Stability of RNA in developing Xenopus embryos and identification of a destabilizing sequence in TFIIIA messenger RNA

Development ◽  
1988 ◽  
Vol 102 (4) ◽  
pp. 837-852 ◽  
Author(s):  
R. Harland ◽  
L. Misher
Keyword(s):  
Xenopus Laevis ◽  
Half Life ◽  
Messenger Rna ◽  
Untranslated Region ◽  
Circular Rna ◽  
Midblastula Transition ◽  
Rna Transcripts ◽  
Xenopus Embryos ◽  
A Site

Synthetic capped RNA transcripts injected into fertilized eggs of Xenopus laevis have a half-life of 3–4 h. Addition of a long (approximately 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 midblastula transition, polyadenylated chloramphenicol transferase (CAT) mRNAs transcribed from injected plasmids have a half-life of 2.5 h. 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 endonuclease.

scholarly journals Dissection of the XChk1 Signaling Pathway in Xenopus laevis Embryos

2000 ◽  
Vol 11 (9) ◽  
pp. 3101-3108 ◽  
Author(s):  
Nicholas C. Kappas ◽  
Pamela Savage ◽  
Katherine C. Chen ◽  
Allan T. Walls ◽  
Jill C. Sible
Keyword(s):  
Cell Cycle ◽  
Xenopus Laevis ◽  
Signaling Pathway ◽  
Embryonic Cell ◽  
Midblastula Transition ◽  
Cell Cycles ◽  
Cyclin Dependent Kinases ◽  
Xenopus Embryos ◽  
Egg Extracts ◽  
Sperm Nuclei

Checkpoint pathways inhibit cyclin-dependent kinases (Cdks) to arrest cell cycles when DNA is damaged or unreplicated. Early embryonic cell cycles of Xenopus laevis lack these checkpoints. Completion of 12 divisions marks the midblastula transition (MBT), when the cell cycle lengthens, acquiring gap phases and checkpoints of a somatic cell cycle. Although Xenopus embryos lack checkpoints prior to the MBT, checkpoints are observed in cell-free egg extracts supplemented with sperm nuclei. These checkpoints depend upon the Xenopus Chk1 (XChk1)-signaling pathway. To understand why Xenopus embryos lack checkpoints,xchk1 was cloned, and its expression was examined and manipulated in Xenopus embryos. Although XChk1 mRNA is degraded at the MBT, XChk1 protein persists throughout development, including pre-MBT cell cycles that lack checkpoints. However, when DNA replication is blocked, XChk1 is activated only after stage 7, two cell cycles prior to the MBT. Likewise, DNA damage activates XChk1 only after the MBT. Furthermore, overexpression of XChk1 inXenopus embryos creates a checkpoint in which cell division arrests, and both Cdc2 and Cdk2 are phosphorylated on tyrosine 15 and inhibited in catalytic activity. These data indicate that XChk1 signaling is intact but blocked upstream of XChk1 until the MBT.

scholarly journals Identification and characterization of a 44 kDa protein that binds specifically to the 3′-untranslated region of CYP2a5 mRNA: inducibility, subcellular distribution and possible role in mRNA stabilization

1996 ◽  
Vol 313 (3) ◽  
pp. 1029-1037 ◽  
Author(s):  
Olivier GENESTE ◽  
Françoise RAFFALLI ◽  
Matti A. LANG
Keyword(s):  
Half Life ◽  
Untranslated Region ◽  
Translational Regulation ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Blocking Agent ◽  
Subcellular Fractionation ◽  
Mrna Stabilization ◽  
Nuclear Extracts

Stabilization of mRNA is important in the regulation of CYP2a5 expression but the factors involved in the process are not known [Aida and Negishi (1991) Biochemistry 30, 8041–8045]. In this paper, we describe, for the first time, a protein that binds specifically to the 3′-untranslated region of CYP2a5 mRNA and which is inducible by pyrazole, a compound known to increase the half-life of CYP2a5 mRNA. We also demonstrate that pyrazole treatment causes an elongation of the CYP2a5 mRNA poly(A) tail, and that phenobarbital, which is transcriptional activator of the CYP2a5 gene that does not affect the mRNA half-life, neither induces the RNA-binding protein nor affects the poly(A) tail size. SDS/PAGE of the UV-cross-linked RNA–protein complex demonstrated that the RNA-binding protein has an apparent molecular mass of 44 kDa. The protein-binding site was localized to a 70-nucleotide region between bases 1585 and 1655. Treatment of cytoplasmic extracts with an SH-oxidizing agent, diamide, an SH-blocking agent, N-ethylmaleimide or potato acid phosphatase abolished complex-formation, suggesting that the CYP2a5 mRNA-binding protein is subject to post-translational regulation. Subcellular fractionation showed that the 44 kDa protein is present in polyribosomes and nuclei, and that its apparent induction is much stronger in polyribosomes than in nuclear extracts. We propose that this 44 kDA RNA-binding protein is involved in the stabilization of CYP2a5 mRNA by controlling the length of the poly(A) tail.

