The cytoplasmic 4S translation inhibitory RNA species of chick embryonic muscle: fractionation of biologically active subspecies by high performance liquid chromatography

A 4S RNA species (iRNA) isolated from chick embryonic muscle which is a potent inhibitor of mRNA translation in vitro shows heterogeneity in the 70-100 nucleotide size range (Sarkar, S., Mukherjee, A.K., and Guha, C. (1981) J. Biol. Chem., 256, 5077-5086). The iRNA was fractionated by HPLC on different size exclusion columns using a variety of elution conditions. Chromatography of iRNA on a TSK 4000 SW column and elution with a low ionic strength buffer gave three components, one of which contained a pure subspecies of about 90-100 nucleotides size, as shown by a single band on PAGE analysis in 99% formamide. The biological activity of this purified subspecies showed that this is a more potent inhibitor of globin mRNA translation than unfractionated iRNA (Sarkar, S., Mukherjee, A.K., and Guha, C., (1981) J. Biol. Chem. 256, 5077-5086). Partial resolution of three additional low molecular weight iRNA subspecies in the 70-80 nucleotide size range in biologically active form was obtained on chromatography of unfractionated iRNA on TSK 4000 SW column in the presence of 0.5 M NaCl or on TSK 3000 SW column in the presence of low salt. The fractionation of iRNA by HPLC appears to be primarily based on size. These results strongly suggest that HPLC may also be useful for the fractionation of a variety of low molecular weight eukaryotic nuclear and cytoplasmic RNAs with retention of biological activity.     

The cytoplasmic 4 S translation inhibitory RNA species of chick embryonic muscle. Effect on mRNA binding to 43 S initiation complex

A cytoplasmic 10 S ribonucleoprotein (iRNP) isolated from chick embryonic muscle is a potent inhibitor of mRNA translation in vitro and contains a 4 S translation inhibitory RNA species (iRNA) (Sarkar, S., Mukherjee, A. K., and Guha, C. (1981) J. Biol. Chem. 256, 5077-5086). Using an in vitro assay system, we show that the iRNA has no effect on the elongation phase of peptide synthesis. iRNA inhibits translation at the initiation step by inhibiting mRNA binding to 43 S initiation complexes. The iRNA does not inhibit the binding of Met-tRNAf to the 40 S ribosomal subunit, but rather causes an increase in the level of 43 S initiation complexes in the reticulocyte lysate. The formation of the 80 S initiation complex from the 43 S complex is specifically blocked in the presence of iRNA. The significance of these results in relation to biological function of iRNA is discussed.     

Regulation of in vitro translation by double-stranded RNA in mammalian cell mRNA preparations.

Polyadenylated mRNA has been purified from a variety of human and mouse cell sources. These preparations are actively translated in the wheat germ cell-free system but have only poor ability to stimulate the nuclease-treated reticulocyte lysate. The translation of endogenous and exogenous globin mRNA is strongly inhibited by the poly(A)+ RNA preparations in reticulocyte lysates. Both polysomal and non-polysomal RNA have similar effects but poly(A)+ RNA is almost 2000-fold more inhibitory than poly(A)-RNA on a weight basis. The inhibition is abolished in the presence a high concentration of poly(I).poly(C). Analysis of endogenous eIF-2 in the lysate reveals that the subunit becomes extensively phosphorylated in the presence of the inhibitory poly(A)+ RNA. Prolonged incubation of lysate with poly(A)+ RNA also causes some nucleolytic degradation of polysomal globin mRNA. These characteristics suggest that some eukaryotic cell mRNAs contain regions of double-stranded structure which are sufficiently extensive to activate translational control mechanisms in the reticulocyte lysate.     

Isolation and partial purification of ceruloplasmin messenger RNA from rat liver

Partially purified ceruloplasmin mRNA was isolated using indirect immunoprecipitation of rat liver polysomes and poly(U)-Sepharose chromatography of polysomal RNA. This RNA programmed the synthesis of ceruloplasmin polypeptides in a cell-free system from mitochondria. Immunochemical analysis of the translation products revealed a 40-fold enrichment of the ceruloplasmin mRNA activity. The purified ceruloplasmin mRNA migrated as a major homogeneous component with an apparent molecular weight about 1×10⁶ daltons in polyacrylamide gels containing sodium dodecyl sulfate. The immunoprecipitated products of the cell-free translation had molecular weights in the range 4.5–5.4×10⁴ daltons as estimated by gel-electrophoresis under denaturating conditions. These values approach the weight of the half-molecule of native ceruloplasmin.     

