Phosphorylation of a 60 kDa polypeptide from Xenopus oocytes blocks messenger RNA translation.

The stored mRNP particles of Xenopus oocytes contain protein kinase activity and two major phosphoproteins of 60 kDa (pp60) and 56 kDa (pp56). These proteins can be phospholabelled in the particles either in vivo or in vitro and then isolated by SDS-PAGE. On renaturing pp60 in the presence of globin mRNA, a stable RNA-protein complex is formed. The complex has a uniform density in Cs salt gradients, corresponding to the binding of about 10 protein molecules to each mRNA, probably at the poly(A) sequence. Compared with uncomplexed mRNA, the RNP complex is translated poorly both in vitro and in vivo. Translation of the complex can be regained after treatment with protein phosphatase. It is shown that dephosphorylation destabilizes the binding of protein to RNA, making the mRNA accessible for translation. Studies with native mRNP particles show that their translation also can be enhanced by dephosphorylation.     

The nature of the 5''-linked 5'' nucleotide sequence at the 5'' end of rabbit globin messenger ribonucleic acid.

Poly(A)-containing messenger RNA isolated from rabbit reticulocytes as estimated by periodate oxidation and condensation with [3H]isoniazid has two oxidizable end groups per molecule of mol. wt. 220000. When the mRNA is subjected to stepwise degradation by beta-elimination, only one oxidizable end-group is found. This indicates that one of the 2',3' hydroxyl end-groups is linked through the normal 3'--5' phosphodiester bond, but that the other is linked in such a way that after stepwise degradation no new 2',3 hydroxyl group is revealed. This structure could be a 5'-linked 5'-phospho di- or tri-ester. On digestion with ribonuclease the isoniazid-labelled RNA produced oligonucleotide hydrazones consistent with a poly(A) sequence at the 3' end plus fragments that are not found after stepwise degradation. These fragments have a charge of --6 and --8 from pancreatic ribonuclease or --7 from ribonuclease T1 digestion. These charges are changed to --3.4 and --4.1 after pancreatic ribonuclease, ribonuclease T2 and alkaline phosphatase digestion. methyl-3H-labelled-poly(A)-containing RNA isolated from late erythroid cells contain a methyl-labelled fragment resistant to endonuclease and phosphodiesterase II digestion. After digestion with phosphodiesterase I this fragment produces methyl-3 H-labelled nucleotides with the electrophoretic mobility of pm7G and pAm. It is concluded that globin mRNA has the 5' sequences m7G(5')ppp'AmpYpGp ... and m7G(5')pppAmpApGpYp.     

Multiple modifications in the phosphoproteins bound to stored messenger RNA in Xenopus oocytes

Messenger RNA molecules accumulated in amphibian oocytes are stabilized and blocked from translation through association with a defined set of phosphoproteins. Phosphoproteins of 60 kDa and 56 kDa (pp60 and pp56) isolated from messenger ribonucleoprotein particles of Xenopus laevis oocytes can be bound in vitro to mRNA sequences. After phospholabelling in vitro, both pp60 and pp56 show a range of ionic forms, which resolve on two-dimensional gel electrophoresis as a series of pairs with identical charge. The similarities between pp60 and pp56 in their ionic properties suggest a common protein primary structure. This suggestion gains further support from proteinase digestion analysis of pp60 and pp56: practically identical size patterns of phospholabelled fragments are generated using a range of different proteinases. However, in spite of their structural similarities, pp60 and pp56 are recognised as antigenically distinct from each other by using polyclonal antibodies. It is concluded from these, and other, observations that pp60 and pp56 are members of a family of structurally similar polypeptides which are subjected to multiple secondary modifications. Of these modifications, phosphorylation appears to be instrumental in establishing tight binding to mRNA, while antigenicity appears to be determined by some other modification. The role of microheterogeneity in the structure of RNA-binding proteins is discussed in relation to the differential activation of mRNA sequences for translation during early development.     

