Resistance of bacterial protein synthesis to double-stranded RNA

Double-stranded RNA fails to inhibit the formation of translation initiation complexes on R17 bacteriophage RNA, overall synthesis of R17 proteins, or the ability of bacterial initiation factor IF-3 to prevent the association of 30S and 50S ribosomal subunits into single ribosomes. Yet, IF-3 can form complexes with double-stranded RNA. However, IF-3 binds to double-stranded RNA with lower apparent affinity than to either R17 RNA or 30S ribosomal subunits; this may explain the resistance of bacterial protein synthesis to double-stranded RNA.     

Role of RNA and protein synthesis and turnover in the heat-induced increase in nuclear protein

The proteins which become associated with nuclei during hyperthermic exposure were characterized by labeled amino acid incorporation. Actinomycin-D (Act-D) or cycloheximide (CHM) pretreatment was used to determine whether concurrent RNA or protein synthesis is required for hyperthermia to induce the increase in nuclear protein content. Prior to heat exposure exponentially growing HeLa cells were (i) pulse labeled for 1 h, (ii) labeled for 36 h, or (iii) labeled for 24 h followed by 17 h chase. The nuclear specific activity (CPM/microgram protein) of [3H]lysine-labeled proteins did not change under any of the labeling conditions, whereas that of [3H]leucine-containing proteins increased significantly with (i) but not with (ii) or (iii), while that of [3H]tryptophan-labeled protein increased significantly with (i) and (ii) but not with (iii). Act-D treatment 1 h prior to and during heating did not affect nuclear protein increase, while CHM-treated cells showed generally less nuclear protein content (70% of control at 60 min) but nevertheless significant nuclear protein increase upon heating (60% increase at 60 min from 0 min). These results suggest that those proteins associated with nuclei following heat exposure are nonhistones with a high turnover rate, and the process dose not require the synthesis of RNA or proteins.     

Nucleotide specificity of the RNA editing reaction in pea chloroplasts

A sensitive in vitro editing assay for the pea chloroplast petB editing site has been developed and utilized to study the mechanism of C-to-U editing in chloroplast extracts. The in vitro editing assay was characterized by several criteria including: linearity with extract amount; linearity over time; dependence on assay components; and specificity of editing site conversion. The increase in the extent C-to-U conversion of the petB editing site was nearly linear with the amount chloroplast protein extract added, although the reaction appeared to decline in rate after approximately 30 min. The assay was tested for the importance of various assay components, and the omission of protease inhibitor and ATP was shown to dramatically reduce the extent of the editing reaction. Sequence analysis of cDNA clones obtained after an in vitro editing reaction demonstrated that 12 of 17 (71%) clones were edited, and that no other nucleotide changes in these cDNAs were detected. Thus, the fidelity and specificity of the in vitro editing system appears to be excellent, and this system should be suitable to study both mechanism of the editing reaction and editing site selection. The in vitro editing reaction was strongly stimulated by the addition of ATP, and all four NTPs and dNTPs stimulated the editing reaction except for rGTP, which had no effect. Thus, the nucleotide specificity of the editing reaction is broad, and is similar in this respect to the mitochondrial editing system. Most enzyme or processes specifically utilize ATP or GTP for phosphorylation and the ability to substitute other NTPs and dNTPs is unusual. RNA helicases have a similar broad nucleotide specificity and this may reflect the involvement of an RNA helicase in plant organelle editing.     

The synthesis and turnover of virus-specific polyadenylated RNA in polyoma-infected cells.