Translational control by messenger RNA competition for eukaryotic initiation factor 2

Translation of globin mRNA in a micrococcal nuclease-treated reticulocyte lysate was studied in the presence of increasing amounts of Mengovirus RNA, under conditions in which the number of translation initiation events remains constant as judged by the transfer of label from N-formyl[35S]methionyl-tRNAf into protein. The translation of globin mRNA is progressively inhibited by low concentrations of Mengovirus RNA, free of detectable traces of double-stranded RNA, concomitant with the increasing synthesis of Mengovirus RNA-directed products. On a molar basis, Mengovirus RNA apparently competes about 35 times more effectively than globin mRNA for a critical component in translation. The competition is relieved by the addition of highly purified eukaryotic initiation factor 2 (eIF-2). Addition of eIF-2 does not stimulate overall protein synthesis, but shifts it in favor of globin synthesis. No stimulation of globin mRNA translation by eIF-2 is seen when Mengovirus RNA is absent. These experiments show that Mengovirus RNA competes, directly or indirectly, with globin mRNA for eIF-2. In direct binding experiments using isolated mRNA and eIF-2, Mengovirus RNA is shown to compete with globin mRNA for eIF-2 and to exhibit a 30-fold higher affinity for this factor. The binding of Mengovirus RNA to eIF-2 is much more resistant to increasing salt concentrations than is the binding of globin mRNA, again reflecting its high affinity. These results reveal a direct correlation between the ability of these mRNA species to compete in translation and their ability to bind to initiation factor eIF-2. They suggest that the affinity of a given mRNA species for eIF-2 is essential in determining its translation, relative to that of other mRNA species. Messenger RNA competition for eIF-2 may contribute significantly to the selective translation of viral RNA in infected cells.     

Poly(A) dependent translation in rabbit reticulocyte lysate

Poly(A) tail has been known to enhance mRNA translation in eukaryotic cells. However, the effect of poly(A) tail in vitro is rather small. Rabbit reticulocyte lysate (RRL) is widely used for studying translation in vitro. Translation in RRL is typically performed in nuclease-treated lysate in which most of the endogenous mRNA have been removed. In this condition, the difference in the translational efficiency between poly(A)⁺ and poly(A)⁻ mRNAs is about two-fold. We studied the effect of poly(A) tail on luciferase mRNA translation in nuclease uncreated reticulocyte lysate, in which endogenous globin mRNAs were actively translated. In the case of capped mRNAs. stimulation of translation by poly(A) addition was about 1.5- to 1.6-fold and the effect of the poly(A) length was small. However, in the case of uncapped mRNAs, the addition of poly(A) tail increased luciferase expression over 10-fold. The effect of the poly(A) tail was dependent on its length. The difference in the translational efficiency was not due to the change of mRNA stability. These data indicate that RRL has the potential to translate mRNA in a poly(A) dependent manner.     

Polysomal and non-polysomal messenger RNA in neuroblastoma cells: Lack of correlation between polyadenylation or initiation efficiency and messenger RNA location

Neuroblastoma cytoplasm was fractionated on sucrose gradients into polysomes (>90 S) and non-polysomal particles (<90 S). Purified RNA from these fractions was translated using a wheat germ lysate and translation products were compared by two-dimensional gel electrophoresis. Non-polysomal messenger RNA directed the synthesis of a specific subset of polysomal mRNA translation products. Careful comparison of individual translation products demonstrated that specific mRNAs were not randomly distributed between polysomes and the non-polysomal fraction.Fractionation of both RNA populations into polyadenylated (poly(A)⁺) and non-adenylated (poly(A)^?) species indicated that specific, abundant non-polysomal mRNAs were not less adenylated than their polysomal counterparts. Furthermore, comparison of translation products from assays of subsaturating and supersaturating RNA concentrations demonstrated that no simple correlation could be made between the relative initiation efficiency of a specific mRNA and its distribution between polysomes and non-polysomal particles.     

In vivo degradation of rat globin messenger RNA during maturation of reticulocytes.

