Relative amounts of newly synthesized poly(A)+ and poly(A)- messenger RNA during development of Xenopus laevis

The relative amounts of newly synthesized poly(A)⁺ and poly(A)^? mRNA have been determined in developing embryos of the frog Xenopus laevis. Polysomal RNA was isolated and fractionated into poly(A)⁺ and poly(A)^? RNA fractions with oligo(dT)-cellulose. In normal embryos the newly synthesized polysomal poly(A)⁺ RNA has a heterodisperse size distribution as expected of mRNA. The labeled poly(A)^? RNA of polysomes is composed mainly of rRNA and 4S RNA. The amount of poly(A)^? mRNA in this fraction cannot be quantitated because it represents a very small proportion of the labeled poly(A)^? RNA. By using the anucleolate mutants of Xenopus which do not synthesize rRNA, it is possible to estimate the percentage of mRNA which contains poly(A) and lacks poly(A). All labeled polysomal RNA larger than 4S RNA which does not bind to oligo(dT)-cellulose in the anucleolate mutants is considered presumptive poly(A)^? mRNA. The results indicate that about 80% of the mRNA lacks a poly(A) segment long enough to bind to oligo(dT). The poly(A)⁺ and poly(A)^? mRNA populations have a similar size distribution with a modal molecular weight of about 7 × 10⁵. The poly(A) segment of poly(A)⁺ mRNA is about 125 nucleotides long. Analysis of the poly(A)^? mRNA fraction has shown that it lacks poly(A)₁₂₅.     

The effect of organ preservation on polysomal messenger RNA

Organ preservation decreases polysomal RNA⁺-poly(A) in mouse kidneys. Warm storage at 37 °C markedly decreased the total polysomal RNA. Moreover, the percentage of ³H-polysomal RNA⁺-poly(A) is decreased 24.1% after 1 hr. Cold storage does not decrease the amount of polysomal RNA, but the decrease in the ³H-polysomal RNA which is poly(A)⁺ was diminished 83.7% after 48 hr of cold storage.     

Androgenic control of polysomal poly(A)-containing RNA in the cultured rat ventral prostate

Testosterone stimulated, at the concentration of 10-7 M and independently of other hormones, the accumulation of polysomal poly(A)-containing RNA (mRNA) in cultured explants of rat ventral prostate and concomitantly also protein synthesis. The hormone-induced accumulation of polysomal mRNA, which reached its maximum at 24 h after testosterone addition, paralleled the preferential labeling of high molecular weight RNA demonstrable with the electrophoretic analysis of the double-isotope labeled RNA after a short pulse (30 min). These findings are consistent with the idea that testosterone activated the synthesis of precursor mRNA leading to an increased amount of polysomal mRNA and eventually an activated protein synthesis. The synthesis and maturation of rRNA appeared to proceed even in the absence of testosterone, which is in contrast to the vivo findings on castrated rats. This partial uncoupling of RNA synthesis from androgenic control may account for the slow and less marked hormonal responses found in protein synthesis and glucose metabolism in cultured explants from normal animals. Because of the lack of uniformity in the suture, routine light microscopic control to assess the viability of cultured explants was found to be a prerequisite for successful biochemical work on prostate culture.     

Diabetes-induced alteration in subcellular distribution of poly(A)-rich RNA from skeletal muscle

Summary Skeletal muscle ribosome preparations from diabetic rats have lower polysome content than those from normal animals. The ratio of poly(A) containing RNA between polysomes and postribosomal supernatant has been measured both in normal and diabetic rats. The results suggest that in diabetic animals there is a greater proportion of free poly(A) containing RNA, in postribosomal supernatant.This work was supported by Grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas (República Argentina), Universidad Nacional de Buenos Aires, and partly under contract of the Ministére de la Politique Scientifique within the framework of the Association Euratom—University of Brussels—University of Pisa.     

Complexity of poly(A+) and poly(A-) polysomal RNA in mouse liver and cultured mouse fibroblasts.

RNA excess hybridization experiments were used to measure the complexity of nuclear RNA, poly(A+) mRNA, poly(A-) mRNA, and EDTA-released polysomal RNA sedimenting at less than 80 S in mouse liver and in cultured mouse cells. With both cell types, poly(A-) RNA was found to contain 30-40% of the sequence diversity of total mRNA. In the case of liver this represents 5,700 poly(A-) molecules and 8,600 poly(A+) molecules for a total of approximately 14,300 different mRNAs. Comparison of the complexity of mRNA with that of nuclear RNA revealed that in liver and in cultured cells, mRNA has only 10-20% of the sequence diversity present in nuclear RNA. This latter observation is consistent with existing data on mammalian cells from this and other laboratories.     

