Two distinct forms of the 64,000 M _r protein of the cleavage stimulation factor are expressed in mouse male germ cells

Abstract

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...

Loss of polyadenylation protein τCstF-64 causes spermatogenic defects and male infertility

“…‡ Micrometer per second Ϯ SD. for spermatogenesis and male fertility because of its expression during male meiosis when CstF-64 is absent (5). In support of this hypothesis, Cstf2t Ϫ/Ϫ males were infertile due to low sperm counts (Table 3), significant developmental defects in spermiogenesis (Fig.…”

“…Although polyadenylation is nearly universal, features of polyadenylation are different in mammalian male germ cells than in other tissues: male germ cell mRNAs exhibit increased alternative polyadenylation (3,4), decreased use of the AAUAAA polyadenylation signal (5,6), and reduced dependence on downstream sequence elements (DSEs) (7). These differences suggest a modified mechanism for polyadenylation in male germ cells.…”

“…CstF-64 is expressed in nuclei of early spermatogenic cells (5,12). However, because it is on the X chromosome, CstF-64 expression halts in pachytene spermatocytes because of meiotic sex chromosome inactivation (MSCI) (13).…”

Tissue-specific autoregulation of Drosophila suppressor of forked by alternative poly(A) site utilization leads to accumulation of the suppressor of forked protein in mitotically active cells

“…Model for tissue-specific su(f) autoregulation+ The Su(f) protein regulates its own level by promoting the utilization of poly(A) site in su(f) intron 4, which leads to the formation of a truncated transcript+ In dividing tissues, this autoregulation is weak, possibly because of the lack or the low amount of another component essential for Su(f) activity on the su(f) intronic poly(A) site+ This allows Su(f) accumulation+ In nondividing tissues, su(f) autoregulation is strong, possibly as a result of a high level of the protein required for efficient Su(f) activity on the su(f) intronic poly(A) site+ This results in the synthesis of a very low level of Su(f) protein+ type expression pattern of the P[su(f)-lacZ]G reporter is restored in the su(f) ts67g mutant+ Therefore, in that experiment, accumulation of the Su(f)-b-galactosidase protein is repressed in nondividing tissues but not in dividing tissues although the Su(f) protein is present at the same level in both tissues+ This suggests that this repression in nondividing tissues depends on another protein that stimulates utilization of the poly(A) site in intron 4 (Fig+ 5)+ This protein would induce a modification of Su(f) activity either via a direct posttranslational modification of Su(f) or by interacting with Su(f)+ Such a Su(f)-interacting protein could be a specific protein or a component of the general 39-end processing machinery+ An obvious candidate is the 64K subunit of CstF known to interact with the 77K subunit in human (Takagaki & Manley, 1994)+ Variations in the level of this 64K subunit contribute to the shift in poly(A) site selection in the immunoglobulin M heavy-chain locus, during B cell differentiation (Takagaki et al+, 1996;Takagaki & Manley, 1998)+ Autoregulation of su(f) appears to be conserved in another Drosophila species, D. virilis , as the structure of the su(f) gene as well as sequences downstream of the intronic poly(A) site, including the GU-rich motif, are conserved in this species+ Interestingly, such autoregulation has been proposed to occur for the yeast homolog of su(f), the RNA14 gene (Mandart, 1998)+ The RNA14 gene also produces truncated RNAs, whose accumulation requires wildtype RNA14 function+ The fact that autoregulation of this gene is conserved from yeast to Drosophila suggests that the level of this protein must be tightly regulated+ Consistent with this, overexpression of su(f) in Drosophila using the UAS/Gal4 system can lead to lethality+ In mammals, several examples document the fact that variations in the general cleavage factor CstF participate in the regulation of poly(A) site choice+ In most cases, the 64K subunit of CstF has been found to be responsible for these variations+ Variations in the activity of 64K have been reported to occur during the shift in poly(A) site choice in adenovirus (Mann et al+, 1993)+ An increase in the 64K level participates in the switch of poly(A) site utilization in the immunoglobulin M heavy-chain locus during B cell maturation (Takagaki et al+, 1996;Takagaki & Manley, 1998)+ The 64K subunit level has also been shown to increase in cultured cells induced to proliferate (Martincic et al+, 1998), and recently a new form of the 64K protein has been described in mouse (Wallace et al+, 1999); this form is specific to testis and brain and has been proposed to replace the 64K form during some steps of spermatogenesi...…”

Protein factors in pre-mRNA 3′-end processing

“…It is expressed in male germ cells and may have important roles in germ-cell-specific polyadenylation and spermatogenesis [144][145][146][147][148].…”