Translational properties of a non-polyadenylated oligo(U)-containing RNA fraction from wheat embryos

A non-polyadenylated oligo(U)-containing RNA (poly(A)- . oligo(U)+ RNA) fraction was isolated from wheat embryo cytoplasm and its properties were compared with those of polyadenylated RNA (poly(A)+ RNA) from the same source. Both RNA preparations were highly heterogeneous and effectively stimulated [14C]leucine incorporation in a wheat germ cell-free translation system. Electrophoretic patterns of the translation products appearing in the non-polyadenylated RNA- and polyadenylated RNA-supplemented translation assays, respectively, differed from each other. The non-polyadenylated RNA-specific translation products included, in particular, a series of high molecular weight polypeptides. It is concluded that a specific class of non-polyadenylated oligo(U)-containing mRNA species (other than histone mRNAs) occurs in the wheat embryo cells.     

Hybridization properties of non-polyadenylated oligo(U)-containing RNA from germinating wheat embryos

• 1.1. Total cytoplasmic RNA of germinating wheat embryos was fractionated by affinity chromatography and separated into non-polyadenylated oligo(U)-containing RNA (A(−)U(+)RNA) and polyadenylated oligo(U)-lacking RNA (A(+)U(−)RNA).
• 2.2. The reassociation kinetics of ³²P-labelled complementary DNA (cDNA) reverse-transcribed from A(−)U(+)RNA shows that this RNA fraction is transcribed from unique DNA sequences of the genome similarly as typical mRNA.
• 3.3. Cross hybridization experiments show no significant sequence homology between the two RNA fractions. Therefore it is concluded that non-polyadenylated oligo(U)-containing RNA of wheat embryo may represent a discrete class of mRNA.

     

Transcription of Ribosomal and Messenger RNAs in Early Wheat Embryo Germination

Germinating wheat embryos (Triticum aestivum L). synthesize both ribosomal and messenger RNA at the earliest times after the onset of germination. The rates of synthesis of these two RNAs are determined at various stages in germination by an analysis of newly synthesized radioactive RNA on oligo(dT)-cellulose. The rate of messenger RNA synthesis is essentially constant throughout 18 hours of germination, while that of ribosomal RNA synthesis increases steadily, particularly after the onset of cell expansion (6 hours), reaching at 16 to 18 hours, a rate of synthesis between 5- and 20-fold greater than that observed at the earliest stages. The net effect is a relative decrease in the fraction of transcribed high molecular weight RNA that is mRNA. Throughout the first 7 hours of germination, mRNA is 25 to 30% of the transcribed fraction, whereas by 16 to 18 hours it has declined to a level of 4 to 8%.     

Covalent circularization of exogenous RNA during incubation with a wheat embryo cell extract

Cell extracts from wheat embryos have been widely used for mRNA-directed protein production. Here, we found that a significant fraction of exogenous linear RNAs are circularized in a wheat embryo extract. The circularization was seen only in uncapped RNAs. The amount of the circular species reached around 1% of the initial RNA and increased along with an increase in the initial concentration more than proportionally. The circular RNAs were stable but unable to be translated in the extract. The circularization was competitively inhibited in the presence of a known substrate of a wheat embryo RNA ligase. Thus, we cloned the RNA ligase cDNAs. Three isoform sequences were homologous to the other plant RNA ligases. An addition of a cell-free synthesized wheat RNA ligase abolished the inhibition, which indicates a participation of its activity in the circularization. A possible role in RNA metabolism, RNA silencing in particular, is discussed.     

Cellular RNA and influenza-virion RNA are synthesized from different pyrimidine-nucleoside-triphosphate pools in chick-embryo cells.

Chick embryo cells infected with an influenza A (fowl plague) virus have been labelled with (3H)-uridine for different lengths of time. Virion RNA and cellular RNA have been separated by specific hybridization with a surplus of unlabelled viral complementary RNA and RNase digestion. The ratio of the specific radioacticity in the UMP and CMP moieties of both types of RNA has been determined. Since the rate of approach to equilibrium of CMP to UMP labelling of both types of RNA is completely different it is concluded that cellular and virion RNA are synthesized using different pyrimidine nucleoside triphosphate pools.     

Uridine 5'-phosphate photohydrate formation in irradiated Escherichia coli 30S ribosomes: photochemistry and relation to ribonucleic acid-protein cross-linking

