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We describe a new RNA binding protein from Xenopus we have named ePABP2 (embryonic poly(A) binding protein type II). Based on amino acid similarity, ePABP2 is closely related to the ubiquitously expressed nuclear PABP2 protein that directs the elongation of mRNA poly(A) tails during pre-mRNA processing. However, in contrast to known PABP2 proteins, Xenopus ePABP2 is a cytoplasmic protein that is predominantly expressed during the early stages of Xenopus development and in adult ovarian tissue. Biochemical experiments indicate ePABP2 binds poly(A) with specificity and that this binding requires the RRM domain. Mouse and human ePABP2 proteins were also identified and mouse ePABP2 expression is also confined to the earliest stages of mouse development and adult ovarian tissue. We propose that Xenopus ePABP2 is the founding member of a new class of poly(A) binding proteins expressed in vertebrate embryos. Possible roles for this protein in regulating mRNA function in early vertebrate development are discussed.
FIG. 3. Xenopus ePABP2 is expressed during oogenesis, in early embryos and in adult ovary tissue. A: Total RNA was isolated from Xenopus embryos at the indicated stages of development (the blot on the left) and various adult tissues (the blot on the right), fractionated by denaturing agarose gel electrophoresis and transferred to filters. Each filter was hybridized with a radiolabeled probe to detect mRNA encoding the Xenopus nuclear PABP2 (the upper panels, 1015 nucleotides). The filters were stripped and reprobed to detect Xenopus ePABP2 mRNA (the lower panels, 1260 nucleotides). B: Total protein was isolated from Xenopus oocytes at the indicated stages (blot on the left), embryos at the indicated stages of development (the blot in the middle), and various adult tissues (the blot on the right), fractionated by SDS polyacrylamide gel electrophoresis and transferred to filters. Five cells worth of protein were analyzed for the stage I, II, and III oocytes, whereas a single cells worth of protein was analyzed for stage IV and VI oocytes. Each filter was probed with the polyclonal -PABP2 antibodies to detect both Xenopus nuclear PABP2 and ePABP2. C: Mouse ePABP2 is expressed in early embryos and ovary tissue. cDNA generated from RNA isolated from mouse oocytes and blastocyst stage embryos was used as the template for PCR with primers for the ubiquitously expressed GAPHD mRNA or mouse ePABP2 mRNA (mePABP2). Both primer sets span one or more introns of the genomic sequence. Amplification was done for 30 and 40 cycles and the products analyzed by agarose gel electrophoresis. mRNA from the mouse tissues indicated was analyzed using RNA filter hybridization and a probe for mouse ePABP2 (the blot on the left, 1100 nucleotides). The same filter was stripped and reprobed for mouse cytoskeletal actin mRNA (the blot on the right). Mouse ePABP2 mRNA is expressed in ovarian tissue (**) but not other adult mouse tissues. Reproduced with permission of the Publisher, John Wiley & Sons.