Stimulation of myofibrillar synthesis by exercise is mediated by more efficient translation of mRNA.

Resistance exercises stimulate protein synthesis in human muscle, but the roles of changes in mRNA concentrations and changes in the efficiency of mRNA translation have not been defined. The present study was done to determine whether resistance exercise affects concentrations of total RNA, total mRNA, actin mRNA, or myosin heavy-chain mRNA (total and isoform specific). Eight subjects, 62-75 yr old, performed unilateral knee extensions at 80% of their one-repetition-maximum capacity on days 1, 3, and 6 of the study. On day 7, biopsies of exercised and nonexercised vastus lateralis muscles were obtained. Myofibrillar synthesis was determined by stable- isotope incorporation, and mRNA concentrations were determined by membrane hybridization and PCR-based methods. The exercise stimulated myofibrillar synthesis [30 +/- 6 (SE)%] without affecting RNA or mRNA concentrations. The effect of exercise on protein synthesis in individual subjects did not correlate with the effect on total RNA and mRNA concentrations. These data suggest that the stimulation of myofibrillar synthesis by resistance exercise is mediated by more efficient translation of mRNA.     

Degradation of messenger RNA, ribosomal RNA and 2-5A-dependent inhibition of protein biosynthesis

In rabbit reticulocyte lysates the addition of exogenous 2-5A leads after 10-20 minutes to the inhibition of protein synthesis. This inhibition can be blocked by rat antiserum to 2-5A. In intact ribosomes the ribosomal RNA is cleaved after 2-5A addition, but this cleavage is not in correlation with the protein synthesis shutoff. Ribosomal 5S RNA and 5,8S RNA are not cleaved even after several hours of incubation with 2-5A. The degradation of polysome associated mRNA correlates with the protein synthesis inhibition as revealed by Northern blot hybridization of globin mRNA with 32P-labelled p beta G plasmid. The addition of 2-5A antiserum to the rabbit reticulocyte lysate also inhibits the degradation of polysome bound globin mRNA.     

shRNA Transcribed by RNA Pol II Promoter Induce RNA Interference in Mammalian Cell

RNA interference is a powerful tool for gene functional analysis in mammals. Permanent gene suppression can be achieved by siRNAs as stem-loop precursors transcribed from RNA Pol III promoter such as H1 and U6 based on vector. This approach, however, has a major limitation: inhibition can not be controlled in a time or tissue specific manner because the RNA Pol III promoter is not time or tissue specific. To overcome these limitations, we designed a strategy that allows synthesis of small hairpin RNAs in a GFP-fused form mediated by RNA Pol II promoter CMV to efficiently and specifically knock down expression of both exogenous and endogenous genes in mammalian cells. As assayed by both fluorescence observing and quantitative RT-PCR, the protein and mRNA products of exogenous gene RFP were efficiently and specifically inhibited; quantitative RT-PCR and western blotting results respectively demonstrated that endogenous lamin B2 mRNA and protein was suppressed without global down-regulation of protein synthesis. Furthermore, GFP-fused shRNA efficacy for RNAi is dependent on target position based on this vector system. This method may provide a novel approach for the application of RNAi technology in suppressing gene expression in mammalian system. Jing Yuan, Xiaobo Wang and Ning Li - These authors contributed equally to this work.     

一种简单、快速、经济的mRNA分离方法

对oligo(dT)纤维素纯化poly(A)RNA的方法进行了改良.缩小了层析柱床的体积,使填料用量仅为常规量的1/50-1/100,即在移液器尖嘴中进行层析.与常规方法相比,它具有实验时间短、洗脱的mRNA可直接合成cDNA和得率较高等优点.     

