The stability of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells varies with growth rate.

The decay rates of eucaryotic elongation factor Tu (eEF-Tu) mRNA and eucaryotic initiation factor 4A (eIF-4A) mRNA in Friend erythroleukemia (FEL) cells were determined under several different growth conditions. In FEL cells which were no longer actively dividing (stationary phase), eEF-Tu mRNA was found to be rather stable, with a t1/2 of about 24 h. In rapidly growing FEL cells eEF-Tu mRNA was considerably less stable, with a t1/2 of about 9 h. In both cases a single rate of mRNA decay was observed. However, when stationary-phase cells resumed growth after treatment with fresh medium, we observed that eEF-Tu mRNA decay followed a biphasic process. The faster of the two decay rates involved approximately 50% of the eEF-Tu mRNA and had a t1/2 of about 1 h. The decay rates for eIF-4A (t1/2 = 2 h) and total poly(A)+ RNA (t1/2 = 3 h) were unaffected by changes in growth conditions. The t1/2 for polysomal eEF-Tu mRNA was found to be about 8 h when stationary FEL cells were treated with fresh medium. Previous work in this laboratory has shown (T. R. Rao and L. I. Slobin, Mol. Cell. Biol. 7:687-697, 1987) that when FEL cells are allowed to grow to stationary phase, approximately 60% of the mRNA for eEF-Tu is found in a nontranslating postpolysomal messenger ribonucleoprotein (mRNP) particle. eEF-Tu mRNP was rapidly cleared from stationary cells after treatment with fresh medium. The data presented in this report indicate that the stability of eEF-Tu mRNP is rapidly altered and the particle is targeted for degradation when stationary FEL cells resume growth.     

Regulation of the utilization of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells.

When Friend erythroleukemia cells were allowed to grow to stationary phase (2 X 10(6) to 3 X 10(6) cells per ml), approximately 60% of the mRNA for eucaryotic elongation factor Tu (eEF-Tu) sedimented at less than or equal to 80S, and most of the remaining factor mRNA was associated with small polysomes. Under the same growth conditions, greater than 90% of the mRNA for eucaryotic initiation factor 4A remained associated with polysomes. The association of eEF-Tu mRNA with polysomes changed dramatically when stationary-phase cells were treated with fresh medium. After 1 h in fresh medium, approximately 90% of eEF-Tu mRNA in Friend cells was found in heavy polysomes. Associated with the shift of eEF-Tu mRNA into heavy polysomes, we found at least a 2.6-fold increase in the synthesis of eEF-Tu in vivo as well as a remarkable 40% decrease in the total amount of eEF-Tu mRNA per cell. Our data raise the possibility that eEF-Tu mRNA that has accumulated in ribonucleoprotein particles in stationary-phase cells is degraded rather than reutilized for eEF-Tu synthesis.     

Protein kinase activity associated with stored messenger ribonucleoprotein particles of Xenopus oocytes

As the oocytes of Xenopus laevis grow and develop they accumulate vast stores of mRNA for use during early embryogenesis. The stored mRNA is stabilized and may be prevented from being translated in oocytes by the binding of a defined set of oocyte-specific proteins to form messenger RNP (mRNP) particles. A key event in the interaction of protein with mRNA is the phosphorylation of those few polypeptides that bind directly to all classes of polyadenylated mRNA. In this study we show that the phosphorylating enzyme (protein kinase), in addition to its target phosphoproteins, is an integral component of the mRNP particles. This association extends through various stages in the formation and use of the mRNP particles. Examination of material from oocytes of an early developmental stage (early stage 1), when the level of accumulated mRNA is low, reveals an excess of protein particles free of RNA, sedimenting at 6-18 S, and containing protein kinase activity and mRNA-binding phosphoproteins. At stages of maximum rate of mRNA accumulation (stages 1 and 2), the phosphoproteins and kinase are found primarily in individual mRNP particles that sediment at 40-80 S. As ribosomes become abundant (stages 2 and 3), the mRNP particles tend to interact with ribosomal subunits, at least in vitro, to form blocked translation initiation complexes that sediment at 80-110 S. These results are compared with observation on stored mRNP in other developmental systems.     

