Department of Cell Biology & Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, USA.
Department of Electrical and Computer Engineering & TEES-AgriLife Center for Bioinformatics and Genomic Systems Engineering, Texas A&M University, College Station, TX, USA.
Andrology. 2018 Jul;6(4):605-615. doi: 10.1111/andr.12488. Epub 2018 Apr 19.
Formation of the 3' ends of mature mRNAs requires recognition of the correct site within the last exon, cleavage of the nascent pre-mRNA, and, for most mRNAs, addition of a poly(A) tail. Several factors are involved in recognition of the correct 3'-end site. The cleavage stimulation factor (CstF) has three subunits, CstF-50 (gene symbol Cstf1), CstF-64 (Cstf2), and CstF-77 (Cstf3). Of these, CstF-64 is the RNA-binding subunit that interacts with the pre-mRNA downstream of the cleavage site. In male germ cells where CstF-64 is not expressed, a paralog, τCstF-64 (gene symbol Cstf2t) assumes its functions. Accordingly, Cstf2t knockout (Cstf2t ) mice exhibit male infertility due to defective development of spermatocytes and spermatids. To discover differentially expressed genes responsive to τCstF-64, we performed RNA-Seq in seminiferous tubules from wild-type and Cstf2t mice, and found that several histone and histone-like mRNAs were reduced in Cstf2t mice. We further observed delayed accumulation of the testis-specific histone, H1fnt (formerly, H1t2 or Hanp1) in Cstf2t mice. High-throughput sequence analysis of polyadenylation sites (A-seq) indicated reduced use of polyadenylation sites within a cluster downstream of H1fnt in knockout mice. However, high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) was not consistent with a direct role of τCstF-64 in polyadenylation of H1fnt. These findings together suggest that the τCstF-64 may control other reproductive functions that are not directly linked to the formation of 3' ends of mature polyadenylated mRNAs during male germ cell formation.
成熟 mRNA 3' 末端的形成需要识别最后一个外显子中的正确位点，切割新生的前体 mRNA，并为大多数 mRNA 添加 poly(A)尾巴。有几个因素参与识别正确的 3' 末端位点。切割刺激因子 (CstF) 有三个亚基，CstF-50（基因符号 Cstf1）、CstF-64（Cstf2）和 CstF-77（Cstf3）。其中，CstF-64 是与切割位点下游的前体 RNA 相互作用的 RNA 结合亚基。在不表达 CstF-64 的雄性生殖细胞中，一个类似物 τCstF-64（基因符号 Cstf2t）承担其功能。因此，Cstf2t 敲除（Cstf2t）小鼠由于精母细胞和精细胞发育缺陷而表现出雄性不育。为了发现对 τCstF-64 有反应的差异表达基因，我们在野生型和 Cstf2t 小鼠的生精小管中进行了 RNA-Seq，发现 Cstf2t 小鼠中的几种组蛋白和组蛋白样 mRNA 减少。我们进一步观察到 Cstf2t 小鼠中睾丸特异性组蛋白 H1fnt（以前称为 H1t2 或 Hanp1）的积累延迟。多聚腺苷酸化位点（A-seq）的高通量序列分析表明，在敲除小鼠中，H1fnt 下游簇内的多聚腺苷酸化位点使用减少。然而，交联免疫沉淀（HITS-CLIP）分离的 RNA 的高通量测序与 τCstF-64 直接参与 H1fnt 的多聚腺苷酸化作用不一致。这些发现共同表明，τCstF-64 可能控制其他生殖功能，这些功能与雄性生殖细胞形成过程中成熟多聚腺苷酸化 mRNA 3' 末端的形成没有直接关系。
