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...
A ribonucleoprotein fraction that contains most of the rapidly labelled hnRNA has been isolated from gently ruptured oocytes of Triturus cristatus. This fraction consists of large aggregates of ribonucleoprotein and has a high (30:1) ratio of protein to RNA. The labelled RNA is contained in ribonucleoprotein particles that have a density of 1.27 g/cm3 in Cs2SO4 gradients (1.39 g/cm3 after formaldehyde fixation in CsCl gradients). Evidence is presented that the particles are associated in vivo with a librillar protein network. When the ribonucleoprotein aggregates are treated with ribonuclease, high salt concentration and nonionic detergent, a fibrillar protein residue is produced which contains many species of protein but a few that have electrophoretic characteristics that are identical to major ribonucleoprotein particle proteins. Isolated labelled hnRNA has been shown to bind specifically polypeptides of molecular weight 60000 and 54000 that are found in both particle and fibril preparations. In binding assays in vitro, these polypeptides are found to interact with mRNA to a lesser extent and not with rRNA. The isolated 60000‐Mr and 54000‐Mr proteins have the dual ability of forming ribonucleoprotein ‘particles’ with hnRNA and of polymerizing to generate 10‐nm fibrillar structures in the absence of RNA. The possible cellular functions of these proteins are discussed.
At early stages of oogenesis in Xenopus laevis most of the ribosomal 5s RNA is complexed with three proteins to form two types of cytoplasmic RNP storage particle. A particle sedimenting at 42s contains 5s RNA and tRNA together with two proteins of Mr 48000 (P48) and M , 43000 (P43) and a second particle sedimenting at 7 s contains 5s RNA plus a protein of M , 40000 (P40, also known as the transcription factor, TFIIIA). In this report we use antibodies monospecific for each protein to follow the movement of 5s RNA from nucleus to cytoplasm to nucleolus to cytoplasm and to determine the fate of each of the proteins that associate with 5s RNA during these transitions. Both P48 and P43 have roles additional to the formation of the 42s RNP storage particle; P48 is detected in the nucleus during early oogenesis and is cleaved to yield an Mr-33000 fragment that remains associated with 5s RNA that is excess to ribosome requirement during late oogenesis; P43 appears to be cleaved to yield fragments of M , 28000 and 17000, the latter being present in ribosomal fractions. Apparently, there is no function for P40 in addition to those already described in transcription of 5s RNA genes and in storage of 5s RNA as a 7s RNP particle.During amphibian oogenesis there is an uncoupled production of ribosomal RNA components. In early oocytes there is maximal production and stabilization of 5s RNA and tRNA, so that by the end of the previtellogenic period as much as 80% of the total RNA consists of these two types [I]. Only later, during vitellogenesis, does the production of ribosomal 18 S + 5.8 S + 28 S RNA reach maximal rate due to activation of transcription in extrachromosomal nucleoli [2]. This phasing of activity relates to the necessity to produce almost equal numbers of products from vastly different numbers of genes. For instance, in the oocytes of Xenopus luevis there are, per genome, lo5 copies of 5s RNA genes [3], 3 x lo4 copies of tRNA genes [4] and, after amplification, 2 x lo6 copies of 18s + 5.8s + 28s RNA genes [5]. The net output from the activity of these genes contributes to the production of 10I2 ribosomes [6] and 2.4 x 10" tRNA molecules [7] by the end of oogenesis.The 5s RNA and tRNA moleciiles stored during previtellogenesis are stabilized through their association with a set of three abundant proteins to form ribonucleoprotein (RNP) particles. Both 5s RNA and tRNA molecules interact with two proteins of M, 48000 (P48) and 43000 (P43) to form an RNP particle that sediments at 42s [6, 81 while 5s RNA alone interacts with a protein of M , 40000 (P40) to form an RNP particle that sediments at 7s [9]. It has been demonstrated previously that 5s RNA can interact specifically with all three proteins and that such complexes occur naturally in oocytes [9, lo].Antibodies raised against isolated P48, P43 and P40 are each completely monospecific within this group [I 11. By using these antibodies in immunoblotting assays we are able to follow the distribution and utilization of the 5 S-RNA-binding
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