The degradation of globin mRNA in rat reticulocytes maturing in the peripheral blood was investigated. Poly(A) and non poly(A) portions of mRNA molecules were determined quantitatively by hybridization with radioactive poly(U) and complementary DNA, respectively. During the degradation of mRNA in vivo, it was shown that (1) globin mRNA and the bulk of RNA decrease in parallel, (2) the average chain length of poly(A) segments in the mRNA does not change, (3) the percentage of poly(A) (-) globin mRNA in total globin mRNA does not change, and (4) fragments of large molecular weight do not accumulate. Possible mechanisms of degradation of globin mRNA in the reticulocytes are discussed on the basis of these observations.     

Isolation of globin messenger RNA of Xenopus laevis

The polypeptide chains of Xenopus laevis hemoglobin have been analyzed by sodium dodecyl sulfate (SDS) and acid-urea gel electrophoresis. Four components can be distinguished, each having an approximate molecular weight of 13,000 daltons. Messenger RNA coding for the globin chains has been isolated and characterized. In a denaturing acrylamide gel the mRNA has an approximate molecular weight of 250,000 daltons. The complexity of the RNA is consistent with the presence of four different mRNA molecules, each of this molecular weight. When the mRNA is assayed in a wheat germ in vitro translation system, four polypeptides are synthesized corresponding to the four globin subunits. The relative proportion of the four synthesized polypeptides appears to vary according to the developmental stage of the red blood cells used for mRNA isolation. Hybridization of a complementary DNA (cDNA) copy of the globin mRNA to Xenopus laevis DNA in DNA excess indicates that each of the globin genes is present in one to three copies per haploid genome.     

Inhibition of protein synthesis in reticulocyte lysates by a double-stranded RNA component in HeLa mRNA

Double-stranded RNA (dsRNA) inhibits protein synthesis initiation in rabbit reticulocyte lysates by the activation of a latent dsRNA-dependent cAMP-independent protein kinase which phosphorylates the α-subunit of the eukaryotic initiation factor eIF-2. In this study, we describe a dsRNA-like component which is present in preparations of HeLa mRNA (poly A⁺) isolated from total cytoplasmic RNA. The inhibitory species in the HeLa cytoplasmic mRNA was detected by (a) its ability to inhibit protein synthesis with biphasic kinetics in reticulocyte lysates translating endogenous globin mRNA, and (b) by the inefficient translation of HeLa cytoplasmic mRNA in a nuclease-treated mRNA-dependent reticulocyte lysate. The inhibitory component was characterized as dsRNA by several criteria including (i) the ability to activate the lysate dsRNA-dependent eIF-2α kinase (dsI); (ii) the prevention of both dsI activation and inhibition of protein synthesis by high levels of dsRNA or cAMP; (iii) the reversal of inhibition by eIF-2; and (iv) the inability to inhibit protein synthesis in wheat germ extracts which lack latent dsI. By the same criteria, the putative dsRNA component(s) appears to be absent from preparations of HeLa mRNA isolated exclusively from polyribosomes.     

Cell-free translation analysis of messenger RNA in echinoderm and amphibian early development

A wheat germ cell-free translation system has been used to analyze populations of abundant messenger RNA from sea urchin eggs and embryos and from amphibian oocytes and ovaries. We show directly that sea urchin eggs and embryos contain translatable mRNA of three general classes: poly(A)⁺ mRNA, poly(A)^? histone mRNA, and poly(A)^? nonhistone mRNA. Additionally, some histone synthesis appears to be promoted by poly(A)⁺ RNA. Sea urchin eggs seem to contain a higher proportion of prevalent poly(A)^? nonhistone mRNAS than do embryos. Some differences in the proteins encoded by poly(A)⁺ and poly(A)^? RNAs are detectable. Many coding sequences in the egg appear to be represented in both poly(A)⁺ and poly(A)^? RNAs, since the translation products of the two RNA classes exhibit many common bands when run on one-dimensional polyacrylamide gels. However, some of this overlap is probably due to fortuitous comigration of nonidentical proteins. Distinct stage-specific changes in the spectra of prevalent translatable mRNAs of all three classes occur, although many mRNAs are detectable throughout early development. Particularly striking is the presence of an egg poly(A)^? mRNA, encoding a 70,000–80,000 molecular weight protein, which is not detected in morula or later-stage embryos. In amphibian (Xenopus laevis and Triturus viridescens) ovary RNA, the translation assay detects the following three mRNA classes: poly(A)⁺ nonhistone mRNA, poly(A)^? histone mRNA, and poly(A)⁺ histone mRNA. Amphibian ovary RNA appearently lacks an abundant poly(A)^? nonhistone mRNA component of the magnitude detectable in sea urchin eggs. mRNA encoding histone-like proteins is found in the very earliest (small stage 1) oocytes of Xenopus as well as in later stage oocytes. During oogenesis there appear to be no striking qualitative changes in the spectra of prevalent translatable mRNAs which are detected by the cell-free translation assay.     