The mouse Y-box protein, MSY2, is associated with a kinase on non-polysomal mouse testicular mRNAs

In male germ cells many mRNAs are sequestered by proteins into translationally silent messenger ribo-nucleoprotein (mRNP) particles. These masked paternal mRNAs are stored and translated at specific times of germ cell development. Little is known about the mammalian testicular mRNA masking proteins bound to non-polysomal mRNAs. In this report, the major proteins binding to non-polysomal testicular mRNAs were isolated and analyzed. The two predominant proteins identified were: a Y-box protein (MSY2), the mammalian homolog to the Xenopus oocyte masking protein FRGY2/mRNP3+4, and a poly(A) binding protein. A kinase activity was also found associated with these non-polysomal RNAs. The kinase co-immunoprecipitates with MSY2 and phosphorylates MSY2 in vitro. The MSY2 associated kinase is not casein kinase 2, the kinase believed to phosphorylate mRNP3+4 in oocytes, but a yet unidentified kinase. MSY2 was found to be phosphorylated in vivo and MSY2 dephosphorylation led to a decrease in its affinity to bind RNA as judged by northwestern blotting. Therefore, testicular masked mRNAs may be regulated by the phosphorylation state of MSY2. Reconstitution experiments in which non-polysomal mRNA-binding proteins are dissociated from their RNAs and allowed to bind to exogenous mRNAs suggest that MSY2 binds RNA in a sequence-independent fashion. Furthermore, association of the non-polysomal derived proteins to exogenous non-specific mRNAs led to their translational repression in vitro.     

Regulated transport of messenger ribonucleic acid from isolated liver nuclei by nucleic acid binding proteins

Rat liver nucleocytosolic messenger ribonucleic acid (mRNA) transport is shown to be regulated by proteins with a high affinity for nucleic acids. In the cell-free system described, the energy-dependent transport of all RNA classes [transfer RNA (tRNA), mRNA, and ribosomal RNA (rRNA)] exhibited a dependence upon the availability of discrete minor sets of cytosol proteins. In addition to having a different level of saturation, only the mRNA "transport protein" activities are increased by adenosine cyclic 3',5'-phosphate (cAMP), an effect most likely mediated by a cAMP-dependent protein kinase. The mRNA transport proteins were isolated from cytosol by precipitation with streptomycin sulfate followed by deoxyribonucleic acid (DNA)-cellulose affinity chromatography, or from oligo-(thymidylate)-cellulose bound cytoplasmic messenger ribonucleoprotein (mRNP) particles by high-salt extraction. Either method yielded a protein fraction which exhibited a 1000-fold increase in mRNA transport activity as compared to cytosol. Over one-half of the mRNA transport activity is associated with the mRNP of the cell. A partial homology between the cytosol and mRNP-derived proteins was demonstrated by polyacrylamide gel electrophoresis. One major (20 000 daltons) and several minor proteins (23 000, 52 000, 54 000, and 72 000 daltons) were in common. Nuclear 4-5S exited from in vitro incubated nuclei in three phases, according to their differential in vivo rates of labeling and intranuclear pool sizes. The amount of nuclear RNA transported in vitro as mRNA (about 1.0%) agrees wtih the in vivo estimates. Additional evidence for in vivo equivalence was provided by the physicochemical characterization and bioassay of the RNA. The transported mRNA sedimented in urea-sucrose gradients as an 8-18S heterodisperse product. This RNA initiated cell-free translation with the synthesis of precursor peptides as diverse in size as those for albumin and alpha 2U-globulin. The relative abundancies of various transported mRNAs were different than the corresponding abundancies of liver cytoplasmic mRNAs.     

CiYB1 is a major component of storage mRNPs in ascidian oocytes: implications in translational regulation of localized mRNAs

In ascidian eggs, the existence of several localized maternal cytoplasmic determinants has been proposed and the importance of localized mRNAs for tissue differentiation has been demonstrated. We previously identified the ascidian Y-box proteins (CiYB1, 2 and 3), homologues of which are known to be involved in the storage of maternal mRNA in oocytes of other organisms. In this study, we found that CiYB1 protein is abundant in the gonad, egg, and embryo. Purification of messenger ribonucleoprotein (mRNP) particles from the gonad revealed that CiYB1 was one of their major components. A significant change in the distribution of CiYB1 protein from stored mRNP particles in the gonad to the ribosome fraction in eggs and embryos was observed. This change correlates most likely with the shift of stored maternal mRNAs to polyribosomes. Moreover, we found that CiYB1 colocalized with Cipem and Ci-macho1 mRNAs, which are localized at the posterior end of the embryo at the cleavage stage. Cipem and Ci-macho1 mRNAs were co-immunoprecipitated with CiYB1 in the oocyte and embryo lysates. The formation of a complex between Cipem mRNA and CiYB1 protein resulted in translational repression in the in vitro translation system. Our results indicate that associating with CiYB1 protein contributes to the translational control of the localized mRNA in eggs and embryos.     

