We have purified and characterized a factor required for accurate polyadenylation of mammalian pre-mRNAs in vitro. This factor, called cleavage-stimulation factor (CstF), is composed of three distinct polypeptide subunits of 77, 64, and 50 kD. Using monoclonal antibodies directed against the 64-and 50-kD subunits, we show that CstF is required for efficient cleavage of polyadenylation substrates. Furthermore, CstF present in unfractionated nuclear extracts interacts with pre-mRNAs containing the signal sequence AAUAAA, but not AAGAAA, in such a manner that the 64-kD subunit can be cross-linked to the RNA by UV light. This polypeptide is thus identical to the previously described 64-kD nuclear protein that binds to AAUAAAcontaining RNAs. Finally, indirect immunofluorescence of fixed cells indicates that CstF is distributed diffusely throughout the nucleus in a granular pattern distinct from the "speckled" pattern displayed by factors involved in pre-mRNA splicing, but similar to that of heterogeneous nuclear ribonucleoproteins. A model is presented in which CstF binds specifically to nascent RNA polymerase II transcripts and, by interacting with other factors, results in a rapid initiation of 3'-end processing of pre-mRNAs.

The CstF polyadenylation factor is a multisubunit complex required for efficient cleavage and polyadenylation of pre-mRNAs. Using an RNase H-mediated mapping technique, we show that the 64-kDa subunit of CstF can be photo cross-linked to pre-mRNAs at U-rich regions located downstream of the cleavage site of the simian virus 40 late and adenovirus L3 pre-mRNAs. This positional specificity of cross-linking is a consequence of CstF interaction with the polyadenylation complex, since the 64-kDa protein by itself is cross-linked at multiple positions on a pre-mRNA template. During polyadenylation, four consecutive U residues can substitute for the native downstream U-rich sequence on the simian virus 40 pre-mRNA, mediating efficient 64-kDa protein cross-linking at the downstream position. Furthermore, the position of the U stretch not only enables the 64-kDa polypeptide to be cross-linked to the pre-mRNA but also influences the site of cleavage. A search of the GenBank database revealed that a substantial portion of mammalian polyadenylation sites carried four or more consecutive U residues positioned so that they should function as sites for interaction with the 64-kDa protein downstream of the cleavage site. Our results indicate that the polyadenylation machinery physically spans the cleavage site, directing cleavage factors to a position located between the upstream AAUAAA motif, where the cleavage and polyadenylation specificity factor is thought to interact, and the downstream U-rich binding site for the 64-kDa subunit of CstF.Polyadenylation of precursor mRNAs involves a series of distinct steps: recognition of pre-mRNA substrates which contain the hexanucleotide sequence AAUAAA (19) by polyadenylation factors; endonucleolytic cleavage of the premRNA at a distinct site five to 40 nucleotides (nt) 3' of the hexanucleotide; and addition of approximately 200 adenosine residues at the 3' end (11,22,29). In vivo, these processes are tightly coupled, but the steps can be readily uncoupled in vitro. Previous studies have identified U-rich or GU-rich regions, located as far as 50 nt downstream of the cleavage site, which influence polyadenylation both in vivo and in vitro (6, 13-15, 20, 21, 23, 24, 34). To date, the mechanism by which these downstream sequence elements influence polyadenylation has not been elucidated.A 64-kDa (64K) polypeptide can be efficiently photo crosslinked to AAUAAA-containing RNA substrates that are undergoing polyadenylation in nuclear extracts of HeLa cells (31,16). The 64K polypeptide is a subunit of cleavage and polyadenylation stimulation factor (CstF [27,28]; also termed CF1 [7,8]). A role for the factor in poly(A) addition was confirmed by the ability of antibodies specific for the 64K polypeptide to deplete CstF and block in vitro polyadenylation (27). The 64K polypeptide will specifically cross-link to AAUAAA-containing mRNAs as part of the CstF complex only in the presence of a second multisubunit factor (33) termed the cleavage and polyadenylation specificity factor (CPSF; formerly...

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...