Translocation of a store of maternal cytoplasmic c-myc protein into nuclei during early development

1989 ◽  
Vol 9 (12) ◽  
pp. 5395-5403
Author(s):  
M Gusse ◽  
J Ghysdael ◽  
G Evan ◽  
T Soussi ◽  
M Méchali
Keyword(s):  
Embryonic Development ◽  
Xenopus Laevis ◽  
Protein Content ◽  
Mature Oocyte ◽  
Gastrula Stage ◽  
Cleavage Stage ◽  
Midblastula Transition ◽  
Phosphorylation State ◽  
Growing Cell ◽  
Delayed Translation

The c-myc proto-oncogene is expressed as a maternal protein during oogenesis in Xenopus laevis, namely, in nondividing cells. A delayed translation of c-myc mRNA accumulated in early oocytes results in the accumulation of the protein during late oogenesis. The oocyte c-myc protein is unusually stable and is located in the cytoplasm, contrasting with its features in somatic cells. A mature oocyte contains a maternal c-myc protein stockpile of 4 x 10(5) to 6 x 10(5) times the level in a somatic growing cell. This level of c-myc protein is preserved only during the cleavage stage of the embryo. Fertilization triggers its rapid migration into the nuclei of the cleaving embryo and a change in the phosphorylation state of the protein. The c-myc protein content per nucleus decreases exponentially during the cleavage stage until a stoichiometric titration by the embryonic nuclei is reached during a 0.5-h period at the midblastula stage. Most of the maternal c-myc store is degraded by the gastrula stage. These observations implicate the participation of c-myc in the events linked to early embryonic development and the midblastula transition.

The origins of primitive blood in Xenopus: implications for axial patterning

Development ◽  
1999 ◽  
Vol 126 (3) ◽  
pp. 423-434 ◽  
Author(s):  
M.C. Lane ◽  
W.C. Smith
Keyword(s):  
Xenopus Laevis ◽  
Marginal Zone ◽  
Cell Stage ◽  
Axial Patterning ◽  
Xenopus Embryos ◽  
Spemann's Organizer ◽  
Dorsal Mesoderm

The marginal zone in Xenopus laevis is proposed to be patterned with dorsal mesoderm situated near the upper blastoporal lip and ventral mesoderm near the lower blastoporal lip. We determined the origins of the ventralmost mesoderm, primitive blood, and show it arises from all vegetal blastomeres at the 32-cell stage, including blastomere C1, a progenitor of Spemann's organizer. This demonstrates that cells located at the upper blastoporal lip become ventral mesoderm, not solely dorsal mesoderm as previously believed. Reassessment of extant fate maps shows dorsal mesoderm and dorsal endoderm descend from the animal region of the marginal zone, whereas ventral mesoderm descends from the vegetal region of the marginal zone, and ventral endoderm descends from cells located vegetal of the bottle cells. Thus, the orientation of the dorsal-ventral axis of the mesoderm and endoderm is rotated 90(degrees) from its current portrayal in fate maps. This reassessment leads us to propose revisions in the nomenclature of the marginal zone and the orientation of the axes in pre-gastrula Xenopus embryos.

scholarly journals Messenger RNA transcripts of the meiotic regulator BOULE in the testis of azoospermic men and their application in predicting the success of sperm retrieval

2005 ◽  
Vol 20 (3) ◽  
pp. 782-788 ◽  
Author(s):  
Yung Ming Lin ◽  
Pao Lin Kuo ◽  
Ying Hung Lin ◽  
Yen Ni Teng ◽  
Johnny Shinn Nan Lin
Keyword(s):  
Messenger Rna ◽  
Sperm Retrieval ◽  
Rna Transcripts
Sign in / Sign up

Export Citation Format

Share Document