Expression of ribosomal-protein genes in Xenopus laevis development

Using probes to Xenopus laevis ribosomal-protein (r-protein) mRNAs, we have found that in the oocyte the accumulation of r-protein mRNAs proceeds to a maximum level, which is attained at the onset of vitellogenesis and remains stable thereafter. In the embryo, r-protein mRNA sequences are present at low levels in the cytoplasm during early cleavage (stages 2-5), become undetectable until gastrulation (stage 10) and accumulate progressively afterwards. Normalization of the amount of mRNA to cell number suggests an activation of r-protein genes around stage 10; however, a variation in mRNA turnover cannot be excluded. Newly synthesized ribosomal proteins cannot be found from early cleavage up to stage 26, with the exception of S3, L17 and L31, which are constantly made, and protein L5, which starts to be synthesized around stage 7. A complete set of ribosomal proteins is actively produced only in tailbud embryos (stages 28-32), several hours after the appearance of their mRNAs. Before stage 26 these mRNA sequences are found on subpolysomal fractions, whereas more than 50% of them are associated with polysomes at stage 31. Anucleolate mutants do not synthesize ribosomal proteins at the time when normal embryos do it very actively; nevertheless, they accumulate r-protein mRNAs.     

Translational regulation of the expression of ribosomal protein genes in Xenopus laevis

The mRNAs coding for ribosomal proteins (rp-mRNA) are subjected to translational control during Xenopus oogenesis and embryogenesis, and also during nutritional changes in Xenopus cultured cells. This regulation, which appears to respond to the cellular need for new ribosomes, operates by changing the fraction of rp-mRNA engaged on polysomes, each translated rp-mRNA molecule always remaining fully loaded with ribosomes. All rp-mRNAs analyzed up to now show this translational behavior, and also share some structural features in their untranslated portions. In particular they all have rather short 5' untranslated regions, similar to each other, and always start at the very 5' end with a stretch of several pyrimidines. Fusion to a reporter-coding sequence of the 5' untranslated region of r-protein S19 has shown that this is involved in the translational regulation.     

Preferential expression of actin genes during oogenesis of Drosophila

The expression of actin genes was examined during oogenesis of Drosophila. Accumulation of actin proteins was quantitated by a two-stage electrophoresis procedure. Egg chambers accumulate actins preferentially, resulting in a twofold enrichment over other nonyolk proteins. RNA gel blot hybridization experiments demonstrated a concomitant twofold selective increase of actin mRNA levels over that of other mRNAs, suggesting regulation of actin genes at the pretranslational level. Despite an abrupt arrest of actin protein accumulation near the end of oogenesis, the bulk of the actin mRNAs remains associated with polysomes of constant size. It appears that this shut-off of actin protein accumulation is due to an overall decrease in translational efficiency, rather than actin mRNA degradation or its dissociation from polysomes.     

Translational discrimination of ribosomal protein mRNAs in the early Drosophila embryo

Most Drosophila mRNAs are actively translated in the early embryo, with the exception of the poorly translated ribosomal protein (r-protein) mRNAs. Two possible mechanisms for this translational discrimination were tested: (1) Translation of r-protein mRNAs is discriminated against by the limited activity of translational initiation factors in the early embryo and (2) translation of r-protein mRNAs is repressed by trans-acting factors that reversibly bind these mRNAs. Exogenously provided initiation factors promoted partial recruitment of r-protein mRNAs into polysomes, suggesting that modulation of initiation factor activity may play a role in the translational discrimination of r-protein mRNAs during embryogenesis. No evidence for involvement of reversibly binding trans-acting factors was obtained, although there are limitations in the interpretation of the latter experiments.     