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.     

Proteins associated with the messenger ribonucleoprotein particle for the estrogen-regulated apolipoprotein II mRNA.

The stability of the mRNA for apolipoprotein (apo) II is regulated by estrogen [Gordon et al. (1988) J. Biol. Chem. 263, 2625-2631]. On the hypothesis tha estrogen regulation of apoII mRNA stability is mediated through mRNA-protein interaction, we have examined the messenger ribonucleoprotein particle (mRNP) for apoII mRNA following release from chicken liver polyribosomes. Polyribosomes containing undegraded apoII mRNA were obtained when tissue was homogenized without detergent, and polyribosomes were isolated following simultaneous addition of detergent and magnesium to a 20000g supernatant. ApoII mRNP released by EDTA sedimented at 12-18 S in sucrose gradients, and banded at rho = 1.4 g/mL in CsCl isopycnic centrifugation, indicative of a 3:1 ratio of protein to mRNA. A fraction in which apoII mRNP was enriched to 40-50% of total mRNP was prepared by successive size fractionation steps on sucrose gradients. Proteins associated with sucrose gradient enriched apoII mRNP were examined by iodination of UV-cross-linked proteins followed by SDS-polyacrylamide gel electrophoresis. Comparisons of proteins in highly enriched apoII mRNP to proteins in mRNP from non-estrogen-treated rooster liver did not reveal any differences. This result suggests that the major proteins associated with apoII mRNA are mRNP proteins also associated with the bulk of liver mRNAs.     

Information relay from gene to protein: the mRNP connection

Eukaryotic messenger RNAs and their binding proteins are organized into structural units called ribonucleoprotein particles (mRNPs). Some mRNP proteins are ubiquitous, and might bind all mRNAs to ensure efficient translation. Other mRNA proteins, however, are cell-specific and bind only certain mRNAs that display regulated translation. This is particularly evident in early development, where some mRNP particles can be sequestered from the translational apparatus for months before they enter polysomes. Recent investigations suggest that these and other mRNP proteins bind specific sequences and regulate translation.     

She2p is a novel RNA binding protein with a basic helical hairpin motif

Selective transport of mRNAs in ribonucleoprotein particles (mRNP) ensures asymmetric distribution of information within and among eukaryotic cells. Actin-dependent transport of ASH1 mRNA in yeast represents one of the best-characterized examples of mRNP translocation. Formation of the ASH1 mRNP requires recognition of zip code elements by the RNA binding protein She2p. We determined the X-ray structure of She2p at 1.95 A resolution. She2p is a member of a previously unknown class of nucleic acid binding proteins, composed of a single globular domain with a five alpha helix bundle that forms a symmetric homodimer. After demonstrating potent, dimer-dependent RNA binding in vitro, we mapped the RNA binding surface of She2p to a basic helical hairpin in vitro and in vivo and present a mechanism for mRNA-dependent initiation of ASH1 mRNP complex assembly.     

Accelerated poly(A) loss and mRNA stabilization are independent effects of protein synthesis inhibition on alpha-tubulin mRNA in Chlamydomonas.