Determination of synthesis rate and lifetime of bacterial mRNAs

A method has been developed to determine the synthesis rate and lifetime of bacterial mRNAs, either bulk mRNA or specific mRNAs, with a minimum of physiological disturbance. The method uses hybridization of pulse-labeled RNA to specific probes followed by an evaluation based on a computer simulation of the labeling kinetics of different classes of RNA. The method was applied to the determination of bulk mRNA in Escherichia coli growing in glucose minimal medium: 60% of the instantaneous rate of RNA synthesis, or 2.3% of the total amount of RNA, was found to be mRNA with an average lifetime of 1.0 +/- 0.2 min (= 0.7 min half-life).     

Studies on the form and synthesis of messenger ribonucleic acid in the rat ventral prostate gland, including its tissue-specific stimulation by androgens

1. When prostate polyribosomes are labelled with radioactive precursors in vivo and subsequently dissociated with sodium dodecyl sulphate, a heterogeneous 6-15S RNA species may be identified that possesses all of the distinctive properties of mRNA. 2. Apart from the selective incorporation of 5'-fluoro-orotic acid into this 6-15S RNA component, it is bound by nitrocellulose filters under experimental conditions where only poly(A)-rich species of RNA are specifically retained. Most importantly, however, only the 6-15S RNA fraction is capable of promoting the incorporation of amino acids into peptide linkage in an mRNA-depleted cell-free system derived from ascites-tumour cells. 3. With the development of a simpler method for labelling the total RNA fraction of the prostate gland in vitro, the poly(A)-enriched RNA fraction may be readily isolated by adsorption and elution from oligo(dT)-cellulose. The synthesis of the poly(A)-enriched 6-15S RNA fraction is stringently controlled by androgens in a highly tissue- and steroid-specific manner. 4. From an analysis of the proteins synthesized in the ascites cell-free system in the presence of the poly(A)-rich RNA fraction, it appears that protein synthesis in the prostate gland is stimulated in a rather general way, even during the earliest phases of the androgenic response. This conclusion may require modification when more specific means of analysis are available than those used in the present investigation. 5. The implications of these findings to the mechanism of action of androgens are discussed.     

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%.     

RNA synthesis during the stationary growth phase in the Bacillus subtilis Cgr4 mutant

RNA synthesis was studied in Bacillus subtilis Cgr4 grown in the mineral sporulation medium enriched with glucose up to 2% and amino acids up to 1%. To study mRNA synthesis, a method of transfer of the 3H-uridine pulse-labeled culture to the supernatant of physiologically identical, not labeled culture, followed by further incubation was used, the amount of 3H-uridine in the supernatant as well as in cells being measured. RNA was also analysed electrophoretically and distribution of the label among the fractions was determined. It is shown that mRNA synthesized in the logarithmic phase degrades up to 12% on the 2nd hour of growth during 10 min; the mRNA in the stationary phase is stable on the 7th hour of growth; no degradation is observed in the course of 2-3 hours. The beginning of degradation coincides in time with secondary induction of the synthesis of serine proteases and with the onset of sharp decrease in incorporation of 3H-uridine in RNA as well as with induction of spore morphogenesis. On the basis of electrophoretical analysis of pulse-labeled RNA, it was demonstrated that, prior to the transfer, labeled uridine was included and preserved in RNA fraction for 2-3 hours after the transfer, this fraction corresponding in mobility with mRNA in polyacrylamide gel. The following conclusion may be drawn: stable mRNAs are synthesized in the stationary phase and may be used for the translation of extracellular serine protease.     

mRNA quantitation by a simple and sensitive RNAse protection assay.