Proteomic characterization of evolutionarily conserved and variable proteins of Arabidopsis cytosolic ribosomes

Analysis of 80S ribosomes of Arabidopsis (Arabidopsis thaliana) by use of high-speed centrifugation, sucrose gradient fractionation, one- and two-dimensional gel electrophoresis, liquid chromatography purification, and mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization) identified 74 ribosomal proteins (r-proteins), of which 73 are orthologs of rat r-proteins and one is the plant-specific r-protein P3. Thirty small (40S) subunit and 44 large (60S) subunit r-proteins were confirmed. In addition, an ortholog of the mammalian receptor for activated protein kinase C, a tryptophan-aspartic acid-domain repeat protein, was found to be associated with the 40S subunit and polysomes. Based on the prediction that each r-protein is present in a single copy, the mass of the Arabidopsis 80S ribosome was estimated as 3.2 MD (1,159 kD 40S; 2,010 kD 60S), with the 4 single-copy rRNAs (18S, 26S, 5.8S, and 5S) contributing 53% of the mass. Despite strong evolutionary conservation in r-protein composition among eukaryotes, Arabidopsis 80S ribosomes are variable in composition due to distinctions in mass or charge of approximately 25% of the r-proteins. This is a consequence of amino acid sequence divergence within r-protein gene families and posttranslational modification of individual r-proteins (e.g. amino-terminal acetylation, phosphorylation). For example, distinct types of r-proteins S15a and P2 accumulate in ribosomes due to evolutionarily divergence of r-protein genes. Ribosome variation is also due to amino acid sequence divergence and differential phosphorylation of the carboxy terminus of r-protein S6. The role of ribosome heterogeneity in differential mRNA translation is discussed.     

Labelling kinetics of RNA containing poly(a) in liver subcellular fractions

Kinetics of incorporation of (3H) uridine into cytoplasmic RNA fractions of rat liver is investigated. The fractions include free and membrane bound polysomes, rough membranes sedimenting with mitochondria and free cytoplasmic RNA particles. (1) Poly(A) containing RNA, isolated by oligo-dT cellulose, amounts to 0.4% of the total RNA in the homogenate, 0.5% in bound polysomes, 3.4% in free polysomes and 16% in free cytoplasmic RNA particles. (2) The rate of (3H) uridine incorporation into RNA lacking poly(A) proceeds uniformly in all subcellular fractions except for free cytoplasmic RNA particles, which accumulate negligible amounts of radioactivity. (3) The initial labelling of RNA containing poly(A) is most active in free cytoplasmic RNA particles supporting their identity as mRNA en route to polysomes. The initial specific radioactivities decrease in the following order: homogenate, bound polysomes, rough membranes sedimenting with mitochondria, free polysomes. The data suggest that mRNA is supplied to free and membrane-bound polysomes via different routes. The kinetic analysis indicates that free cytoplasmic RNA particles may be a precursor of mRNA of free polysomes rather than that of bound polysomes. (4) The kinetic differences of free and membrane bound polysomes are also demonstrated by comparing the radioactivity of RNA containing poly(A) to the total radioactivity at various incorporation times. In bound polysomes this decreases from 31% at 1 h to 10% at 25 h, whereas in free polysomes the corresponding ratio increases from 10 to 13%. RNA containing poly(A) of free cytoplasmic RNA particles represents 64% of the total radioactivity throughout the experiment.     

CHARACTERIZATION OF BRAIN RIBONUCLEOPROTEIN PARTICLES

Abstract— Brain RNP particles were characterized to determine whether they play a role in the regulation of brain protein synthesis. RNP particles were isolated from the postribosomal supernatant of cerebral hemispheres of young rabbits, employing conditions which minimize adventitious protein-RNA interactions. Brain RNP particles consist of a different set of proteins compared to proteins associated with either 40 and 60s ribosomal subunits or polysomal mRNA. Poly(A+)mRNA from brain RNP particles stimulates the incorporation of [³⁵S]methionine in a wheat embryo cell-free system and codes for a different set of proteins compared to poly(A+)mRNA isolated from polysomes (with some overlap; i.e. mRNA coding for brain-specific S100 protein is present in both RNP particles and polysomes).
Addition of total brain RNP particles to a cell-free wheat embryo system inhibits the endogenous incorporation of [³⁵S]methionine. Total RNP particles were fractionated by sucrose density gradient centrifugation into a'light'and a'heavy'fraction. The light RNP fraction inhibited while the heavy RNP fraction stimulated protein synthesis in the wheat embryo cell-free system. Analysis of the protein composition of fractionated RNP particles revealed that the light and heavy RNP particles contained different sets of proteins. Together these results suggested that one class of brain RNP particles may contain a translational inhibitor and may be involved in the regulation of protein synthesis in the brain.     