Escherichia coli poly(A)-binding proteins that interact with components of degradosomes or impede RNA decay mediated by polynucleotide phosphorylase and RNase E

The multifunctional ribonuclease RNase E and the 3'-exonuclease polynucleotide phosphorylase (PNPase) are major components of an Escherichia coli ribonucleolytic "machine" that has been termed the RNA degradosome. Previous work has shown that poly(A) additions to the 3' ends of RNA substrates affect RNA degradation by both of these enzymes. To better understand the mechanism(s) by which poly(A) tails can modulate ribonuclease action, we used selective binding in 1 m salt to identify E. coli proteins that interact at high affinity with poly(A) tracts. We report here that CspE, a member of a family of RNA-binding "cold shock" proteins, and S1, an essential component of the 30 S ribosomal subunit, are poly(A)-binding proteins that interact functionally and physically, respectively, with degradosome ribonucleases. We show that purified CspE impedes poly(A)-mediated 3' to 5' exonucleolytic decay by PNPase by interfering with its digestion through the poly(A) tail and also inhibits both internal cleavage and poly(A) tail removal by RNase E. The ribosomal protein S1, which is known to interact with sequences at the 5' ends of mRNA molecules during the initiation of translation, can bind to both RNase E and PNPase, but in contrast to CspE, did not affect the ribonucleolytic actions of these enzymes. Our findings raise the prospect that E. coli proteins that bind to poly(A) tails may link the functions of degradosomes and ribosomes.     

Isolation of cDNA for a Xenopus sperm-specific basic nuclear protein (SP4) and evidence for expression of SP4 mRNA in primary spermatocytes

A cDNA library was prepared in lambda gt 11 from poly(A)+ mRNA isolated from a pure population of Xenopus round spermatids and screened with an antibody against SP3-5 (sperm-specific proteins) of Xenopus sperm. Positive clones were sequenced and an arginine-rich clone, designated pXSP531, was obtained. The 473-nucleotide sequence of pXSP531 contained an open reading frame of 237 nucleotides which was preceded by a 5' untranslated region of 67 nucleotides. The 3' untranslated region contained 149 nucleotides, including a consensus polyadenylation signal (AAATAAAA). Twenty nucleotides of a poly(A) tail was contained in the pXSP531. SP3-5 were separated from each other by reverse-phase chromatography and sequenced. The amino acid sequence of the peptide fragments which were obtained by digestion of SP4 with V8 protease and separated by reverse-phase chromatography was identical to the sequence of the N-terminal 43 and C-terminal 15 amino acids deduced from the nucleotide sequence of pXSP531. This result demonstrates that pXSP531 encodes SP4. Northern hybridization of RNA extracted from primary spermatocytes and round spermatids on Days 0 and 6 with SP4 cDNA probe (pXSP531) showed that SP4 mRNA is present both in primary spermatocytes and in round spermatids as is protamine mRNA in the rainbow trout. The size of the SP4 mRNA in round spermatids on Day 0 was longer by 60 nucleotides compared to that in primary spermatocytes and that in spermatids on Day 6 was shorter by 30 nucleotides compared to that on Day 0. These size differences were due to differences in the length of the poly(A) tracts because digestion of poly(A) with ribonuclease H resulted in the shortening of mRNA to the same size for three stages.     

A small nuclear ribonucleoprotein associates with the AAUAAA polyadenylation signal in vitro

RNAs containing the polyadenylation sites for adenovirus L3 or E2a mRNA or for SV40 early or late mRNA are substrates for cleavage and poly(A) addition in an extract of HeLa cell nuclei. When polyadenylation reactions are probed with ribonuclease T1 and antibodies directed against either the Sm protein determinant or the trimethylguanosine cap structure at the 5' end of U RNAs in small nuclear ribonucleoproteins, RNA fragments containing the AAUAAA polyadenylation signal are immunoprecipitated. The RNA cleavage step that occurs prior to poly(A) addition is inhibited by micrococcal nuclease digestion of the nuclear extract. The immunoprecipitation of fragments containing the AAUAAA sequence can be altered, but not always abolished, by pretreatment with micrococcal nuclease. We discuss the involvement of small nuclear ribonucleoproteins in the cleavage and poly(A) addition reactions that form the 3' ends of most eukaryotic mRNAs.     

Translational control by cytoplasmic polyadenylation during Xenopus oocyte maturation: characterization of cis and trans elements and regulation by cyclin/MPF.