In Chlamydomonas, the usual rapid degradation of tubulin mRNAs induced by flagellar amputation is prevented by inhibition of protein synthesis with cycloheximide. Evidence is presented that the ability of cycloheximide to stabilize alpha-tubulin mRNA depends on the time of addition. Addition of cycloheximide to cells before induction strongly stabilizes the induced mRNAs, while addition after their synthesis stabilizes them only transiently. Moreover, cycloheximide inhibition does not stabilize the same alpha-tubulin mRNA species in uninduced cells. These results suggest that cycloheximide is not acting to stabilize the induced alpha-tubulin mRNAs simply by preventing ribosome translocation. The stabilized state of tubulin mRNA was found to correlate with its occurrence on smaller polysomes but larger EDTA-released mRNP particles than the unstable state. A second effect of cycloheximide on the metabolism of induced tubulin mRNAs is to accelerate complete poly(A) removal. This effect of cycloheximide inhibition, unlike stabilization, occurs whenever cycloheximide is added to cells, and appears unrelated to stabilization. The effect is shown to be mRNA-specific; poly(A)-shortening on the rbcS2 mRNA is not altered in the presence of cycloheximide, nor do completely deadenylated molecules accumulate. Experiments in which cells were released from cycloheximide inhibition suggest that deadenylated alpha-tubulin mRNAs may be less stable than their polyadenylated counterparts during active translation.     

The expression of a gene for eukaryotic elongation factor Tu in Artemia during development. Translation of poly(A)+ RNA and the use of a synthetic oligonucleotide to detect the presence of eukaryotic elongation factor Tu-specific mRNA

Total in vivo proteins from Artemia embryos at different developmental stages were examined by two-dimensional gel electrophoresis. A variety of peptides change during development, with one of them, the eukaryotic elongation factor Tu (eEF-Tu), presenting a dramatic increase from dormant embryos to nauplii. When poly(A)+ RNA is translated in vitro, the same relative increase is seen for eEF-Tu during development. Based on the amino acid sequence for Artemia eEF-Tu (Amons, R., Pluijms, W., Roobol, K., and M?ller, W. (1983) FEBS Lett. 153, 37-42), a synthetic oligodeoxynucleotide was prepared and used to prime the synthesis of cDNA with poly(A)+ RNA from 12-h developing embryos as template. Direct sequence analysis of the 900-base primary cDNA product shows it to be specific for the 5' end of Artemia eEF-Tu mRNA. Hybridization of a "Northern" blot of denatured (poly(A)+ RNA from different developmental stages with this cDNA reveals a major band migrating at about 1800 bases, which increase in intensity as development proceeds, paralleling the increase in eEF-Tu seen by in vitro translation. When poly(A)+ RNA is separated on a nondenaturing gel, blotted to poly(U) paper, and hybridized with the eEF-Tu cDNA, a single band is observed migrating faster than 18 S. Elution and in vitro translation of this band results in a major product migrating with eEF-Tu in a dodecyl sulfate-polyacrylamide gel and which is precipitable with eEF-Tu-specific antibodies.     

Possible involvement of messenger RNA-associated proteins in protein synthesis

Two distinct forms of globin messenger RNA were isolated from mouse spleen cells infected with Friend erythroleukemia virus: polyribosomal messenger ribonucleoprotein particles (15S mRNP), and their corresponding protein-free mRNAs obtained by chemical deproteinization. The translation efficiencies of both messenger forms were assayed in a Krebs II ascites cell-free system. Selective removal of RNA-binding proteins from the ascites cell lysate did not affect globin synthesis when the mRNA was supplied as 15S mRNP; deproteinized mRNA however was not translated. Only in the presence of two fractions of RNA-binding proteins was the protein-free mRNA translated. Some of the RNA-binding proteins have the same molecular weights and isoelectric points as the principal proteins of 15S mRNP.     

The C-terminal extension of Lsm4 interacts directly with the 3′ end of the histone mRNP and is required for efficient histone mRNA degradation

Metazoan replication-dependent histone mRNAs are the only known eukaryotic mRNAs that lack a poly(A) tail, ending instead in a conserved stem–loop sequence, which is bound to the stem–loop binding protein (SLBP) on the histone mRNP. Histone mRNAs are rapidly degraded when DNA synthesis is inhibited in S phase in mammalian cells. Rapid degradation of histone mRNAs is initiated by oligouridylation of the 3′ end of histone mRNAs and requires the cytoplasmic Lsm1-7 complex, which can bind to the oligo(U) tail. An exonuclease, 3′hExo, forms a ternary complex with SLBP and the stem–loop and is required for the initiation of histone mRNA degradation. The Lsm1-7 complex is also involved in degradation of polyadenylated mRNAs. It binds to the oligo(A) tail remaining after deadenylation, inhibiting translation and recruiting the enzymes required for decapping. Whether the Lsm1-7 complex interacts directly with other components of the mRNP is not known. We report here that the C-terminal extension of Lsm4 interacts directly with the histone mRNP, contacting both SLBP and 3′hExo. Mutants in the C-terminal tail of Lsm4 that prevent SLBP and 3′hExo binding reduce the rate of histone mRNA degradation when DNA synthesis is inhibited.     

