Pramanik S K, Walsh R W, Bag J
Eur J Biochem. 1986 Oct 15;160(2):221-30. doi: 10.1111/j.1432-1033.1986.tb09960.x.
The distribution of mRNA between the detergent-soluble and insoluble (cytoskeleton) fractions in rat L6 myoblast and myotube cells was examined. Approximately 85% of cytoplasmic mRNA in both myoblasts and myotubes was found associated with the cytoskeletal framework. The cytoskeleton-bound mRNA was present as polysomes. In contrast, the mRNA of the detergent-soluble fraction was not associated with ribosomes and was thus considered to be the repressed population. The association of mRNA with the cytoskeletal framework was not affected by treatments leading to dissociation of polysomes. Differential distribution of mRNA between the soluble and cytoskeleton-bound fractions was analyzed by in vitro translation. The mRNAs coding for polypeptides of molecular masses 40 kDa and 60 kDa were preferentially enriched in the soluble fraction. The nature of binding between mRNA and the cytoskeletal framework was examined following in vivo cross-linking of RNA and protein by irradiating muscle cells with ultraviolet light. It was observed that this treatment covalently linked RNA and the neighbouring protein moieties without any detectable damage to the cytoskeletal framework, as measured by the distribution of RNAs and proteins between the cytoskeleton and soluble fractions. Analysis of the polypeptide moieties cross-linked to the mRNA have shown that a large number of polypeptides of molecular masses between 15-220 kDa were associated with both cytoskeleton-bound and soluble mRNAs. The polypeptide moieties of these mRNA-protein complexes were not only similar in the cytoskeleton and soluble mRNA-protein complexes but also were similar between myoblasts and myotubes. However, one polypeptide of 165 kDa was preferentially associated with the cytoskeleton-bound mRNA-protein complexes. Interestingly this 165-kDa polypeptide was also preferentially enriched in the total proteins from the cytoskeleton fraction. This result suggests a possible role of the 165-kDa polypeptide in association between mRNA and the cytoskeletal framework. To examine the mechanism of interaction between mRNA and the cytoskeletal framework we have reported here a ghost monolayer transcription system from myotubes. This transcription system was able to synthesize rRNA and mRNA. The mRNA transcribed in vitro was preferentially associated with the cytoskeleton structure present in the ghost monolayer system.
研究了大鼠L6成肌细胞和肌管细胞中mRNA在去污剂可溶性和不溶性（细胞骨架）组分之间的分布。在成肌细胞和肌管细胞中，约85%的细胞质mRNA与细胞骨架框架相关联。细胞骨架结合的mRNA以多核糖体形式存在。相比之下，去污剂可溶性组分中的mRNA不与核糖体结合，因此被认为是受抑制的群体。mRNA与细胞骨架框架的关联不受导致多核糖体解离的处理的影响。通过体外翻译分析了mRNA在可溶性和细胞骨架结合组分之间的差异分布。编码分子量为40 kDa和60 kDa多肽的mRNA优先富集于可溶性组分中。在用紫外线照射肌肉细胞使RNA和蛋白质在体内发生交联后，研究了mRNA与细胞骨架框架之间的结合性质。观察到这种处理使RNA与相邻的蛋白质部分共价连接，且根据RNA和蛋白质在细胞骨架和可溶性组分之间的分布情况测量，对细胞骨架框架没有任何可检测到的损伤。对与mRNA交联的多肽部分的分析表明，大量分子量在15 - 220 kDa之间的多肽与细胞骨架结合的mRNA和可溶性mRNA都相关联。这些mRNA - 蛋白质复合物的多肽部分不仅在细胞骨架和可溶性mRNA - 蛋白质复合物中相似，而且在成肌细胞和肌管细胞之间也相似。然而，一种165 kDa的多肽优先与细胞骨架结合的mRNA - 蛋白质复合物相关联。有趣的是，这种165 kDa的多肽在细胞骨架组分的总蛋白中也优先富集。这一结果表明165 kDa多肽在mRNA与细胞骨架框架的关联中可能发挥作用。为了研究mRNA与细胞骨架框架之间的相互作用机制，我们在此报道了一种来自肌管的空壳单层转录系统。该转录系统能够合成rRNA和mRNA。体外转录的mRNA优先与空壳单层系统中存在的细胞骨架结构相关联。