Translational control of globin chain ontogeny in hamster yolk sac erythroid cells.

Prior research has demonstrated that globin ontogeny of hamster proceeds nearly to completion during the several days that yolk sac erythroid cells (YSEC) circulate in the embryo; synthesis of embryonic globin chains gives way to synthesis of adult globin chains in these primitive cells. In the present study, we translated total cell RNA extracted from YSEC on days 9-13 of gestation in wheat germ cell-free extract, expecting to observe the same progressive rise that occurs in vivo in rates of translation of alpha- and beta-globin mRNA during ontogeny. The opposite occurred; translation rates of both globins decreased sharply. This disparity between synthesis of alpha- and beta-globins in vivo and in vitro suggested an element of control of translation attributable to the YSEC cytoplasm. We therefore assayed the effect of RNA-free clarified YSEC cytoplasm on cell-free translation of YSEC RNA. A repression of translation was detected of alpha- and beta-globin mRNA (not of embryonic globin mRNA), exercised strongly by cytoplasm from YSEC early in ontogeny (gestational day 9), and weakening as ontogeny progressed. The same effect was noted on alpha- and beta-globin mRNA of adult hamster and of rabbit. Heat treatment of cytoplasm abolished the greater part of the translation regulation, suggesting that the active agent is protein. Further characterization of this translational regulator included: (a) it binds to globin poly(A) mRNA but not to poly(A), (b) it was not detected in cell lysate of adult hamster brain, lung, or erythrocytes, and (c) it did not inhibit translation of adult hamster brain and liver RNA. We conclude that hamster globin ontogeny is substantially modulated by this translational regulation of alpha- and beta-globin expression.     

Evidence that XR family interspersed RNA may regulate translation in Xenopus oocytes

It has been shown that about two thirds of Xenopus oocyte or sea urchin egg cytoplasmic poly(A)⁺ RNA contains interspersed repetitive sequences. The functional significance of this interspersed RNA has remained unknown. Here the function of a subfamily of interspersed RNA (XR family; McGrew and Richter, 1989: Dev Biol 134:267–270) in Xenopus oocytes was studied. We found that the elimination of T7 XR (one of the two complementary strands of the XR repeat) interspersed RNA by complementary oligodeoxynucleotides significantly inhibited protein synthesis. On the other hand, the injection of in vitro synthesized T7 XR RNA stimulated translation. Moreover, the insertion of the T7 XR RNA sequence into globin mRNA repressed the translation of the globin mRNA. In order to explain these results, we analyzed interactions between the XR interspersed RNA and oocyte proteins. We found that the major XR RNA binding proteins were p56 and p60, which could be the known mRNA “masking” proteins that bind mRNA and inhibit translation. Further, a 42 kD protein has been identified that appears to bind T7 XR RNA relatively specifically, although it interacts with mRNA with a lower affinity. Based on all of these data, we have proposed that interspersed RNA may be involved in regulating translation by competing with mRNA to interact with certain proteins that can regulate translation. © 1995 Wiley-Liss, Inc.     

