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Messenger RNA competition experiments were performed in an mRNA-dependent reticulocyte lysate using three kinds of mRNA: rabbit globin mRNA, calf eye lens mRNA and RNA of turnip yellow mosaic virus. Our results indicate that at supersaturating concentrations of mRNA preferential translation of certain mRNA species can be observed. Furthermore, the pattern of mRNA selection by the translational apparatus suggests that the rate of translation of different mRNA species is limited by different components of the reticulocyte lysate. Our observations n the cell-free system are strikingly different from our previously published mRNA competition experiments in Xenopus oocytes using the same preparations of lens and globin mRNA, in which no selective translation was observed [Asselbergs et al., Eur. J. Biochem. 94, 249-254 (1979)]. The restraints on mRNA translation in vitro are apparently different from those in vivo, i.e. in oocytes.

In this study irradiation of intact cells with 244-nm ultraviolet light was used to cross-link hnRNA to proteins that are closely associated with it. In this way proteins interacting specifically with hnRNA could be identified excluding the possibility of non-specific binding of proteins to the RNA during cell fractionation. In uninfected HeLa cells two polypeptides of 41500 and 43000 molecular weight can be cross-linked very efficiently to hnRNA. Other proteins, with molecular weights of 36000 and 37000, were covalently linked less effectively. Irradiation of adenovirus-infected cells results in the cross-linking of the same polypeptides to hnRNA. It was found, however, that hnRNA from adenovirus-infected cells contains both viral and host transcripts. Both classes of transcripts are quantitatively associated with the nuclear matrix and can be cross-linked by irradiation with equal efficiency. To determine whether both adenoviral and cellular transcripts are associated with the same proteins, the cross-linked adenoviral hnRNA-protein complexes were isolated by preparative hybridization to adenoviral DNA immobilized on Sepharose. The results show that the purified cross-linked adenoviral hnRNA-protein complexes also contain the host 41500-Mr and 43000-Mr proteins as major components. This suggests that in the infected cell adenoviral-specific hnRNA is associated with host proteins and that the structural organization of viral hnRNA-protein complexes in infected cells probably is similar to the organization of host hnRNA in uninfected cells.

Ehrlich ascites tumor cells were irradiated with ultraviolet light to cross-link intracellular RNAs to their tightly bound proteins. The efficiency of such cross-linking in vivo was measured by two independent methods, namely by phenol/chloroform extraction and by isopycnic centrifugation. After short irradiation times more than 70% of the ribosomal RNAs and more than 80% of the mRNA were found to be cross-linked to protein. The size of the cross-linked RNAs was essentially unchanged, indicating that the irradiation does not lead to extensive fragmentation of the RNA. Covalent poly(A)-containing mRNA-protein complexes were isolated by oligo(dT)-cellulose chromatography in the presence of sodium dodecylsulphate. The complexed proteins were freed from RNA by extensive nuclease degradation and analysed by polyacrylamide gel electrophoresis. The results show that some specific proteins are associated with poly(A)-containing RNA in vivo.

Native large ribosomal subunits in cultured Ehrlich ascites honor cells analyzed by high-resolution CsCl isopycnic centrifugation consist of at least two classes of particles with densities of 1.57 g/cm3 (L1) and 1.59 g/cm3 (L11), respectively. A wash with 0.5 M KCI converts L1 into particles with the density of L11 particles. Incubation of derived large subunits (density 1.59 g/cm3) with 0.5 M KCI wash of reticuiocyte ribosomes leads to the formation of particles with the density of L1 particles. A protein with a molecular weight of 57000 present in the high-KCl wash of 60-S native subunits was virtually absent in the KCI wash of 40-S subunits or polyribosomes suggesting that specific protein factors may be present on some native 60-S subunits. Possible functions of these protein factors are discussed.

HeLa cells, prelabeled with [3H]leucine for several generations, were pulse-labeled with [14C]-leucine. The 14C/3H ratio of the leucine in the medium, in the total amino acid pool and bound to transfer RNA was determined. It was found that the 14C/3H ratio of the leucyl-tRNA was identical with that of the extracellular medium while that of the amino acid pool was much lower. These results confirm previous reports that selection of amino acids for protein synthesis takes place before the amino acids are released intracellularly. Further, it was concluded that amino acids resulting from intracellular proteolysis are not reutilized directly under the conditions of our experiments. A possible model of intracellular amino acid pathways is discussed.

