Polyadenylation in male germ cells differs from that in somatic cells. Many germ cell mRNAs do not contain the canonical AAUAAA in their 3 ends but are efficiently polyadenylated. To determine whether the 64,000 M r protein of the cleavage stimulation factor (CstF-64) is altered in male germ cells, we examined its expression in mouse testis. In addition to the 64,000 M r form, we found a related Ϸ70,000 M r protein that is abundant in testis, at low levels in brain, and undetectable in all other tissues examined. Expression of the Ϸ70,000 M r CstF-64 was limited to meiotic spermatocytes and postmeiotic spermatids in testis. In contrast, the 64,000 M r form was absent from spermatocytes, suggesting that the testis-specific CstF-64 might control expression of meiosis-specific genes. To determine why the 64,000 M r CstF-64 is not expressed in spermatocytes, we mapped its chromosomal location to the X chromosome in both mouse and human. CstF-64 may, therefore, be absent in spermatocytes because the X chromosome is inactivated during male meiosis. By extension, the testis-specific CstF-64 may be expressed from an autosomal homolog of the X chromosomal gene.Polyadenylation, the process of 3Ј end formation in eukaryotic mRNAs, is required for synthesis, transport, translation, and stability of most mRNAs (1-4). The sequence AAUAAA specifies accurate and efficient addition of poly(A) to the mRNA 3Ј end (5, 6). Substitutions at any position in this sequence diminish polyadenylation in vivo and in vitro (7-9), and 90-95% of sequenced mRNAs have AAUAAA in their 3Ј ends (1, 2). However, many mRNAs expressed in male germ cells do not have AAUAAA (refs. 10-13 and Table 1), but are nevertheless efficiently polyadenylated. Sequences such as UAUAAA, AUUAAA, UACAAA, and GAUAAA might substitute for the normal polyadenylation signal (10, 11), but no experimental evidence support these assertions.One hypothesis to account for the use of non-AAUAAA signals is that a protein involved in polyadenylation is altered in germ cells. A candidate is the 64,000 M r subunit of the cleavage stimulation factor (CstF-64) (14, 15), one of three polypeptides of CstF (16). CstF-64 has been shown to be essential for growth and viability of avian cells (17) and acts by binding to a U-or G͞U-rich region downstream of the cleavage site (18) whereas a 160,000 M r protein of the cleavage and polyadenylation specificity factor binds to the AAUAAA signal (19,20). CstF-64 governs polyadenylation site choice in adenovirus (21) and is involved in the immunoglobin switch from a membrane to secretory form during B-cell maturation (22)(23)(24). CstF also is involved in the cooperation of polyadenylation with splicing (25), and recent results show that it interacts with the C-terminal domain of RNA polymerase II and thus couples polyadenylation with transcription (26).Because of its essential role in somatic cell polyadenylation, we examined CstF-64 expression in male germ cells to test the hypothesis that it might contribute to polyadenylation of non-AA...