Mouse embryo cells infected with the 3049 strain of polyoma virus contain several fold more virus-specific, polyadenylated RNA beginning between 4 and 8 hours after the onset of viral DNA synthesis than do cells infected with wild-type virus (lpS). Following infection with either virus strain, there is an identical small but significant enhancement of the level of total polyadenylated RNA measured by binding of ¹²⁵I-labeled RNA to poly(dT)cellulose. The polyadenylation of “early” virus-specific RNA is inhibited 85–90% by cordycepin resulting in an “early” RNA preparation which competes fully with polyadenylated “early” virus-specific RNA in the ternary complex assay. Utilizing the nonpolyadenylated “early” RNA, competition hybridization demonstrated that approximately 78% of the enlarged pool of “late” 3049 polyadenylated RNA and 72% of the “late” lpS pool consisted of sequences unique to the “late” period. No significant difference in the rate of decay of 3049 and lpS-specific, “late” polyadenylated RNA following actinomycin D block was found. Infection by either strain of polyoma virus did not alter the rate of decay of total polyadenylated RNA.     

Lysosomal enzyme activities and RNA turnover rates in growing and nongrowing WI-38 and HeLa cells

Summary Activities of three lysosomal enzymes—acid RNase,N-acetyl-β-D-glucosaminidase and acid phosphatase—were determined during the growth cycles of WI-38 and HeLa cells, as well as in radiation-arrested WI-38 cells. In confluent and growth-arrested cultures of WI-38 cells, the lysosomal RNase increased six- to sevenfold; glucosaminidase, four- to five-fold; and phosphatase, two- to threefold. In HeLa cells, the lysosomal enzymes also increased in confluent cultures, but less than twofold; and the RNase level increased only transiently. In both WI-38 and HeLa cells, the rate of RNA breakdown, also increased as cultured approached confluency. The rate of turnover of RNA, like the level of acid RNase, was higher in WI-38 cells than in HeLa cells (4 d half-life compared to 8 d). The increase in acid RNase could be prevented by incubation of cells in NH₄Cl, but the rate of turnover in the presence of NH₄Cl increased just as much when cells became confluent or stopped growth. The content of acid RNase could be changed more than 10-fold without altering the rate of RNA turnover. It is suggested that the increase in enzyme level is more important for possible autophagy or increased digestion of engulfed RNA, rather than for normal RNA turnover, when growth stops. This study was supported by Grant GM-21357 from the National Institutes of Health.     

Planarian regeneration: quantitative differences in RNA and protein synthesis related to age

A comparative study of RNA and protein synthesis during regeneration of immature and adult planarians reveals fundamental differences in the regeneration process. Young planarians, which contain about 20 times more RNA/protein in their tissues than adults, actively synthesize RNA prior to any wound. A single stimulation of RNA synthesis is observed after 24 h following sectioning. The electrophoretic pattern of labelled RNA extracted either from intact or regenerating young planarians does not change significantly and shows, besides ribosomal RNA, an important fraction of RNA of heterogeneous molecular weights. This pattern is similar to that observed with extracts of RNA from regenerating adults but only after 24 h following sectioning (Martelly, I. and Le Moigne, A., Reprod. Nutr. Dev., 20 (1980) 1527–1537). Indeed, in adults, a preliminary phase of RNA metabolism is observed during the first day of regeneration. Young and adult planarians differ also in their time course of stimulation of protein synthesis after sectioning. While a lag time of more than 6 h is necessary in adults, protein synthesis is stimulated immediately after sectioning in the young. These differences in the pattern of macromolecular synthesis related to age are discussed in relation with the idea of cellular activation during the regeneration process.     

Elimination and Utilization of Oxidized Guanine Nucleotides in the Synthesis of RNA and Its Precursors

