Polyadenylation of mRNAs encoding secreted proteins by TENT5 family of enzymes is essential for gametogenesis in mice

Cytoplasmic polyadenylation plays a vital role in gametogenesis, however, the participating enzymes and substrates in mammals remain unclear. Using knockout and knock-in mouse models, we describe the essential role of 4 TENT5 poly(A) polymerases in mice fertility and gametogenesis. TENT5B and TENT5C play crucial, but redundant roles in oogenesis, with double knockout of both genes leading to oocyte degeneration. Additionally, TENT5B-GFP females display gain-of-function infertility effect with multiple chromosomal aberrations in ovulated oocytes. TENT5C and TENT5D both regulate different stages of spermatogenesis, shown by sterility of males with either genes knockout mutation. Finally, Tent5a knockout significantly lowers fertility, although the underlying mechanism is not directly related to gametogenesis. Through Direct RNA sequencing we discovered that TENT5 proteins polyadenylate mRNAs encoding endoplasmic reticulum-targeted proteins essential for gametogenesis. Sequence motives analysis and reporter mRNA assay revealed that the presence of endoplasmic reticulum-leader represents the primary determinant of TENT5-mediated regulation. mRNA profiles from Tent5b-/- Tent5c -/- and WT female mice, generated by deep sequencing, in duplicate or triplicate, using Illumina NovaSeq 6000细胞质多聚腺苷酸化在配子发生过程中发挥关键作用，但目前哺乳动物中参与该过程的酶与底物仍未明确。本研究借助基因敲除与基因敲入小鼠模型，阐明了4种TENT5家族聚腺苷酸聚合酶在小鼠生育力与配子发生中的核心作用。TENT5B与TENT5C在卵子发生中发挥关键但功能冗余的作用，二者同时敲除会导致卵母细胞退化。此外，携带TENT5B-绿色荧光蛋白（Green Fluorescent Protein, GFP）的雌性小鼠呈现功能获得性不育表型，其排卵卵母细胞存在多种染色体畸变。TENT5C与TENT5D分别调控精子发生的不同阶段，单一敲除其中任一基因的雄性小鼠均会出现不育。最终，Tent5a基因敲除会显著降低小鼠生育力，尽管其潜在机制并不直接参与配子发生过程。通过直接RNA测序，我们发现TENT5蛋白可对编码配子发生必需的内质网靶向蛋白的mRNA进行多聚腺苷酸化修饰。序列基序分析与报告mRNA实验结果显示，内质网引导序列的存在是TENT5介导调控的核心决定因素。本研究利用Illumina NovaSeq 6000测序平台，通过双重复或三重复实验，获取了Tent5b⁻/⁻Tent5c⁻/⁻与野生型（Wild Type, WT）雌性小鼠的mRNA表达谱。