Dreyfuss G, Adam S A, Choi Y D
Mol Cell Biol. 1984 Mar;4(3):415-23. doi: 10.1128/mcb.4.3.415-423.1984.
Exposure of intact cells to UV light brings about cross-linking of polyadenylated mRNA to a set of cytoplasmic proteins which are in direct contact with the mRNA in vivo. Substantial amounts of an additional protein of molecular weight 38,000 (38K) become cross-linked to the mRNA when cells are treated with inhibitors of mRNA synthesis (actinomycin D, camptothecin, and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole) or after infection with vesicular stomatitis virus. Cordycepin, which inhibits polyadenylation but not mRNA synthesis, has no such effect. Inhibitors of protein synthesis and of rRNA synthesis are also without effect on 38K cross-linking to mRNA. The onset of the effect of inhibitors of mRNA synthesis on the UV cross-linkable interaction between mRNA and 38K is rapid and reaches a maximal level in less than 60 min, and it is completely and rapidly reversible. In cells treated with actinomycin D, the amount of 38K which becomes cross-linked to mRNA is proportional to the extent of inhibition of mRNA synthesis. The association of 38K with mRNA during transcriptional arrest does not require protein synthesis because simultaneous treatment with the protein synthesis inhibitor emetine does not interfere with it. The effectors which promote the interaction of 38K with mRNA do not affect the proteins which are in contact with polyadenylated heterogeneous nuclear RNA and do not markedly affect protein synthesis in the cell. The 38K protein can be isolated with the polyribosomal polyadenylated fraction from which it was purified, and monoclonal antibodies against it were prepared. Immunofluorescence microscopy shows mostly cytoplasmic and some nuclear staining. These observations demonstrate that commonly used inhibitors of transcription affect the physical state of messenger ribonucleoproteins in vivo.
完整细胞暴露于紫外线下会导致多聚腺苷酸化的mRNA与一组细胞质蛋白发生交联，这些蛋白在体内与mRNA直接接触。当细胞用mRNA合成抑制剂（放线菌素D、喜树碱和5,6-二氯-1-β-D-呋喃核糖基苯并咪唑）处理或感染水泡性口炎病毒后，大量额外的分子量为38000（38K）的蛋白质会与mRNA交联。抑制多聚腺苷酸化但不抑制mRNA合成的虫草素没有这种作用。蛋白质合成抑制剂和rRNA合成抑制剂对38K与mRNA的交联也没有影响。mRNA合成抑制剂对mRNA与38K之间紫外线可交联相互作用的影响起效迅速，在不到60分钟内达到最大水平，并且完全且迅速可逆。在用放线菌素D处理的细胞中，与mRNA交联的38K的量与mRNA合成的抑制程度成正比。转录停滞期间38K与mRNA的结合不需要蛋白质合成，因为同时用蛋白质合成抑制剂依米丁处理不会干扰这种结合。促进38K与mRNA相互作用的效应物不会影响与多聚腺苷酸化异质核RNA接触的蛋白质，也不会显著影响细胞中的蛋白质合成。38K蛋白可以从纯化它的多核糖体多聚腺苷酸化部分中分离出来，并制备了针对它的单克隆抗体。免疫荧光显微镜检查显示主要是细胞质染色，还有一些核染色。这些观察结果表明，常用的转录抑制剂会影响体内信使核糖核蛋白的物理状态。
