Stage VI oocyte nuclei were obtained as described (Guille, 1999). a cytoplasmic anchor for CBTF122. Destruction of endogenous RNA by microinjection of RNase promotes premature nuclear translocation of CBTF122. Thus, the nuclear translocation of CBTF122 at the MBT is likely to be coupled to the degradation of maternal mRNA that occurs at that stage. (Sykes et al., 1998). The CBTF binding site in the GATA-2 promoter is evolutionarily conserved and is necessary for the correct regulation of GATA-2 transcription in amphibian embryos and in avian and murine haematopoietic cells (Brewer et al., 1995; Fleenor et al., 1996; Minegishi et al., 1998; Nony et al., 1998). CBTF activity is controlled, at least in part, by its subcellular localization (Brewer et al., 1995; Orford et al., 1998). CBTF activity is present in the oocyte nucleus where GATA-2 is initially transcribed (Zon et al., 1991; Walmsley et al., 1994; Partington et al., 1997; Orford et al., 1998). However, after fertilization, CBTF activity is cytoplasmic and re-localizes to the nucleus only at the gastrula stage when zygotic GATA-2 transcription commences (Brewer et al., 1995). Biochemical evidence indicates that CBTF122 is an essential subunit of CBTF (Orford ovarian expression library in a screen to identify double-stranded (ds) RNA binding proteins (Bass et al., 1994; Wormington et al., 1996). We independently isolated a partial-length cDNA encoding CBTF122 in a similar expression library screen, to identify proteins that bind to translational control elements located within the 5-untranslated region (5-UTR) of ribosomal protein mRNAs (Avni and Shama, 1996). Additional screening using this partial-length cDNA as a hybridization probe and for 5 RACE PCRs resulted in the isolation of a full-length CBTF122 cDNA. The cDNA-derived amino acid sequences for CBTF122 and CBTF98 are shown in Figure?1. Metoprolol tartrate The CBTF98 coding region is 99.8% identical to the corresponding coding region in the CBTF122 cDNA. At present, we cannot determine APRF whether two separate genes encode the 4 kb CBTF122 and 3 kb CBTF98 mRNAs (Figure?2A) or if these mRNAs are derived by alternative splicing of a single transcript. The correspondence between these mRNA species and the two Metoprolol tartrate cDNA sequences was shown by probing of northern blots using 3-UTR-specific probes (data not shown). Although the predicted sizes of CBTF122 and CBTF98 are 95 and 74 kDa, respectively, they migrate aberrantly as 122 and 98 kDa polypeptides in SDSCpolyacrylamide gels (Figure?2B). Open in a separate window Fig. 1. Amino acid alignment of CBTF122 and CBTF98, human NF90 and mouse Spnr proteins. The amino acid sequences shown were aligned using the Pileup program of the GCG software package. NLS, dsRBD1, dsRBD2 and RGG box are boxed in red, orange and yellow, respectively. Open in a separate window Fig. 2. CBTF122 and CBTF98 mRNA and protein levels during development. (A)?Northern blot of total RNA isolated from two oocytes and embryos was probed with 32P-labelled antisense RNA transcribed from the CBTF122 cDNA. The stage of oogenesis (Dumont, 1972) or embryogenesis (Neiuwkoop and Faber, 1967) is indicated Metoprolol tartrate at the top of each lane. Lane M shows progesterone-matured oocytes. (B)?A western blot of total soluble proteins from five oocytes at the indicated stages (lanes 1C5) was probed with affinity-purified anti-CBTF122/98 antibody. A separate western blot (lanes 6 and 7) of total soluble protein from two stage VI or progesterone-matured oocytes (M) was probed with affinity-purified anti-CBTF122/98 antibody. Both CBTF122 and CBTF98 have two regions with significant homology to the dsRNA binding domain (dsRBD) initially delineated for staufen (Figure?1; St Johnston et al., 1991). An arginine/glycine rich region (RGG box) is present in the C-terminal portions of both CBTF122 and CBTF98 (Figure?1). The RGG box is responsible for the RNA binding activities of proteins such as hnRNP U and nucleolin (for reviews see Dreyfuss et al., 1993; Burd and Dreyfuss, 1994). A bipartite nuclear localization signal (NLS) (Robbins et al., 1991) precedes the first dsRBD, consistent with Metoprolol tartrate the nuclear localization of both CBTF122 and CBTF98 (Orford et al., 1998; also.