Development 102, 837-852 (1988) Printed in Great Britain © The Company of Biologists Limited 1988 837 Stability of RNA in developing Xenopus embryos and identification of a destabilizing sequence in TFIIIA messenger RNA RICHARD HARLAND and LYNDA MISHER Department of Molecular Biology, University of California, Berkeley, CA 94720, USA Summary Synthetic capped RNA transcripts injected into ferti- lized eggs of Xenopus laevis have a half-life of 3—4 h. Addition of a long (~200 nucleotide) poly(A) tail increases the half-life to 6-8 h which approaches the half-life of natural polyadenylated globin RNA injected into embryos. Since exonucleolytic action alone could account for the degradation of RNA, we tested whether circular RNA is stable after injection and find that circles are exceptionally stable (half-life greater than 40 h). After the mid blast ula transition, polyadenylated chloramphenicol transferase (CAT) mRNAs transcribed from injected plasmids have a half-life of 2 5h. Insertion of a 1000 nucleotide 3' untranslated region from the Xhox-36 gene into the transcripts does not affect the half-life. In contrast to the finding that internal sequences do not affect stability, we find that sequences from the TFIIIA message reduce the half-life of CAT mRNA from 2 5 h to less than 30 min. We conclude that most RNAs are degraded exonucleolytically from the 3' end, but specialized internal sequences can greatly destabilize the RNA, possibly by acting as a site for an endonu- clease. Key words: RNA stability, Xenopus development, TFIIIA, poly(A). Introduction The regulation of mRNA stability plays an important part in the control of gene expression; the numerous examples where stability is controlled have been recently reviewed (Shapiro et al. 1987). It is of particular interest to us to identify mechanisms that control RNA stability in the developing Xenopus embryo, not only in general, but also in special cases where stability of individual RNAs is controlled. A mechanism for differential RNA stability must be in effect throughout early Xenopus development since there are cases of RNAs that were stable in the oocyte being degraded either early in the blastula (King et al. 1986) or at different times around the beginning of gastrulation (Rebagliati et al. 1985; Dworkin et al. 1985). In these examples, total RNA was examined, thus excluding the possibility that deadenylation led to apparent absence of mRNA on the basis of oligo(dT) selectability. In Drosophila, it has been proposed that one mechanism for the generation of asymmetry in the homogeneously dis- tributed caudal (cad) mRNA is differential RNA stability (Macdonald & Struhl, 1986; Mlodzik & Gehring, 1987). The possibility that differential RNA stability is important to regional specification in the amphibian embryo is also attractive. Although prelocalized RNAs exist along the animal-vegetal axis (Rebagliati etal. 1985; Melton, 1987; Weeks & Melton, 1987), the capacity for dorsal and ventral development is not prelocalized in the radially symmetrical amphibian egg (reviewed by Gerhart & Keller, 1986). The transduction of early cytoplasmic reorganization and inductive interactions into changes in gene expression may well involve the differential stabilization of maternal RNA in different regions of the egg. Despite the work that has been done, we still do not understand what RNA sequences and nucleolytic enzymes control RNA stability in Xenopus oocytes and embryos. Interpretations from earlier work, employing natural RNAs, stressed the importance of a poly(A) tail for stability in oocytes (reviewed by Littauer & Soreq, 1982; Nevins, 1983) though in some cases a fraction of nonadenylated RNA was known to be stable for an extended period (Woodland & Wilt,