Communication between the 5 and 3 ends is a common feature of several aspects of eukaryotic mRNA metabolism. In the nucleus, the pre-mRNA 5 end is bound by the nuclear cap binding complex (CBC). This RNA-protein complex plays an active role in both splicing and RNA export. We provide evidence for participation of CBC in the processing of the 3 end of the message. Depletion of CBC from HeLa cell nuclear extract strongly reduced the endonucleolytic cleavage step of the cleavage and polyadenylation process. Cleavage was restored by addition of recombinant CBC. CBC depletion was found to reduce the stability of poly(A) site cleavage complexes formed in nuclear extract. We also provide evidence that the communication between the 5 and 3 ends of the pre-mRNA during processing is mediated by the physical association of the CBC͞cap complex with 3 processing factors bound at the poly(A) site. These observations, along with previous data on the function of CBC in splicing, illustrate the key role played by CBC in pre-mRNA recognition and processing. The data provides further support for the hypothesis that pre-mRNAs and mRNAs may exist and be functional in the form of ''closed-loops,'' due to interactions between factors bound at their 5 and 3 ends.The biosynthesis of most eukaryotic nuclear mRNAs requires the modification of the 5Ј end of the RNA by the cotranscriptional addition of an m 7 G(5Ј)ppp(5Ј)N cap structure (1, 2), the removal of introns by splicing (3), and the modification of the 3Ј end by endonucleolytic cleavage and poly(A) addition (4, 5).Polyadenylation in vertebrates requires two cis-acting RNA sequence elements which straddle the cleavage site and embody the core poly(A) site: the AAUAAA hexamer 10-30 nucleotides upstream of the cleavage site, and an amorphous U-or GϩU-rich element downstream of the cleavage site. Six factors, comprised of at least 13 proteins, are required for pre-mRNA cleavage and polyadenylation (4, 5). Cleavage and polyadenylation specificity factor (CPSF) (6, 7) binds the pre-mRNA upon recognition of the AAUAAA hexamer, while cleavage stimulatory factor (CstF) (8) binds the downstream element. Together, CPSF and CstF form a relatively stable pre-mRNA-protein complex (9) that allows for the recruitment of cleavage factors I m and II m (10), and poly(A) polymerase (11,12). Following the endonucleolytic cleavage of the pre-mRNA, poly(A) addition requires both CPSF and poly(A) polymerase (7, 13). Poly(A) binding protein II, however, confers both processivity and tail length control to the poly(A) addition reaction (14, 15).The 5Ј cap structure has been shown to influence the efficiency of 3Ј processing in vitro (16)(17)(18). The addition of the cap analog m 7 GpppG to HeLa cell nuclear extract resulted in a reduction in poly(A) site cleavage, although even at high levels of the analog, processing was not completely abolished (16,18). Uncapped pre-mRNAs were found to be poorly processed in nuclear extract, and have been shown to compete less efficiently for 3Ј processing factors...

Gene expression during oocyte maturation, fertilization, and early embryo development until zygotic gene activation is regulated mainly by translational activation of maternally derived mRNAs. This process requires the presence of a poly(A)-binding protein. However, the cytoplasmic somatic cell poly(A)-binding protein (PABP1) is not expressed until later in embryogenesis. We recently identified an embryonic poly(A)-binding protein (ePAB) in Xenopus. ePAB is the predominant cytoplasmic PABP in Xenopus oocytes and early embryos and prevents deadenylation of mRNAs, suggesting its importance in the regulation of gene expression during early Xenopus development. Here we report the identification of the mouse ortholog of Xenopus ePAB. The mouse ePAB gene on chromosome 2 contains 14 exons that specify an alternatively spliced mRNA encoding a protein of 608 or 561 aa with Ϸ65% identity to Xenopus ePAB. Mouse ePAB mRNA is expressed in ovaries and testis but not in somatic tissues. In situ hybridization localizes ePAB RNA to oocytes and confirms its absence from surrounding somatic cells in the mouse ovary. During early development, mouse ePAB is expressed in prophase I and metaphase II oocytes and one-cell and two-cell embryos and then becomes undetectable in four-or-more-cell embryos. In contrast, PABP1 mRNA expression is minimal in oocytes and early embryos until the eight-cell stage when it increases, becoming predominant at the blastocyst stage. The expression of mouse ePAB before zygotic gene activation argues for its importance in translational activation of maternally derived mRNAs during mammalian oocyte and early preimplantation embryo development.translational activation ͉ embryogenesis ͉ oogenesis M echanisms for establishing the germline and carrying out oogenesis in evolutionarily distant animals exhibit common themes. Gametes develop from primordial germ cells that are set aside during early embryogenesis (1). In most metazoans, primordial germ cells have an extragonadal origin and migrate to reach the somatic gonad, where they proliferate by mitosis to form oocytes (1). Oocytes, in turn, enter meiosis only to be arrested at the prophase of the first meiotic division (2, 3). This first meiotic arrest may last up to a few years in Xenopus and several decades in humans and is characterized by synthesis and storage of large quantities of dormant mRNA (4, 5). The resumption of meiosis is stimulated by progesterone in Xenopus (6, 7) or by gonadotropins in mouse and human (8, 9) and marks the onset of oocyte maturation.Oocyte maturation is accompanied by a complex network of translational activation and repression of dormant maternal mRNAs (10-13), whereas transcription is limited at best. These maternal mRNAs drive the oocyte's reentry into meiosis and control the rate of mitosis during the early cleavage divisions of the embryo (13-16). The transcriptional silencing that begins with oocyte maturation persists during the initial mitotic divisions of the embryonic cells. In Xenopus, after 12 rapid synchronous cle...