Differential repression of specific mRNA in erythroblast cytoplasm: a possible role for free mRNP proteins.

Two types of in vivo untranslated 'free' mRNA-protein particles (mRNP) were isolated from duck erythroblast cytoplasm and characterised. Both types, namely the highly purified globin mRNA-specific '20S' mRNP and the '35S' mRNP containing a heterogenous non-globin mRNA population, are not translatable in rabbit reticulocyte lysates, but yield active mRNA upon deproteinisation. In vivo, 90% of globin mRNA is translated, but the majority of mRNA types are found in the inactive mRNP fraction, including fully repressed mRNA species. Searching for the factors controlling differential mRNA repression, we characterised and compared the protein composition of globin and '35S' mRNP using two dimensional gel electrophoresis, in vivo labelling with [35S]methionine and in vivo phosphorylation. The major proteins ubiquitously bound to globin or any other mRNA in the polyribosomes (e.g., the 73 K mol. wt. poly(A) binding protein) were not detected in purified inactive mRNP. In the latter some polypeptides appear to be associated with only one of the two inactive mRNA types while some others are common to both mRNPs. Furthermore, different rates of synthesis and phosphorylation characterize the protein populations of the two types of repressed mRNP. The specificity in composition and metabolism of the populations of polypeptides associated with different subpopulations of inactive cytoplasmic mRNA, as shown here, argues in favour of a role of mRNP proteins in mRNA recognition and selective translational repression, possibly in association with the ScRNA previously found as components of the free mRNP and able to inhibit protein synthesis.     

The estrogen-regulated destabilization of Xenopus albumin mRNA is independent of translation

Protein synthesis inhibitors have been shown to increase the stability of a number of labile mRNAs. In Xenopus laevis serum albumin mRNA is destabilized in the liver cell cytoplasm following estrogen administration. The present study examined the effect of translation inhibitors on this process. The initiation inhibitor 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide causes accumulation of albumin mRNA in 20-80S mRNP particles whereas the elongation inhibitor cycloheximide causes albumin mRNA to accumulate in polysomes. Neither inhibitor blocked the disappearance of albumin mRNA from liver cell cytoplasm when added with estradiol to the medium of liver explant cultures. We conclude that unlike a number of labile mRNAs the instability of Xenopus albumin mRNA following estradiol is independent of translation.     

Studies on a nonpolysomal ribonucleoprotein coding for myosin heavy chains from chick embryonic muscles.

A messenger ribonucleoprotein (mRNP) particle containing the mRNA coding for the myosin heavy chain (MHC mRNA) has been isolated from the postpolysomal fraction of homogenates of 14-day-old chick embryonic muscles. The mRNP sediments in sucrose gradient as 120 S and has a characteristic buoyant density of 1.415 g/cm3, which corresponds to an RNA:protein ratio of 1:3.8. The RNA isolated from the 120 S particle behaved like authentic MHC mRNA purified from chick embryonic muscles with respect to electrophoretic mobility and ability to program the synthesis of myosin heavy chain in a rabbit reticulocyte lysate system as judged by multi-step co-purification of the in vitro products with chick embryonic leg muscle myosin added as carrier. The RNA obtained from the 120 S particle was as effective as purified MHC mRNA in stimulating the synthesis of the complete myosin heavy chains in rabbit reticulocyte lysate under conditions where non-muscle mRNAs had no such effect. Analysis of the protein moieties of the 120 S particle by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows the presence of seven distinct polypeptides with apparent molecular weights of 44,000, 49,000, 53,000, 81,000, 83,000, and 98,000, whereas typical ribosomal proteins are absent. These results indicate that the 120 S particles are distinct cellular entities unrelated to ribosomes or initiation complexes. The presence of muscle-specific mRNAs as cytoplasmic mRNPs suggests that these particles may be involved in translational control during myogenesis in embryonic muscles.