The expression of certain maternal mRNAs during oocyte maturation is regulated by cytoplasmic polyadenylation. To understand this process, we have focused on a maternal mRNA from Xenopus termed G10. This mRNA is stored in the cytoplasm of stage 6 oocytes until maturation when the process of poly(A) elongation stimulates its translation. Deletion analysis of the 3' untranslated region of G10 RNA has revealed that two sequence elements, UUUUUUAU and AAUAAA were both necessary and sufficient for polyadenylation and polysomal recruitment. In this communication, we have defined the U-rich region that is optimal for polyadenylation as UUUUUUAUAAAG, henceforth referred to as the cytoplasmic polyadenylation element (CPE). We have also identified unique sequence requirements in the 3' terminus of the RNA that can modulate polyadenylation even in the presence of wild-type cis elements. A time course of cytoplasmic polyadenylation in vivo shows that it is an early event of maturation and that it requires protein synthesis within the first 15 min of exposure to progesterone. MPF and cyclin can both induce polyadenylation but, at least with respect to MPF, cannot obviate the requirement for protein synthesis. To identify factors that may be responsible for maturation-specific polyadenylation, we employed extracts from oocytes and unfertilized eggs, the latter of which correctly polyadenylates exogenously added RNA. UV crosslinking demonstrated that an 82 kd protein binds to the U-rich CPE in egg, but not oocyte, extracts. The data suggest that progesterone, either in addition to or through MPF/cyclin, induces the synthesis of a factor during very early maturation that stimulates polyadenylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A KH domain RNA binding protein, KSRP, promotes ARE-directed mRNA turnover by recruiting the degradation machinery

Inherently unstable mRNAs contain AU-rich elements (AREs) in their 3' untranslated regions that act as mRNA stability determinants by interacting with ARE binding proteins (ARE-BPs). The mechanisms underlying the function of ARE and ARE-BP interactions in promoting mRNA decay are not fully understood. Here, we demonstrate that KSRP, a KH domain-containing ARE-BP, is an essential factor for ARE-directed mRNA decay. Some of the KH motifs (KHs) of KSRP directly mediate RNA binding, mRNA decay, and interactions with the exosome and poly(A) ribonuclease (PARN). The ability of KHs to promote mRNA decay correlates with their ability to bind the ARE and associate with RNA-degrading enzymes. Thus, KHs promote rapid mRNA decay by recruiting degradation machinery to ARE-containing mRNAs.     

Photocrosslinking of proteins to maternal mRNA in Xenopus oocytes

Ultraviolet irradiation was used to covalently crosslink poly(A) RNA and associated proteins in Xenopus oocytes and reticulocytes. Each cell type contained similar as well as unique crosslinked proteins. The somatic cells contained a single 78-kDa 3' poly(A) tract binding protein while oocyte poly(A), however, was bound by this protein and at least three additional proteins. Based on the mass of poly(A) RNA, oocytes in their earliest stages of growth contained crosslinked proteins that were generally more prevalent than in fully grown oocytes. An investigation of possible messenger RNA-specific proteins was undertaken by a series of RNA injection experiments. Two radiolabeled SP6-derived mRNAs were injected into oocytes; the first, globin mRNA, assembled into polysomes, while the second, a maternal mRNA termed G10, entered a nontranslating ribonucleoprotein compartment. Following the induction of oocyte maturation, additional globin mRNA was recruited onto polysomes while G10 mRNA remained a nontranslating mRNP. The proteins that can be crosslinked to these injected mRNAs were detected by 32P nucleotide transfer. Each mRNA associated with shared as well as unique proteins, some of which were detected only in mature oocytes. The possible function of these proteins is discussed.     

The location of the globin mRNA sequence within its 16S precursor.

The coding sequence of globin mRNA has been located at or very close to the 3' end of its poly(A)-containing 16S precursor. The 16S RNA was annealed to globin cDNA and the hybrid digested with ribonuclease H. The undigested fragment did not bind to oligo(dT)-cellulose and its size was that expected for the intact 5' portion of the precursor.     

The mechanism and regulation of deadenylation: identification and characterization of Xenopus PARN.

In Xenopus oocytes, the deadenylation of a specific class of maternal mRNAs results in their translational repression. Here we report the purification, characterization, and molecular cloning of the Xenopus poly(A) ribonuclease (xPARN). xPARN copurifies with two polypeptides of 62 kDa and 74 kDa, and we provide evidence that the 62-kDa protein is a proteolytic product of the 74-kDa protein. We have isolated the full-length xPARN cDNA, which contains the tripartite exonuclease domain conserved among RNase D family members, a putative RNA recognition motif, and a domain found in minichromosome maintenance proteins. Characterization of the xPARN enzyme shows that it is a poly(A)-specific 3' exonuclease but does not require an A residue at the 3' end. However, the addition of 25 nonadenylate residues at the 3' terminus, or a 3' terminal phosphate is inhibitory. Western analysis shows that xPARN is expressed throughout early development, suggesting that it may participate in the translational silencing and destabilization of maternal mRNAs during both oocyte maturation and embryogenesis. In addition, microinjection experiments demonstrate that xPARN can be activated in the oocyte nucleus in the absence of cytoplasmic components and that nuclear export of deadenylated RNA is impeded. Based on the poly(A) binding activity of xPARN in the absence of catalysis, a model for substrate specificity is proposed.     

