Identification of mRNA‐binding proteins during development: Characterization of Bufo arenarum cellular nucleic acid binding protein

Ultraviolet irradiation was used to covalently cross‐link poly(A)+RNA and associated proteins in eggs and embryos of the toad Bufo arenarum. Four major proteins with apparent sizes of 60, 57, 45 and 30–24 kDa were identified. It was observed that the same mRNA‐binding proteins were isolated from eggs to gastrula and neural stages of development. The 30 kDa polypeptide, p30, appeared as the main ultraviolet (UV) cross‐linked protein in the developmental stages analyzed. By means of polyclonal antibodies, it was determined that this polypeptide has a cytoplasmic localization and it was detected in liver, eggs and embryos. The presence of p30 was also analyzed by western blot during oogenesis and development. The 30 kDa polypeptide was present in all stages analyzed but it could not be detected in stages I–II of oogenesis. At the neural stage, the relative amount of p30 began to decrease, reaching its lowest levels after stages 26–30 (tail‐bud in Bufo arenarum). On the basis of purification, immunoprecipitation and western blot assays the 30 kDa protein was identified as the Bufo arenarum cellular nucleic acid binding protein.

[1]  J. Richter,et al.  Photocrosslinking of proteins to maternal mRNA in Xenopus oocytes. , 1988, Developmental biology.

[2]  A. Spirin.  Chapter 1 On “Masked” Forms of Messenger Rna in Early Embryogenesis and in Other Differentiating Systems , 1966 .

[3]  M. Wormington.  Unmasking the role of the 3' UTR in the cytoplasmic polyadenylation and translational regulation of maternal mRNAs. , 1994, BioEssays : news and reviews in molecular, cellular and developmental biology.

[4]  J. Richter,et al.  Reversible inhibition of translation by Xenopus oocyte-specific proteins , 1984, Nature.

[5]  J. Richter.  Translational control during early development , 1991, Critical reviews in eukaryotic gene expression.

[6]  C. Crosio,et al.  Structure and expression of ribosomal protein genes in Xenopus laevis. , 1995, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[7]  E. Baum,et al.  Coordinate expression of ribosomal protein genes during Xenopus development. , 1985, Developmental biology.

[8]  Coordinate translational regulation in the syntheses of elongation factor 1 alpha and ribosomal proteins in Xenopus laevis. , 1993, Nucleic acids research.

[9]  R. Jackson,et al.  Do the poly(A) tail and 3′ untranslated region control mRNA translation? , 1990, Cell.

[10]  J. Sportsman,et al.  Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose , 1984 .

[11]  A. Nairn,et al.  Rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[12]  J. Richter.  Molecular Mechanisms of Translational Control during the Early Development of Xenopus laevis , 1987 .

[13]  A. Wolffe,et al.  Masking mRNA from translation in somatic cells. , 1993, Genes & development.

[14]  R. Johnson,et al.  Identification of a 60-kDa phosphoprotein that binds stored messenger RNA of Xenopus oocytes. , 1985, European journal of biochemistry.

[15]  H. Koepsell,et al.  Proteolytic enzymes of microorganisms; evaluation of proteinases produced by molds of the Aspergillus flavus-oryzae group in submerged culture. , 1952, Archives of biochemistry and biophysics.

[16]  P. Mariottini,et al.  The 5' untranslated region of mRNA for ribosomal protein S19 is involved in its translational regulation during Xenopus development , 1990, Molecular and cellular biology.

[17]  W. Franke,et al.  Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos , 1991, The Journal of cell biology.

[18]  I. Bozzoni,et al.  Expression of ribosomal-protein genes in Xenopus laevis development , 1982, Cell.

[19]  A. Bird,et al.  Purification, sequence, and cellular localization of a novel chromosomal protein that binds to Methylated DNA , 1992, Cell.

[20]  R. Jackson,et al.  Translational Regulation: Y the message is masked? , 1994, Current Biology.

[21]  R. Bachvarova.  A maternal tail of poly(a): The long and the short of it , 1992, Cell.

[22]  A Jeavons,et al.  Microchannel array detector for quantitative electronic radioautography. , 1995, Cellular and molecular biology.

[23]  A. Wolffe,et al.  Masking and Unmasking Maternal mRNA , 1996, The Journal of Biological Chemistry.

[24]  M. Wormington.  Poly(A) and translation: development control. , 1993, Current opinion in cell biology.

[25]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[26]  J. Sommerville,et al.  Phosphorylation of a 60 kDa polypeptide from Xenopus oocytes blocks messenger RNA translation. , 1987, Nucleic acids research.

[27]  Eric H. Davidson,et al.  Gene activity in early development , 1968 .

[28]  K. Svenson,et al.  Identification of a zinc finger protein that binds to the sterol regulatory element. , 1989, Science.

[29]  E. Morkin,et al.  Alternatively Processed Isoforms of Cellular Nucleic Acid-binding Protein Interact with a Suppressor Region of the Human β-Myosin Heavy Chain Gene (*) , 1995, The Journal of Biological Chemistry.

[30]  R. Mans,et al.  Measurement of the incorporation of radioactive amino acids into protein by a filter-paper disk method , 1961 .

[31]  J. Dumont.  Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in laboratory maintained animals , 1972, Journal of morphology.

[32]  A. Wolffe,et al.  A role for transcription and FRGY2 in masking maternal mRNA within Xenopus oocytes , 1994, Cell.

[33]  N. Standart,et al.  Unmasking mRNA in clam oocytes: role of phosphorylation of a 3' UTR masking element-binding protein at fertilization. , 1996, Developmental biology.

[34]  H. Krutzsch,et al.  Cellular Nucleic Acid Binding Protein Regulates the CT Element of the Human c- myc Protooncogene (*) , 1995, The Journal of Biological Chemistry.

[35]  M. Salpeter,et al.  RESOLUTION IN ELECTRON MICROSCOPE RADIOAUTOGRAPHY , 1969, The Journal of cell biology.

[36]  O. Meyuhas,et al.  The translational cis-regulatory element of mammalian ribosomal protein mRNAs is recognized by the plant translational apparatus. , 1996, European journal of biochemistry.

[37]  L. Solnica-Krezel.  Pattern formation in zebrafish--fruitful liaisons between embryology and genetics. , 1999, Current topics in developmental biology.

[38]  O. H. Lowry,et al.  Protein measurement with the Folin phenol reagent. , 1951, The Journal of biological chemistry.

[39]  P. Ford,et al.  Identification in Xenopus laevis of a class of oocyte-specific proteins bound to messenger RNA. , 1981, European journal of biochemistry.

[40]  L. Pellizzoni,et al.  A Xenopus laevis homologue of the La autoantigen binds the pyrimidine tract of the 5' UTR of ribosomal protein mRNAs in vitro: implication of a protein factor in complex formation. , 1996, Journal of molecular biology.

[41]  M. Di Cristina,et al.  Interaction of proteins with the mRNA for ribosomal protein L1 in Xenopus: structural characterization of in vivo complexes and identification of proteins that bind in vitro to its 5'UTR. , 1993, Nucleic acids research.

[42]  G. Thomas,et al.  Rapamycin selectively represses translation of the "polypyrimidine tract" mRNA family. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[43]  L. Vuillard,et al.  Silver-staining of proteins in polyacrylamide gels: a general overview. , 1994, Cellular and molecular biology.

[44]  L. Pellizzoni,et al.  Cellular nucleic acid binding protein binds a conserved region of the 5' UTR of Xenopus laevis ribosomal protein mRNAs. , 1997, Journal of molecular biology.