Template activity of poly(A+) and poly(A-) messenger RNA of pathogenic arenaviruses

The total RNA from cells infected with Machupo and Lassa viruses as well as poly(A+) and poly(A-) fractions of the RNA were translated in the cell-free protein synthesizing system from rabbit reticulocytes. The translated products were treated with specific antibodies and analyzed in polyacrylamide gel electrophoresis. Only poly(A-) fraction of RNA coded for the synthesis of NP protein in vitro. The mRNAs for NP protein of Machupo and Lassa viruses are supposed to contain no poly(A) sequences at 3'end, or if they really do, the size of the sequences is not adequate for binding with oligo(dT)-cellulose.     

"Cap" sequences in poly(A)-rich RNA from dry wheat embryos

Although template-active RNA in dry seeds and embryos has attracted widespread interest, there have been no published reports about 5'-terminal "capping" sequences in such RNA. Boro[3H]hydride labeling of periodate-oxidized termini and high performance liquid chromatography of cap oligonucleotides have been used to compare terminal sequences in poly(A)-rich RNA from dry and germinating embryos. As is the case in germinating embryos, poly(A)-rich RNA from dry embryos contains only "type 0" cap sequences, i.e., m7G(5')ppp(5')N, in which m7G is the 7-methylguanosine cap and N is any of the classical ribonucleosides: adenosine (A), guanosine (G), cytidine (C),a nd uridine (U). Striking differences between the cell-free translational capacities of bulk messenger RNA (mRNA) populations from dry and germinating embryos are not reflected in signal differences in their proportions of "type 0" cap structures: in general, there is approximately 40% m7G(5')ppp(5')A, with roughly equivalent amounts of m7G(5')ppp(5')G and m7G(5')ppp(5')C accounting for most of the remaining sequences. The findings with mRNA from dry plant embryos serve to emphasize interesting differences between patterns of methylation in the capped and uncapped RNA molecules in higher plants and animals; the differences have not been previously noted in the literature and are the subject of brief comment in this paper.     

Synthesis and assembly of ribulosebisphosphate carboxylase enzyme during greening of barley plants

The synthesis and activation of ribulosebisphosphate carboxylase was studied in etiolated barley leaves during increasing periods of light irradiation. Comparisons were made among enzymatic activity, ¹⁴C-amino acid incorporation into anti-ribulosebisphosphate carboxylase precipitable and 16S protein, and total mass of enzyme. A major portion of newly synthesized anti-ribulosebisphosphate carboxylase specific protein preceded light-induced increase in enzyme activity by a significant period of time. These findings are consistent with a model in which both subunits of ribulosebisphosphate carboxylase are synthesized in response to an early event in greening and subsequently become associated to active oligomeric carboxylase species.     

Growth factor regulated expression of poly(A) binding protein messenger RNA

A 72,000 mol wt protein designated PABP binds to the poly(A)+ track of messenger RNAs with high affinity and has been suggested to play an important role in mRNA metabolism in eucaryotic cells. We have employed a human PABP cDNA probe to study the expression of this gene at the mRNA level in BALB/c3T3 mouse cells under different growth conditions and in exponentially growing HeLa cells throughout the cell division cycle. We describe experiments which establish that in BALB/c3T3 cells the expression of this gene is growth factor regulated. Moreover, the gene behaves like a primary response gene in that its induction in quiescent cells does not require the prior synthesis of other growth factor-regulated proteins. In exponentially growing HeLa cells PABP mRNA is expressed throughout the cell division cycle indicating that the expression of this gene is not limited to a specific phase of the cell cycle.     

Methylated constituents of Aedes albopictus poly (A)-containing messenger RNA.

Poly (A)-containing mRNA prepared from cultured mosquito (Aedes albopictus) cells was found to contain methylated 5'-terminal "caps" as well as internal m6A residues. Both type I [m7G(5')ppp(5')Xmp] and type II [m7G(5')ppp(5')XmpYmp] caps were present, at molar ratio of ca five to one. All four common RNA bases were represented in the second position (Xm) of the caps, adenine being the most abundant and N6-methyladenine being absent. The four bases were also represented in the third position (Ym), but here uracil was the predominant base. There was approximately one internal m6A residue for every three caps. These studies demonstrate that mRNA from an invertebrate source can have a methylation pattern comparable with that of mammalian cells in it complexity.     

