Increased messenger RNA from protein synthesis inhibited human fibroblasts
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Abstract Numerous reports have demonstrated that specific protein synthesis in response to specific inducers is markedly stimulated by a simultaneous brief exposure to protein synthesis inhibitors such as cycloheximide. This phenomenon is known as “superinduction” and is most often attributed to the accumulation of cytoplasmic messenger RNA during the inhibition period. Messenger RNA, as defined by rapid labeling, oligo (dt)‐cellulose binding, and cell free protein synthesis stimulation was measured in cycloheximide treated human fibroblasts. In spite of a consistent 40% decrease in total polysomal 3 H‐uridine labeled RNA, a 1.5‐ to 2‐fold increase in extractable mRNA was observed. These data provide direct evidence that protein synthesis inhibition stimulates the appearance of cytoplasmic mRNA and/or completely blocks its degradation and, are consistent with the hypothesis that mRNA accumulation partly underlies the superinduction phenomena.Keywords:
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Experiments have been designed to test the possibility that, as with messenger RNA from animal cells, 7-methylguanosine may be bonded through a pyrophosphate bridge to the 5'-termini of messenger RNA molecules from the cells of a higher-plant organism. It is concluded that 7-methylguanosine is probably bonded through a pyrophosphate bridge to the 5'-termini of messenger RNA molecules from wheat leaves.
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The regulation of cell growth can be achieved at many levels but ultimately the regulatory factors must alter protein synthesis since growing cells always exhibit an increased rate of protein synthesis compared to resting cells. Some studies using growing and nongrowing mammalian cells have shown that the rate of protein synthesis is directly dependent on mRNA content. Other studies have shown that growing the resting cells have similar amounts of mRNA and that protein synthesis is regulated by the proportion of mRNA in polysomes. We have analyzed mRNA content in growing and resting epithelial cells of Xenopus laevis. Quantitation of poly(A)+ mRNA by uniform labeling with 3H-uridine and by 3H-poly(U) hybridization demonstrated a direct relationship between mRNA content and the relative rate of protein synthesis in growing and resting cells. Likewise, after serum stimulation of resting cells the increase in mRNA content closely paralleled the increase in protein synthesis. Our results suggest that control of protein synthesis in growing and nongrowing cells is exerted before the translational level.
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Abstract Numerous reports have demonstrated that specific protein synthesis in response to specific inducers is markedly stimulated by a simultaneous brief exposure to protein synthesis inhibitors such as cycloheximide. This phenomenon is known as “superinduction” and is most often attributed to the accumulation of cytoplasmic messenger RNA during the inhibition period. Messenger RNA, as defined by rapid labeling, oligo (dt)‐cellulose binding, and cell free protein synthesis stimulation was measured in cycloheximide treated human fibroblasts. In spite of a consistent 40% decrease in total polysomal 3 H‐uridine labeled RNA, a 1.5‐ to 2‐fold increase in extractable mRNA was observed. These data provide direct evidence that protein synthesis inhibition stimulates the appearance of cytoplasmic mRNA and/or completely blocks its degradation and, are consistent with the hypothesis that mRNA accumulation partly underlies the superinduction phenomena.
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Summary The synthesis of RNA in rat spleen cells was stimulated by immunization. Analyses of the RNA associated with ribosomes extracted from these cells which had been immunized 24 hr previously, revealed that during the 1st hr after injection of P32, the RNA synthesized in greatest amounts had messenger-like sedimentation characteristics. Over the next 24 hr, there was an extensive accumulation of stable ribosomal RNA, but the messenger RNA accumulated relatively slowly. The base composition of the 6 to 12 S RNA, measured from the P32 distribution in the various nucleotides, differed from the nucleotide composition of ribosomal and soluble RNA, and resembled more closely that of DNA in the GMP + CMP to AMP + UMP ratios. Particularly high UMP:AMP ratios also characterized this RNA. The base ratios of the messenger RNA which accumulated were different from those of the rapidly labeled but unstable 6 to 12 S RNA. On the basis of these results, it is apparent that the synthesis of at least two major species of messenger RNA are stimulated by immunization. One species is labile and may code for structural proteins and enzymes. One other is relatively stable and may be the message for antibody synthesis.
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Abstract The so-called “cap” structure of the 5′-terminus of eukaryotic messenger RNA (mRNA) is of importance for protein biosynthesis on ribosimes. We have examined the chemical synthesis and structural requirement of the 5′-terminus of mRNA. This paper describes a brief summary of the synthesis of the cap structure and the 5′-terminus of mRNA bearing the leader sequences.
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Abstract Messenger RNAs (mRNAs) with phosphorothioate modification (PS‐mRNA) to the phosphate site of A, G, C, and U with all 16 possible combinations were prepared, and the translation reaction was evaluated using an E. coli cell‐free translation system. Protein synthesis from PS‐mRNA increased in 12 of 15 patterns when compared with that of unmodified mRNA. The protein yield increased 22‐fold when the phosphorothioate modification at A/C sites was introduced into the region from the 5′‐end to the initiation codon. Single‐turnover analysis of PS‐mRNA translation showed that phosphorothioate modification increases the number of translating ribosomes, thus suggesting that the rate of translation initiation (rate of ribosome complex formation) is positively affected by the modification. The method provides a new strategy for improving translation by using non‐natural mRNA.
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