Abstract
Many mRNAs in male germ cells lack the canonical AAUAAA but are normally polyadenylated (Wallace, A. M., Dass, B., Ravnik, S. E., Tonk, V., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., and MacDonald, C. C. (1999) Proc. Natl. Acad Sci. U. S. A. 96, 6763-6768). Previously, we demonstrated the presence of two distinct forms of the M(r) 64,000 protein of the cleavage stimulation factor (CstF-64) in mouse male germ cells and in brain, a somatic M(r) 64,000 form and a variant M(r) 70,000 form. The variant form was specific to meiotic and postmeiotic germ cells. We localized the gene for the somatic CstF-64 to the X chromosome, which would be inactivated during male meiosis. This suggested that the variant CstF-64 was an autosomal homolog activated during that time. We have named the variant form "tau CstF-64," and we describe here the cloning and characterization of the mouse tauCstF-64 cDNA, which maps to chromosome 19. The mouse tauCstF-64 protein fits the criteria of the variant CstF-64, including antibody reactivity, size, germ cell expression, and a common proteolytic digest pattern with tauCstF-64 from testis. Features of mtauCstF-64 that might allow it to promote the germ cell pattern of polyadenylation include a Pro --> Ser substitution in the RNA-binding domain and significant changes in the region that interacts with CstF-77.
Highlights
Polyadenylation is the process of eukaryotic mRNA processing in which 3Ј end cleavage occurs, followed by the addition of as many as 250 adenosine residues [1, 2]
Cstf2 or CSTF2 mapped to the X chromosome in either mouse or human [24], supporting the hypothesis that inactivation of the X chromosome during male meiosis results in inactivation specificity factor; CstF, cleavage stimulation factor; RNA-binding domains (RBDs), RNA-binding domain; RRM, RNA recognition motif; UTR, untranslated region; bp, base pair; kb, kilobase pair; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco’s modified Eagle’s medium; RFLPs, restriction fragment length polymorphisms
Since the gene for the somatic form of CstF-64 (Cstf2) was located on the X chromosome and most X-linked genes are inactivated in male meiosis, CstF-64 was expressed from an autosomal paralog of CstF-64
Summary
8044 –8050, 2001 Printed in U.S.A. The Gene for a Variant Form of the Polyadenylation Protein CstF-64 Is on Chromosome 19 and Is Expressed in Pachytene Spermatocytes in Mice*. We showed that in mice the principal Mr 64,000 form of CstF-64 is found in all somatic cells, and in premeiotic and postmeiotic (but not meiotic) germ cells [24]. This form of CstF-64 is recognized by the monoclonal antibody 3A7 [25]. Cstf2t, the gene for mCstF-64 maps to an autosome, chromosome 19 in mouse These data suggest that mCstF-64 encodes the variant form of CstF-64 seen in meiotic and postmeiotic male germ cells
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