Two zinc finger proteins from Xenopus laevis bind the same region of 5S RNA but with different nuclease protection patterns.

Immature oocytes from Xenopus laevis contain a 42S ribonucleoprotein particle (RNP) containing 5S RNA, tRNA, a 43 kDa protein, and a 48 kDa protein. A particle containing 5S RNA and the 43 kDa protein (p43-5S) liberated from the 42S particle upon brief treatment with urea can be purified by anion exchange chromatography. The purified p43-5S RNA migrates as a distinct species during electrophoresis on native polyacrylamide gels. Radiolabeled 5S RNA can be incorporated into the p43-5S complex by an RNA exchange reaction. The resulting complexes containing labeled 5S RNA have a mobility on polyacrylamide gels identical to that of purified p43-5S RNPs. RNP complexes containing 5S RNA labeled at either the 5' or 3' end were probed with a variety of nucleases in order to identify residues protected by p43. Nuclease protection assays performed with alpha-sarcin indicate that p43 binds primarily helices I, II, IV, and V of 5S RNA. This is the same general binding site observed for TFIIIA on 5S RNA. Direct comparison of the binding sites of p43 and TFIIIA with T1 and cobra venom nucleases reveals striking differences in the protection patterns of these two proteins.     

A finger protein structurally similar to TFIIIA that binds exclusively to 5S RNA in Xenopus

A 5S RNA binding protein (p43) in Xenopus is a major constituent of oocytes and comprises part of a 42S ribonucleoprotein storage particle. We have cloned and sequenced p43 cDNA from X. laevis and X. borealis as well as the cDNA for X. borealis TFIIIA. Like TFIIIA, p43 has nine zinc fingers, seven of which are exactly the same size as their counterparts in TFIIIA. Amino acid homology between the two proteins is restricted mainly to conserved residues characteristic of zinc fingers. In contrast to TFIIIA, which binds specifically to both 5S RNA and 5S RNA genes, p43 binds exclusively to 5S RNA.     

Purification of a p47 phosphoprotein from Xenopus laevis oocytes and identification as an in vivo and in vitro p34cdc2 substrate

This paper describes the purification of a 47 kDa protein from Xenopus laevis oocytes that becomes phosphorylated when the oocytes undergo meiotic maturation. This protein (p47) is part of a high molecular mass complex containing at least two other proteins of molecular mass 30 and 36 kDa. This complex can be isolated from stage VI oocytes before maturation. We obtained a pattern for phosphopeptides in p47 phosphorylated in vivo very similar to that of the purified protein phosphorylated in vitro by p34cdc2 (a H1 kinase which is a component of the M-phase promoting factor) and [gamma-32P]ATP. Therefore, the purified p47, already described as a marker of MPF activity, is the first reported in vivo substrate for the cell division control kinase.     

Disappearance of a Novel Protein Component of the 26S Proteasome duringXenopusOocyte Maturation

We have prepared polyclonal antibodies againstXenopus20S proteasomes. The antibodies cross-react with several proteins that are common to 20S and 26S proteasomes and with at least two proteins that are unique to 26S proteasomes. The antibodies were used to analyze changes in the components of proteasomes during oocyte maturation and early development ofXenopus laevis.A novel protein with a molecular weight of 48 kDa, p48, was clearly detected in immature oocytes, but was found at very low levels in mature oocytes and ovulated eggs. p48 was reduced to low levels during oocyte maturation, after maturation-promoting factor was activated. The amount of p48 in eggs remained low during early embryonic development, but increased again after the midblastula transition. These results show that at least one component of 26S proteasomes changes during oocyte maturation and early development and suggest that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles, of the early embryo.     

Disappearance of a novel protein component of the 26S proteasome during Xenopus oocyte maturation

We have prepared polyclonal antibodies against Xenopus 20S proteasomes. The antibodies cross-react with several proteins that are common to 20S and 26S proteasomes and with at least two proteins that are unique to 26S proteasomes. The antibodies were used to analyze changes in the components of proteasomes during oocyte maturation and early development of Xenopus laevis. A novel protein with a molecular weight of 48 kDa, p48, was clearly detected in immature oocytes, but was found at very low levels in mature oocytes and ovulated eggs. p48 was reduced to low levels during oocyte maturation, after maturation-promoting factor was activated. The amount of p48 in eggs remained low during early embryonic development, but increased again after the midblastula transition. These results show that at least one component of 26S proteasomes changes during oocyte maturation and early development and suggest that alterations in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycles, of the early embryo.     

Three genes under different developmental control encode elongation factor 1-alpha in Xenopus laevis.

We have cloned cDNAs encoding two variants of the elongation factor for protein synthesis in Xenopus laevis, called EF-1 alpha. One of these (42Sp50) is expressed exclusively in immature oocytes. It is one of two protein components of a 42S RNP particle that is very abundant in previtellogenic oocytes. The 42S RNP particle consists of various tRNAs, 5S RNA, 42Sp50 and a 5S RNA binding protein (42Sp43). A major function served by 42Sp50 appears to be the storage of tRNAs for later use in oogenesis and early embryogenesis. The second EF-1 alpha variant (EF-1 alpha O) is expressed mainly in oocytes but transiently in early embryogenesis as well. Its mRNA cannot be detected after neurulation in somatic cells. EF-1 alpha O is closely related to a third EF-1 alpha (EF-1 alpha S), discovered originally by Krieg et al. (1). EF-1 alpha S is expressed at low levels in oocytes but actively in somatic cells. The latter two proteins are very similar to known eukaryotic EF-1 alpha from other organisms and presumably function in their respective cell types to support protein synthesis.     

Biochemical research on oogenesis. Comparison between the proteins of the ribosomes and the proteins of the 42 S particles from small oocytes of Xenopus laevis

Previtellogenic oocytes of Xenopus laevis synthesize large amounts of 5 S RNA and transfer RNA, but very little, if any, 28 S and 18 S RNA. About half of the RNA of these oocytes is stored in nucleoprotein particles sedimenting at 42 S. These particles contain 5 S RNA, transfer RNA, and several proteins, the function of which remains so far unknown.The proteins of the 42 S particles were analyzed by two-dimensional electrophoresis on polyacrylamide gel. The resulting fingerprints displayed one major and two minor basic spots. None of these coincided with any of the 37 spots produced by the 60 S subunit of the ribosomes and with the 30 spots produced by the 40 S subunit. We conclude that no ribosomal component other than 5 S RNA is present in the 42 S particles.The fingerprints of 40 S and 60 S ribosomal proteins from X. laevis coincided almost completely with the corresponding fingerprints from the rat and the rabbit.     

The N-terminal K homology domain of the poly(rC)-binding protein is a major determinant for binding to the poliovirus 5'-untranslated region and acts as an inhibitor of viral translation

The poly(rC)-binding proteins (PCBP1 and PCBP2) are RNA-binding proteins whose RNA recognition motifs are composed of three K homology (KH) domains. These proteins are involved in both the stabilization and translational regulation of several cellular and viral RNAs. PCBP1 and PCBP2 specifically interact with both the 5'-element known as the cloverleaf structure and the large stem-loop IV RNA of the poliovirus 5'-untranslated region. We have found that the first KH domain of PCBP2 (KH1) specifically interacts with the viral RNAs, and together with viral protein 3CD, KH1 forms a high affinity ternary ribonucleoprotein complex with the cloverleaf RNA, resembling the full-length PCBP protein. Furthermore, KH1 acts as a dominant-negative mutant to inhibit translation from a poliovirus reporter gene in both Xenopus laevis oocytes and HeLa cell in vitro translation extracts.