Membrane-bound ribosomes of myeloma cells. VI. Initiation of immunoglobulin mRNA translation occurs on free ribosomes

Immunoglobulin heavy (Ig H) and light (Ig L) chain mRNA molecules have been released from the endoplasmic reticulum (ER) membranes as free (F) mRNP particles when MOPC 21 (P3K) mouse myeloma cells are exposed to a hypertonic initiation block (HIB). The subsequent fate of these mRNA sequences has been examined when the cells are returned to normal growth medium. Upon return to isotonicity, all previously translated mRNA molecules reassociate with ribosomes and form functional polysomes. Ig H mRNA is found incorporated first into F polysomes and then into membrane-bound (MB) polysomes. Kinetic studies indicate that the time of passage of Ig H mRNA in F polysomes is approximately 30 s, during which a nascent polypeptide chain of approximately 80 amino acids would have been completed. When the rate of polypeptide elongation is depressed with emetine during the recovery from HIB, both Ig H and L mRNA molecules accumulate in small F polysomes. These results indicate that the formation of Ig-synthesizing polysomes proceeds in the sequence: mRNA leads to F polysomes leads to MB polysomes. With the additional observation that during HIB recovery puromycin completely prevents the reassociation of Ig mRNA with the ER, these findings support a model of MB polysome formation in which the specificity of membrane attachment is determined by the nature of the N- terminal amino acid sequence of the nascent polypeptide chain.     

Membrane-bound ribosomes of myeloma cells. V. Subcellular distribution of immunoglobulin mRNA molecules

The subcellular distribution of the most abundant mRNA sequences, particularly those of the immunoglobulin heavy (Ig H) and light (IG L) chain mRNA sequences, of MOPC 21 (P3K) mouse myeloma cells has been examined by translating the mRNA of various subcellular fractions in a messenger-dependent reticulocyte lysate (MDL) and by identifying Ig products with the use of a specific antiserum. Analyses of the distribution of the mRNA template activity and the translation products by SDS polyacrylamide gel electrophoresis reveal that approximately 85% of the mRNA present in the free ribosomal fraction is incorporated into polysomes and that the remainder is present as mRNP particles. On the endoplasmic reticulum (ER) the mRNA is found entirely in polysomes. In general, the size class of free (F) and membrane-bound (MB) polysomes corresponds to the size of their translation products. Thus, mRNAs coding Ig H (5.0 x 10(5) daltons in size) and Ig L (2.5 x 10(5) daltons in size) are incorporated into polysomes formed of 12 and 6 ribosomes, respectively. About 10% of the Ig mRNAs are not bound to membranes. A third of these are associated with mRNPs and the remainder incorporated into F polysomes of the same size as the Ig-synthesizing MB polysomes.     

Quantitative changes in protein synthesis during oogenesis in Xenopus laevis

Protein synthesis rates in Xenopus laevis oocytes from stage 1 through stage 6 were measured. In addition, the translational efficiencies, total RNA contents, and percentages of ribosomes in polysomes in growing oocytes at several stages were determined. Stage 1 oocytes synthesize protein at a mean rate of 0.18 ng hr⁻¹ while stage 6 oocytes make protein at a rate of 22.8 ng hr⁻¹. Polysomes from growing and full-grown oocytes sedimented in a sucrose gradient with a peak value of 300 S, corresponding to a weight-average packing density of 10 ribosomes per mRNA. Ribosome transit times of endogenous mRNAs were essentially unchanged at all stages examined. While the oocyte's total ribosomal RNA content was observed to increase about 115-fold during oogenesis, the percentage of ribosomes in polysomes remained constant at approximately 2%. Taken together, the data suggest that the 127-fold increase in protein synthesis which occurs during Xenopus oogenesis involves the progressive recruitment onto polysomes of mRNA from the maternal stockpile.     

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.