Albrecht G, Krowczynska A, Brawerman G
J Mol Biol. 1984 Oct 5;178(4):881-96. doi: 10.1016/0022-2836(84)90317-6.
Masked and exposed sites in rabbit beta-globin messenger RNA were identified through S1 nuclease mapping of RNase T1 cleavage sites. Sites exposed to this enzyme were compared in deproteinized polysomal RNA and in mRNA in its native configuration in reticulocyte extracts. The analysis showed that most of the 3' non-coding region is well accessible to the enzyme, both in deproteinized RNA and in the cell extract. A possible protecting function for the poly(A) sequence is suggested by the fact that molecules with very short poly(A) segments were cleaved preferentially in this region. The G residues in the 5' non-coding region were inaccessible to RNase T1. A highly sensitive site adjacent to the initiation AUG codon was evident in the deproteinized RNA. This site was far less accessible to the enzyme in the mRNA associated with ribosomes in the cell extract. The first 150 nucleotides in the coding region showed very little susceptibility to digestion by the enzyme, in deproteinized RNA as well as in the cell extracts. Preparations of untreated mRNA showed the occurrence of truncated molecules, apparently generated by cleavage by endogenous nucleases. These cleavages were most prevalent in the two non-coding regions. They occurred at sites containing A-U sequences in the 3' non-coding region, and at sites with different sequences in the 5' non-coding region. Incubation of cell extracts at 37 degrees C did not cause any increase in these endogenous cleavages. It is suggested that they may have been generated in the intact cells, possibly as part of the mRNA degradation process in maturing reticulocytes.
通过核糖核酸酶T1切割位点的S1核酸酶图谱鉴定了兔β-珠蛋白信使核糖核酸中的隐蔽位点和暴露位点。在脱蛋白的多聚核糖体核糖核酸和网织红细胞提取物中天然构象的信使核糖核酸中，比较了暴露于该酶的位点。分析表明，在脱蛋白的核糖核酸和细胞提取物中，大部分3'非编码区都能很好地被该酶识别。多聚腺苷酸（poly(A)）序列具有可能的保护功能，这一推测基于以下事实：具有非常短的poly(A)片段的分子在该区域优先被切割。核糖核酸酶T1无法识别5'非编码区的鸟嘌呤（G）残基。在脱蛋白的核糖核酸中，靠近起始甲硫氨酸（AUG）密码子处有一个高度敏感位点。在细胞提取物中与核糖体结合的信使核糖核酸中，该位点对酶的可及性要低得多。在脱蛋白的核糖核酸以及细胞提取物中，编码区的前150个核苷酸对该酶的消化作用敏感性很低。未经处理的信使核糖核酸制剂显示存在截短分子，显然是由内源性核酸酶切割产生的。这些切割在两个非编码区最为普遍。它们发生在3'非编码区含有A-U序列的位点，以及5'非编码区具有不同序列的位点。将细胞提取物在37℃孵育不会导致这些内源性切割增加。推测它们可能在完整细胞中产生，可能是成熟网织红细胞中信使核糖核酸降解过程的一部分。