Reactive oxygen species are produced as side products of oxygen utilization and can lead to the oxidation of nucleic acids and their precursor nucleotides. Among the various oxidized bases, 8-oxo-7,8-dihydroguanine seems to be the most critical during the transfer of genetic information because it can pair with both cytosine and adenine. During the de novo synthesis of guanine nucleotides, GMP is formed first, and it is converted to GDP by guanylate kinase. This enzyme hardly acts on an oxidized form of GMP (8-oxo-GMP) formed by the oxidation of GMP or by the cleavage of 8-oxo-GDP and 8-oxo-GTP by MutT protein. Although the formation of 8-oxo-GDP from 8-oxo-GMP is thus prevented, 8-oxo-GDP itself may be produced by the oxidation of GDP by reactive oxygen species. The 8-oxo-GDP thus formed can be converted to 8-oxo-GTP because nucleoside-diphosphate kinase and adenylate kinase, both of which catalyze the conversion of GDP to GTP, do not discriminate 8-oxo-GDP from normal GDP. The 8-oxo-GTP produced in this way and by the oxidation of GTP can be used for RNA synthesis. This misincorporation is prevented by MutT protein, which has the potential to cleave 8-oxo-GTP as well as 8-oxo-GDP to 8-oxo-GMP. When ¹⁴C-labeled 8-oxo-GTP was applied to CaCl₂-permeabilized cells of a mutT⁻ mutant strain, it could be incorporated into RNA at 4% of the rate for GTP. Escherichia coli cells appear to possess mechanisms to prevent misincorporation of 8-oxo-7,8-dihydroguanine into RNA.     

RNA synthesis in maturing avian erythrocyte nuclei

The rate of RNA synthesis and its inhibition by α-amanitin in the nuclei of mature and immature avian erythrocytes are increased with the increase in ionic strength of incubation medium. Polyacrylamide gel electrophoresis indicates that heterogeneous species of RNAs are synthesised in the mature and immature erythrocyte nuclei. However, a large number of high molecular weight RNAs are synthesised in the nuclei of immature erythrocytes. Elution profiles on poly(U)-sepharose chromatography indicate that the RNAs synthesised in the nuclei of two types of cell contain poly(A) segments. Sixteen per cent of mature erythrocyte nuclear RNA syntbesised are polyadenylated, while it is 13% in immature erythrocyte nuclei. However, the total RNA synthesised is 2–3 fold higher in immature erythrocyte nuclei than that in mature erythrocyte nuclei.     

A single C. elegans PUF protein binds RNA in multiple modes

PUF proteins specifically bind mRNAs to regulate their stability and translation. Here we focus on the RNA-binding specificity of a C. elegans PUF protein, PUF-11. Our findings reveal that PUF-11 binds RNA in multiple modes, in which the protein can accommodate variable spacings between two distinct recognition elements. We propose a structural model in which flexibility in the central region of the protein enables the protein to adopt at least two distinct structures, one of which results in base flipping.     

Monitoring differential synthesis of RNA in individual cells by capillary electrophoresis

A capillary electrophoresis (CE)-based technique is reported here to monitor differential RNA synthesis in individual Chinese hamster ovary cells at distinct stages of the cell proliferation cycle. Cell synchronization was achieved by the shake-off method, in which mitotic (M) cells were dislodged, and cells at G(1), S, and G(2) phases were harvested 2.5, 10, and 13 h, respectively, after synchronizing the mitotic cells. Thirty-two cells (eight from each phase) were analyzed by injecting each cell into the capillary, lysing it with dilute surfactant, separating the RNA by capillary electrophoresis, and detecting the peaks with laser-induced fluorescence. The results from single cells show that the total amount of RNA increased at each successive stage (from G(1) to M), while the relative synthetic rates of different RNA fractions varied with progression through the cycle. There was a threefold increase in the synthetic rate of total RNA from S to G(2), compared with G(1) to S. In addition, differential accumulation of specific RNA fractions was observed, with the low-molecular-mass fraction exhibiting a much higher synthetic rate from G(2) to M, relative to the rates of the larger ribosomal RNA (rRNA) fractions. Comparison of the large rRNA fractions with one another reveals that at S phase more 28S rRNA was accumulated than 18S rRNA, and at G(1) and M phases, the synthetic rate of 28S rRNA was slowed compared with that of 18S. Minimal sample preparation, combined with the separation power of CE and single-cell detection sensitivity of laser-induced fluorescence, results in a simple method for assessing differential accumulation of RNA from distinct individual cells.