[No authors listed]
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, ÏCstF-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 ÏCstF-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 ÏCstF-64 in polyadenylation of H1fnt. These findings together suggest that the ÏCstF-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.
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