protein expression in signaling pathway in rare cells are shown. Empirically, an EGFP‐TSPAN8 fusion plasmid is transfected into MCF‐7 breast cancer cells and the expressions of two cancer stemness biomarkers (ALDHA1 and SOX2) are analyzed. The scTAC method clearly reveals an interesting phenomenon that transfected adherent MCF‐7 cells exhibit some stem cell characteristics, but they do not have stem cell appearance.