Association of the polyadenylate segment of messenger RNA with other polynucleotide sequences in mouse sarcoma 180 polyribosomes.

Limited digestion of polysomal RNA with pancreatic ribonuclease releases a structure consisting of poly(A) associated with other polyribonucleotide sequences. This complex can be purified by oligo(dT)-cellulose chromatography. Heating for formamide treatment causes the dissociation of fragments free of poly(A) from the poly(A)-containing components. The two types of fragments tend to reassociate under annealing conditions, and this association is prevented by poly(U). Control experiments indicate that this structure is not an artifact generated during the manipulations. The same structure can be obtained by limited RNase digestion of polyribosomes, followed by deproteinization. The results suggest that the mRNA in polyribosomes may have a defined configuration caused by the interaction of the poly(A) sequence with another segment of the RNA.     

A 60-kDa protein from rabbit reticulocytes specifically recognizes the capped 5' end of beta-globin mRNA

The binding of proteins from rabbit reticulocyte lysate to in-vitro-generated beta-globin mRNA and its defined segments was investigated using ultraviolet-cross-linking experiments as well as gel-retardation assays. Under stringent conditions, only three proteins (72, 60 and 50 kDa) were found associated with full-length beta-globin mRNA at different positions. The 72-kDa protein is most likely the poly(A)-binding protein and binds, as expected, to the poly(A) tail, whereas the 50-kDa protein exhibits affinity for the trailer region of beta-globin mRNA. The binding region of the 60-kDa protein is located at the 5' end of beta-globin mRNA. The interaction of this protein is dependent on the presence of the 5' cap structure, as indicated by competition experiments using an uncapped beta-globin-mRNA leader segment. Further competition experiments with beta-globin mRNA, deleted in part in the leader region, suggest that, besides the cap structure, certain sequence elements are necessary for the interaction of the 60-kDa protein and the beta-globin mRNA leader.     

Isolation and characterization of polyadenylate-containing RNA from Bacillus brevis.

A substantial fraction (30--40%) of pulse-labeled RNA from exponentially growing cells of Bacillus brevis contains polyadenylate sequences, as measured by adsorption to oligo(dT)-cellulose. The weight-average length of poly(A) tracts obtained after digestion with pancreatic and T1 ribonucleases is 60 nucleotide residues. Susceptibility to degradation by snake venom phosphodiesterase after ribonuclease degradation indicates that the poly(A) sequences are located near the 3' ends of the RNA chains, but that in 40% of the material at least one internal pyrimidine nucleotide residue intervenes between the poly(A) tract and the 3'-hydroxyl terminus. These pyrimidine nucleotides consist of 65% cytidylate and 35% uridylate residues. In the remaining RNA chains, the poly(A) sequence is directly at the 3'-terminus, but the possibility cannot be excluded that a small fraction of this material may contain a 3'-hydroxyl terminal guanylate residue. The weight-average sedimentation coefficient of poly(A)-containing RNA is 12.5 S, corresponding to a polynucleotide chain length of 800--900 residues. This is in a size range expected for messenger RNA, a possibility which is also supported by the observation that pulse-labeled RNA has a considerably higher poly(A) content than long-term labeled RNA.     

The occurrence and distribution of poly(a) ribonucleic Acid in soybean

The occurrence and distribution of poly(A) sequences in the RNA of soybean (Glycine max var. Wayne) have been studied. Only one of the two species of AMP-rich RNA contains poly(A). D-RNA does not contain detectable poly(A) sequences. The TB-RNA is the poly(A) RNA in this system. At least a part (up to 50% or more) of the mRNA in polyribosomes contains a poly(A) sequence. The poly(A) RNA is heterodisperse in size but has a mean size of approximately 18S (2,000 nucleotides) in urea and formamide gels. The poly(A) fragment resulting from ribonuclease A and T₁ digestion migrates as a broad band overlapping the 4 to 5.8S regions of the gels with a mean size of somewhat greater than 5S. No evidence was found for the occurrence of a discrete oligo(A) fragment in the poly(A) RNA; however, oligonucleotides which migrate faster than the poly(A) fraction were observed in preparations which were not bound to oligo(dT) cellulose prior to electrophoresis. This oligonucleotide region was enriched in AMP (up to about 65%) as would be expected after ribonuclease A and T₁ digestion.