The Role of the poly(A) sequence in mammalian messenger RNA

The poly(A) sequence is added to 3' termini of nuclear RNA segments destined to become part of the mRNA, and may play an essential role in the selection of these segments. It appears to be required for at least some of the splicing events involved in mRNA processing. In the cytoplasm, the poly(A) segment is the target of a degradation process which causes its gradual shortening, and leads to a heterogeneous steady-state poly(A)-size distribution. Complete loss of the poly(A) is probably followed by inactivation of the mRNA, since chains depleted of poly(A) do not accumulate in the cells. A role for this sequence in the promotion of mRNA stability is suggested by the behavior of globin mRNA depleted of poly(A) after injection into frog oocytes. The poly(A) shortening process may be part of the mRNA inactivation mechanism, as indicated by the greater sensitivity to degradation of the poly(A) of some short-lived mRNAs. However, the stochastic mRNA decay implies that new and old mRNA chains, with long and short poly(A) segments, respectively are equally susceptible to inactivation. The poly(A)-lacking histone mRNAs are stable only in cells engaged in DNA replication. Present knowledge favors a role for poly(A) in the control of mRNA stability. Loss of this sequence could be controlled through modulation of poly(A)-protein interactions or through masking of a sequence directly adjacent to the poly(A). In the nucleus, the poly(A) sequence could also serve as stabilizing agent, but, in addition, it might interact with the splicing machinery.     

Size of the poly(A) region in mouse globin messenger RNA

A 9S RNA fraction from mouse reticulocytes, containing the active - and -globin mRNAs, has been isolated by hybridization of the polyadenylate regions in the mRNAs to oligo(dT)-cellulose. The adenylate-rich sequence isolated by limited RNase digestion of the globin mRNAs migrates between 4S and 5S RNA standards when co-electrophoresed on 12% polyacrylamide gels. Poly(A) standards, 28 and 84 nucleotides in length, showed anomolous migration relative to the 4S and 5S RNAs. The average size of the adenylate-rich sequence, estimated by its migration relative to the poly(A) standards, is about 50 nucleotides. The polyadenylate stretch in mouse globin mRNA is therefore much shorter than those found in other mRNAs.     

Peptide coding capacity of polysomal and non-polysomal messenger RNA during growth of animal cells.

The major peptides encoded by cytoplasmic RNA preparation translated in vitro in extracts of wheat germ were displayed by two-dimensional electrophoresis on polyacrylamide gels. With this assay system polysomal and non-polysomal RNA preparations were found to differ in coding capacity. These differences tended to be greater in RNA preparations from stationary phase cells than in those from exponential phase cells. These differences were maintained when the concentrations of potassium and magnesium were above or below the optimal concentrations for incorporation. Most of the messenger RNA activities preferentially in the post-polysomal region could be driven into the polysomal region in the presence of cycloheximide. We conclude that these measurements are valid measurements of concentrations of individual functional mRNA species in these RNA preparations.     

A measurement of the sequence complexity of polysomal messenger RNA in sea urchin embryos

The first measurement has been made of the number of diverse mRNA sequences (mRNA sequence complexity) in the total polysomes of a eucaryotic system, the sea urchin gastrula. mRNA was purified of nuclear RNA and any other heterogeneous RNA contaminants by release from polysomes with puromycin. Trace quantities of labeled nonrepetitive DNA fragments were hybridized with an excess of mRNA. The hybridization reaction followed ideal first order kinetics in mRNA concentration. At completion of the hybridization reaction, 1.35% of the nonrepetitive DNA was present as mRNA-DNA hybrid. The hybridized DNA was extracted and was at least 70% hybridizable with mRNA, demonstrating a 50-fold purification of the expressed sequences. This purified DNA fraction reassociated with excess unfractionated sea urchin DNA at a rate identical to that of the total nonrepetitive DNA tracer. The mRNA had therefore been hybridized to nonrepetitive DNA sequence, and the amount of hybrid could be used as a direct measure of the mRNA sequence complexity.The complexity of the gastrula mRNA can be calculated as about 17 million nucleotides, sufficient to comprise some 14,000 distinct structural genes. This result also provides an estimate of the number of diverse proteins being translated in the gastrula. From the rate of mRNA-DNA hybrid formation, we estimate that about 8% of the mRNA belongs to this complex class, and that less than 500 copies of each species of message in this class exist per embryo. Most of the mRNA population consists of a relatively small number of diverse species represented a much larger number of times.