Regulation of messenger RNA stability in mouse erythroleukemia cells

The decay rates of several messenger RNA species were determined in mouse erythroleukemia cells. The t1/2 values for the actin and tubulin mRNAs were 16 to 26 hours and about seven hours, respectively. The globin mRNA, and two mRNA species subject to translation repression, the P40 and P21 mRNAs, were about as stable as the ribosomal RNA. A stable tubulin mRNA component also appeared to be present in the cells. Exposure of the cells to dimethylsulfoxide for 48 hours led to considerable increases in the rates of decay of all but the globin mRNA. The induction of erythroid differentiation caused by the drug appears to lead to activation of a mRNA-degradation process that affects individual species to different degrees. The newly synthesized actin and tubulin mRNAs lost their poly(A) rather rapidly. This was accompanied by accumulation of poly(A)-deficient mRNA chains, particularly in the case of actin mRNA. The steady-state distribution of mRNA components, determined by Northern blot analysis, also showed that the actin mRNA and one tubulin mRNA species have a high proportion of poly(A)-deficient molecules. The globin, P40 and P21 mRNAs showed little tendency to lose their poly(A) sequence. The steady-state globin and P40 mRNAs also had a low proportion of chains depleted of poly(A). For all five species, the proportions of poly(A)-deficient chains in newly synthesized mRNA were about the same in uninduced and induced cells, in spite of the large decreases in mRNA stability in the induced cells. The lack of correlation between tendency to lose poly(A) and rate of mRNA decay, and the large accumulation of poly(A)-deficient molecules in the cases of the actin and tubulin mRNAs suggest that the stability of mRNA is not determined solely by the presence of poly(A) on the RNA chains. The behavior of the untranslated species in induced and uninduced cells also fails to support the notion of a relationship between translation and mRNA decay.     

Partial purification and characterization of the mRNA for alpha-amylase from barley aleurone layers

The poly(A)-containing mRNA from barley aleurone layers pretreated with gibberellic acid has been purified by phenol-chloroform extraction and repeated oligo[d(pT)]-cellulose chromatography. This RNA has been translated in both the wheat germ and reticulocyte lysate in vitro translation systems with greater than 50% of the synthesized protein being α-amylase. The mRNA for α-amylase has been further purified by dimethylsulfoxide-formamide-sucrose density gradient centrifugation and by gel electrophoresis. By these methods, its molecular weight has been determined to be 580,000.     

Changes in translatable poly(A) RNA from differentiated potato tissues transformed with shoot-inducing Ti TL-DNA of Agrobacterium tumefaciens

Summary Two dimensional gel electrophoresis was used to examine differences in steady state total poly(A) RNA from untransformed potato (Solanum tuberosum cv. Maris Bard) and potato transformed with shoot-inducing T_L-DNA from A. tumefaciens. RNA was compared from phenotypically very distinct in vitro cultured shoots, more similar grafted plants and tubers. In each case between 200–400 translation products were identified representing the more abundant poly(A) mRNA's. In general, poly(A) RNA from the transformed tissues gave more high molecular weight products. This increase was most evident in poly(A) RNA from shoot cultures. Depending on the tissue examined, 1–5% of the translation products with a molecular weight <43 KD were observed to increase or decrease in abundance. The influence of T-DNA on cellular gene expression in the different transformed potato tissues is discussed in relation to previously determined changes in T-DNA gene expression (particularly of the T-DNA cytokinin gene) and the corresponding changes in endogenous hormone concentrations. It is concluded that some of the specific changes in low molecular weight products are either directly caused by the increased cytokinin levels or are indirectly involved in maintaining the transformed phenotype. re]19850530 rv]19851206 ac]19851210     

Cell-free translation of messenger RNA from oocytes of Xenopus laevis

The poly(A)⁺ RNA which accumulates during oogenesis in the amphibian Xenopus laevis is shown to be functional mRNA; the RNA was active in the mRNA-dependent “shift assay” for initiation sites in the rabbit reticulocyte lysate, and was an efficient template for protein synthesis in the wheat-germ cell-free system. Analysis of the in vitro protein products showed no differences between the coding properties of poly(A)⁺ RNA extracted from oocytes at all stages of development from previtellogenesis to maturity. In previtellogenic oocytes, the in vitro products of polysomal and of mRNP-associated poly(A)⁺ RNA were also identical. Neither was there any evidence for changes in the coding properties of the poly(A)⁺ mRNA of the oocyte. However, the patterns of oocyte in vivo protein synthesis changed markedly during early vitellogenesis. We conclude that the mRNP-associated poly(A)⁺ RNA present in mature oocytes constitutes the stored maternal mRNA, and that during oogenesis the coding composition of the poly(A)⁺ mRNA synthesised does not change markedly, while some form of translational control operates to direct the changing pattern of protein synthesis.