An RNAse protection assay is described that increases substantially the degree of precision with which one can measure the mRNA levels in cells and tissues through the use of the internal standard. The assay can be used to measure any mRNA for which the corresponding cDNA is available. We describe here the use of the assay to measure the apolipoprotein (apo)-A-I, apo-B, and apo-E mRNA levels in tissues from the cynomolgus monkey. cDNA fragments derived from each mRNA were subcloned into pGEM-9Zf(-), a vector containing a polylinker that is flanked by the SP6 and T7 RNA polymerase promoters. That series of plasmids, called RNA quantitation vectors (pRQV-AI, B, or E), permitted the synthesis of a sense RNA strand and an antisense RNA strand for the gene of interest. The sense stand was used as the internal standard and added to the RNA to be analyzed just prior to initiation of the assay. The radiolabeled antisense strand served as the probe. By including some nucleotides derived from the vector, we were able to design both the internal standard and the probe such that, after solution hybridization and RNAse digestion, the size of the protected internal standard-probe fragments was different from that of the authentic mRNA-probe fragments. Those fragments were then separated by gel electrophoresis, and the radioactivity in the authentic mRNA band was compared to that in the internal standard band. The mass of the authentic mRNA could then be calculated from the ratio of the radioactivity in each band and the mass of the internal standard.     

Temperature-sensitive Yeast Mutant Defective in Ribonucleic Acid Production

A single, recessive mutation in a nuclear gene confers a temperature-sensitive growth response in a mutant of Saccharomyces cerevisiae, ts(-) 136. The mutant grows normally at 23 C, but exhibits a rapid and preferential inhibition of ribonucleic acid (RNA) accumulation after a shift to 36 C, demonstrating a defect in stable RNA production. Cultures of the mutant which were shifted from 23 to 36 C display the following phenomena which indicate that messenger RNA (mRNA), as well as stable RNA production, is defective. The entrance of pulse-labeled RNA into cytoplasmic polyribosomes is even more strongly inhibited than is net RNA accumulation. The rate of protein synthesis, at first unaffected, decreases slowly; this decrease is paralleled by the decay of polyribosomes to monoribosomes with a half-time of 23 min. The polyribosomes which remain after a 30-min preincubation of the mutant at 36 C are active in polypeptide synthesis in vivo, whereas the monoribosomes which accumulate are not. Furthermore, ribosomes isolated from a culture of the mutant preincubated for 1 hr at 36 C are inactive in polypeptide synthesis in vitro, but can be restored to full activity by the addition of polyuridylic acid as mRNA. We conclude that mutant ts(-) 136 is defective either in the synthesis of all types of cytoplasmic RNA, or in the transport of newly synthesized RNA from the nucleus to the cytoplasm, and that the mRNA of a eucaryotic organism (yeast) is metabolically unstable, having a half-life of approximately 23 min at 36 C.     

Plant enzyme synthesis: Hormonal regulation of invertase and peroxidase synthesis in sugar cane

Summary Using sugar-cane internodal tissue in which RNA synthesis was ratelimiting for invertase of peroxidase synthesis, measurements were made of enzymeforming-capacity after blocking further RNA synthesis with actinomycin D or 6-methylpurine. In this way it was possible to determine whether added auxin (naphthaleneacetic acid) or gibberellic acid (GA₃) affected steps prior or subsequent to synthesis of the RNA fractions specifically required for synthesis of either enzyme. Both auxin and GA₃ increased the enzyme-forming-capacity for invertase but not for peroxidase. The effects of the two hormones are interpreted as causing stabilization of mRNA for invertase.Abscisic acid (ABA) increased the rate of synthesis of invertase but not peroxidase. ABA did not change the rate of loss of invertase when peptide-bond formation was blocked with cycloheximide, but stimulated its synthesis when RNA synthesis was blocked with 6-methyl purine. Hence, the site of action of ABA is subsequent to invertase-mRNA formation and prior to invertase destruction.Kinetin had no short-term effects when RNA synthesis was limiting for invertase production, and does not appear to directly modulate mRNA synthesis or stabilization, or amino-acid-polymerization steps. In treatments longer than 5 hours, kinetin inhibited synthesis of all three enzymes studied, so that its effect on enzyme synthesis in this tissue appears to be unspecific.Abbreviations used throughout text ABA (±)-abscisic acid (abscisin II, dormin) - GA₃ gibberellic acid - NAA -naphthaleneacetic acid