Irradiation of aqueous buffered solutions of Escherichia coli 30S ribosomes with doses of 254-nm radiation greater than 10(19) quanta causes formation of uridine 5'-phosphate (UMP) photohydrates in ribosomal 16S RNA (rRNA). The number of molecules of UMP photohydrate formed at doses less than 2 x 10(20) quanta is linearly dependent on dose of absorbed 254-nm radiation. Maximum UMP photohydrate formation is dependent on initial ribosome concentration. When solutions containing 1 A260 unit of 30S ribosomes/mL were irradiated with greater than 2 x 10(20) quanta of 254-nm radiation, maximum photohydrate formation was equal to 47 residues/ribosome. Irradiation of solutions containing 2 A260 units/mL with greater than 7 x 10(20) quanta caused formation of 102 UMP photohydrates/ribosome. These values correspond to conversion of either 15 or 33%, respectively, of the total UMP content of 30S ribosome 16S rRNA to photohydrates. Target theory analysis of UMP photohydration in 30S ribosomes showed that UMP photohydrates are formed by single-hit kinetics from two photochemically distinct precursors. Of the total 16S rRNA UMP residues, 10% was included in the most rapidly (low dose) reacting fraction. The respective photohydration cross sections are 0.014 (low dose) and 0.0095 cm2/muEinstein (high dose) for ribosome solutions containing 2 A260 units/mL. UMP photohydrate content of irradiated 30S ribosomes was compared with that of previous data for the extent of RNA-protein cross-linking at equivalent doses of absorbed 254-nm radiation. This comparison showed that at least two UMP photohydrates form per RNA-protein cross-linking event in 30S ribosomes irradiated with a dose of 254-nm radiation (1.5 x 10(19) quanta), which causes cross-linking of only three ribosomal proteins to 16S rRNA.     

Nucleic acid and protein synthesis and loss of vigour in germinating wheat embryos

A study has been made of the RNA and protein synthesising systems of wheat embryos isolated from seed lots having high viability but differing in vigour. The rate of RNA and protein synthesis in wheat embryos during the early hours of germination is related to the vigour of the seed lot. The imposition of a stress factor, in the nature of a sub-optimal germination temperature, during germination of isolated wheat embryos magnifies the differences in rates of protein and RNA synthesis between high and low vigour seed. Using cell-free protein synthesising systems it has been demonstrated that an important difference between high and low vigour embryos lies in the relative levels of messenger RNA in the embryo. High vigour embryos contain relatively higher levels of poly A⁺-RNA (i.e. potential mRNA species) than lower vigour embryos and furthermore the level of poly A⁺-RNA in high vigour embryos increases during early germination whilst in lower vigour embryos the level decreases. The difference in poly A⁺-RNA levels accounts, at least partially, for the differences in rates of protein synthesis observed between embryos from high and low vigour wheat seed during early germination at both optimal and sub-optimal germination temperatures.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - poly A⁺-RNA polyadenylated RNA - GM germination medium - PMS post-mitochondrial supernatant fraction     

Kinetic Changes of Nucleic Acids and Protein During Embryogenesis of Wheat (Triticun vulgaris L.)

Per embryonic total nucleic acid, RNA content and per cell RNA content increased during embryogenesis, reached maximun at 21 day after anthersis. The per embryo and per cell protein content also increased concomitantly. But the protein content continued to increase up to 24 days after anthersis. On the basis of dry weight, RNA content decreased in the early stage of embryogenesis, but then increased over the period of later developmental stage. The protein content on the basis of dry weight also changed in similar way. It was likely the protein and RNA content changes concomitantly during the developmental process of wheat embryo. As to per embryo DNA content, it increased in early developmental stage, but then remained in a similar level during the later stage. The relationship between the changes of RNA content and protein synthesis, embryonie develope is also discussed in present paper.     

A highly specific terminal uridylyl transferase modifies the 3'-end of U6 small nuclear RNA.

HeLa cell extracts contain significant amounts of terminal uridylyl transferase (TUTase) activity. In a template-independent reaction with labeled UTP, these enzymes are capable of modifying a broad spectrum of cellular RNA molecules in vitro . However, fractionation of cell extracts by gel filtration clearly separated two independent activities. In addition to a non-specific enzyme, an additional terminal uridylyl transferase has been identified that is highly specific for cellular and in vitro synthesized U6 small nuclear RNA (snRNA) molecules. This novel TUTase enzyme was also able to select as an efficient substrate U6 snRNA species from higher eucaryotes. In contrast, no labeling was detectable with purified fission yeast RNA. Using synthetic RNAs containing different amounts of transcribed 3'-end UMP residues, high resolution gel electrophoresis revealed that U6 snRNA species with three terminal U nucleotides served as the optimal substrate for the transferase reaction. The 3'-end modification of the optimal synthetic substrate was identical to that observed with endogenous U6 snRNA isolated from HeLa cells. Therefore, we conclude that the specific addition of UMP residues to 3'-recessed U6 snRNA molecules reflects a recycling process, ensuring the functional regeneration for pre-mRNA splicing of this snRNA.     

Changes in Soluble RNA and Ribonuclease Activity During Germination of Wheat

Gross changes in protein and nucleic acid were studied in germinating wheat seeds. The nucleic acid fraction was separated on columns of methylated albumin-keiselguhr. It was found that more than 50% of the transfer RNA was lost from the embryo in the first 10 to 15 hours of germination. This was followed by a period of rapid resynthesis of transfer RNA, to the normal level at about 20 hours. The decline and increase in transfer RNA was accompanied by a change in the ratios of certain amino acid acceptor species. Evidence is also presented that an embryo ribonuclease is lost during the first 10 to 15 hours, followed by the appearance of a second seedling ribonuclease between 15 and 30 hours of germination.     

Biochemical characterization of a U6 small nuclear RNA-specific terminal uridylyltransferase.