Lens mRNA was translated in reticulocyte lysate predominantly into monomeric alpha-crystallin chains. Lens polyribosomes added to the cell-free system produced the same polypeptides, but these were detected predominantly in alpha-crystallin aggregates. Lens mRNA, after microinjection into Xenopus laevis oocytes, produced alpha-crystallin subunits that were exclusively found in the form of high-molecular-weight complexes. Also after injection of the purified 14-S mRNA, coding for the alphaA subuint, the synthesized alpha-A polypeptides were incorporated into high-molecular-weight aggregates. In contrast, the synthesis of alphaB subunits, directed by a 10-S mRNA, did not result in aggregate formation. The experiments thus suggest that aggregate formation of alpha-crystallin is triggered by its alphaA subunits, which are then joined by the alphaB subunits. This process occurs partly in the cell-free system and completely in Xenopus oocytes.

Native small ribosomal subunits in cultured Ehrlich ascites tumor cells, analyzed by high-resolution CsCl isopycnic centrifugation, consist of at least five classes of particles. These particles with buoyant densities of 1.39, 1.42, 1.45, 1.49 and 1.51 g/cm3 were designated as SI, SII, SIII, SIV and SV, respectively. They were different from the ribosome-derived 40-S subunits which have a density of 1.54 g/cm3. Native large ribosomal subunits consist of at least two classes of particles with densities of 1.57 g/cm3 (LI) and 1.59 g/cm3 (LII), respectively. The ribosome-derived 60-S subunits have the same buoyant density as LII particles. Labeling of Ehrlich ascites cells with radioactive uridine and a subsequent chase in the presence of RNA-synthesis inhibitors shows that radioactivity was incorporated first in precursor particles with a density of 1.48 g/cm3 and then subsequently appeared in SIII and SII particles. Met-tRNAf is found exclusively on native 40-S particles with densities of 1.42 and 1.49 g/cm3. This result has been observed in cells labeled with [35S] methionine in vivo as well as with tRNA charged in vitro. The possibility that SII particles contain 40-S initiation complexes is discussed

The membrane-bound polyribosomes in Ehrlich ascites tumor cells can be separated into a loosely bound and a tightly bound fraction by means of a high salt treatment. Both membrane fractions as well as the free polyribosomes in the supernatant synthesize about the same set of proteins, suggesting a close relationship between these polyribosome fractions in the Ehrlich cell. Relatively high concentrations of cycloheximide do not prevent newly synthesized poly(A)-containing mRNA from entering the tightly bound polyribosome fraction. Nor had treatment of the cells with puromycin in the presence of cycloheximide, which released about 70% of the nascent chains, any significant effect on the entrance of newly synthesized mRNA into tightly bound polyribosomes. These results suggest that in Ehrlich ascites tumor cells nascent polypeptide chains are not involved in the binding of polyribosomes to membranes.

In a nuclease-treated reticulocyte lysate reinitiation of protein biosynthesis was less inhibited by 7-methylguanosine 5′-monophosphate (m7GMP), whereas primary-initiation could be totally blocked by cap analogues. We suggest that a cap binding factor binds to mRNA as a first step in initiation of protein synthesis and that this factor remains bound during the subsequent reinitiation cycles. Primarily because of the greater relative importance of the reinitiation process, globin synthesis in the untreated lysate was less inhibited by m7GMP.

When calf lens crystallin mRNA and rabbit globin mRNA are competing for factors limiting protein synthesis in living Xenopus oocytes, no mRNA species is preferentially selected for translation. Differences in the intrinsic translational efficiency of the mRNA species exist, but the relative efficiencies are the same at high and low mRNA concentrations. mRNAs already being translated, in particular endogenous oocyte mRNAs, are less sensitive to competitive inhibition by injected mRNAs. As injected mRNAs gradually become incorporated into the protein-synthesizing machinery of the oocyte, they acquire the same status as the oocyte's own active mRNAs. Exogenous mRNAs this become endogenous mRNAs. These results, together with previous estmates of the translational efficiency of injected heterologous mRNA species, are compatible with the assumption that a large proportion of the endogenous mRNAs is not competing for the translational apparatus of the oocyte and, therefore, probably is present in the temporarily inactivated form.