Identification of a 60‐kDa phosphoprotein that binds stored messenger RNA of Xenopus oocytes

Abstract

Rapidly labelled, polyadenylated RNA is contained in three distinct fractions isolated from homogenized amphibian oocytes: (a) in ribonucleoprotein particles that are associated with a fibrillar matrix, the complexes sedimenting at > 1500s; (b) in ribonucleoprotein particles that sediment at 20 -120s and have the characteristics of stored (maternal) messenger ribonucleoprotein (mRNP) and (c) in polyribosomes that sediment at 120 ~ 360 S. We have compared the RNA and protein components of the first two of these RNP fractions. The polydenylated RNA extracted from the two RNP fractions differs in that the RNA from fibril-associated RNP contains a much higher content of repeat sequences than does the RNA from mRNP. In other words, the RNA from fibrilassociated RNP is largely unprocessed and may constitute a premessenger state, which for convenience is referred to as premessenger RNP (pre-mRNP). RNA-binding experiments demonstrate that the polypeptide most tightly bound in pre-mRNP is a 54-kDa component (p54), whereas the polypeptide most tightly bound in mRNP is a 60-kDa component (p60). Antibodies raised against p60 are used to show that this polypeptide is a common major component of pre-mRNP and mRNP and that it is also located in oocyte nuclei. However the state of p60 is modified between the premessenger and stored message levels: the polypeptide in mRNP is heavily phosphorylated whereas the equivalent polypeptide in pre-mRNP is completely unphosphorylated. The relative roles of the presence of repeat sequences and phosphorylation of mRNA-associated protein in blocking translation are discussed.Both messenger RNA molecules and premessenger RNA molecules of amphibian oocytes are associated with classes of oocyte-specific proteins [l, 21. That oocytes should have categories of ribonucleoprotein (RNP) distinct from somatic cells probably relates to the special function of oocytes in accumulating mRNA throughout oogenesis. Most of the stored (maternal) mRNA is not utilized until egg maturation and early embryogenesis [3] and is somehow stabilized and prevented from being translated in oocytes. It is of general interest to understand how the block to translation occurs for it may represent a situation that arises, albeit less frequently, in other cell types.Recently it has been demonstrated that a mixture of oocyte mRNP proteins, when combined with globin mRNA in vitro to form reconstituted RNP particles, is effective in blocking its translation [4]. How this finding relates to the endogenous stored mRNA of oocytes is uncertain for, unlike mature globin mRNA, much of the stored mRNA of Xenopus (and sea urchin) oocytes is unprocessed : most molecules contain sequences transcribed from short repetitive DNA elements of the genome [ 5 , 61. The relevance of repeat sequences, possibly lying adjacent to coding sequences, to the block in translation of stored mRNA has recently been assessed. Up to 70% of the mass of polyadenylated RNA in Xenopus oocytes contains repeat sequences [7] and has been shown to be untranslatabl...

PUB1 is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae.

“…Many investigators have speculated on the function of free mRNP complexes in normal mRNA metabolism; however, direct evidence for the existence of these structures in vivo and identification of the functions that they perform have remained elusive. Stored mRNAs, such as the maternal mRNAs of amphibian oocytes, are generally considered a special class of nonpolyribosome-bound mRNP (16). The identification of authentic mRNPs and elucidation of their roles in eukaryotic protein synthesis have proven difficult (17).…”

Identification and characterization of the poly(A)-binding proteins from the sea urchin: a quantitative analysis.

“…The literature documents many cases of mRNP protein phosphorylation (6,10,13,15,29,38,49). This group includes HD40 from Artemia salina, a helix-destabilizing protein with poly(A)-binding activity (49).…”

Binding of a phosphoprotein to the 3' untranslated region of the mouse protamine 2 mRNA temporally represses its translation.

“…8). Phosphorylation has been proposed to regulate translation in Xenopus oocytes (7,23,45). One of RNAbinding proteins in Xenopus oocytes, the phosphoprotein pp6O, forms a stable complex with mRNA and inhibits translation of in vitro-assembled mRNPs, whereas after dephosphorylation, binding affinity of this protein is reduced and translation occurs.…”

“…We have analyzed protein binding to transcripts derived from subclones of the 3' UTR of mP2, which contains three highly conserved cis-acting elements, Y, H, and Z (34). RNase T1 mapping assays demonstrated that the Y and H elements specifically bind protein present in testicular cytoplasmic extracts from adult mice (34) 22-day-old mice (1 to 2 ml containing up to 50 mg of protein) were applied to columns packed with 1 to 2 ml of DEAESepharose 6B equilibrated in buffer C (20 mM HEPES [pH 7.6], 3 mM MgCl2, 40 mM KCl, 1 mM DTT, 10% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 0.5 ,ug of leupeptin per ml, 0.7 ,ug of pepstatin per ml, 2 ,ug of aprotinin per ml). After the samples were loaded, columns were washed with 5 to 10 ml of buffer C. The flowthrough fraction contained approximately 30% of the protein loaded.…”