The HeLa cell terminal uridylyltransferase (TUTase) that specifically modifies the 3'-end of mammalian U6 small nuclear RNA (snRNA) was characterized with respect to ionic dependence and substrate requirements. Optimal enzyme activity was obtained at moderate ionic strength (60 mm KCl) and depended on the presence of 5 mm MgCl2. In vitro synthesized U6 snRNA without a 3'-terminal UMP residue was not accepted as substrate. In contrast, U6 snRNA molecules containing one, two or three 3'-terminal UMP residues were filled up efficiently, generating the 3'-terminal structure with four UMP residues observed in newly transcribed cellular U6 snRNA. In this reaction, the addition of more than one UMP nucleotide depended on higher UTP concentrations. The analysis of internally mutated U6 snRNA revealed that the fill-in reaction by the U6-TUTase was not controlled by opposite-strand nucleotides, excluding an RNA-dependent RNA polymerase mechanism. Furthermore, electrophoretic mobility-shift analyses showed that the U6-TUTase was able to form stable complexes with the U6 snRNA in vitro. On the basis of these findings, a protocol was developed for affinity purification of the enzyme. In agreement with indirect labeling results, PAGE of a largely purified enzyme revealed an apparent molecular mass of 115 kDa for the U6-TUTase.     

Wheat Storage Proteins : ISOLATION AND CHARACTERIZATION OF THE GLIADIN MESSENGER RNAs

A total RNA extract was prepared from developing wheat seeds using guanidine-HCl to eliminate endogenous RNase activity. The RNA preparation, substantially free of protein, carbohydrate and DNA, was chromatographed on either a poly uridylic acid-agarose or poly guanylic acid-agarose column to yield a gliadin-enriched mRNA fraction. Only slight differences were observed for the products synthesized in a wheat germ cell-free translation system when either poly adenylic acid-enriched or cytosine-rich RNA was used as a template. These results are consistent with the high proline content of the gliadins and indicate that a large proportion of the mRNA activity in these RNA preparations is directed toward gliadin synthesis. After a second affinity chromatography step, the gliadin-enriched mRNA fraction was fractionated by two cycles on sucrose-density gradient centrifugation under denaturing conditions. The RNA sedimented as a broad band with a peak at 14S and a shoulder at the 11S region of the sucrose gradient. RNA from the peak 14S fraction translated predominantly the two major gliadin polypeptides which had molecular weights of 34,000 and 36,000. Analysis of the 14S RNA by methylmercury hydroxide-agarose gel electrophoresis revealed the presence of a predominant RNA species with a molecular size of 415,000 (1,200 nucleotides).     

Developmental changes in the activity of messenger RNA isolated from germinating castor bean endosperm

The capacity of polyadenylated RNA from developing castor bean endosperm to program protein synthesis in a wheat germ cell-free translational system has been examined. Although the use of micrococcal nuclease-treated wheat germ extracts demonstrated a low but significant content of translatable mRNA in dry seeds, a large scale increase in total translational capacity was observed during germination. The cellular content of translatable mRNA peaked on the 4th day of germination and subsequently declined. It is concluded that protein synthesis in castor bean endosperm cells during germination is directed by newly transcribed mRNA.     

Stimulation of encephalomyocarditis virus RNA translation in wheat embryo extracts by rabbit reticulocyte ribosomal wash.

Encephalomyocarditis virus RNA induced a low level of amino acid incorporation in wheat embryo extracts, but the incorporation did not result in the formation of any viral proteins. However, if wheat embryo extracts were supplemented with a ribosomal wash fraction from rabbit reticulocytes, a large stimulation of amino acid incorporation and the formation of encephalomyocarditis virus-specific proteins were obtained. Among the proteins synthesized were capsid precursor proteins which can be processed by an encephalomyocarditis virus-coded protease to produce the viral capsid proteins.     

Characterization of the U3 and U6 snRNA genes from wheat: U3 snRNA genes in monocot plants are transcribed by RNa polymerase III

We have demonstrated recently that the genes encoding the U3 small nuclear RNA (snRNA) in dicot plants are transcribed by RNA polymerase III (pol III), and not RNA polymerase II (pol II) as in all other organisms studied to date. The U3 gene was the first example of a gene transcribed by different polymerases in different organisms. Based on phylogenetic arguments we proposed that a polymerase specificity change of the U3 snRNA gene promoter occurred during plant evolution. To map such an event we are examining the U3 gene polymerase specificity in other plant species. We report here the characterization of a U3 gene from wheat, a monocot plant. This gene contains the conserved promoter elements, USE and TATA, in a pol III-specific spacing seen also in a wheat U6 snRNA gene characterized in this report. Both the U3 and the U6 genes possess typical pol III termination signals but lack the cis element, responsible for 3-end formation, found in all plant pol II-specific snRNA genes. In addition, expression of the U3 gene in transfected maize protoplasts is less sensitive to -amanitin than a pol II-transcribed U2 gene. Based on these data we conclude that the wheat U3 gene is transcribed by pol III. This observation suggests that the postulated RNA polymerase specificity switch of the U3 gene took place prior to the divergence of angiosperm plants into monocots and dicots.