Tanzer M M, Meagher R B
Department of Genetics, University of Georgia, Athens 30602-7223.
Mol Cell Biol. 1994 Apr;14(4):2640-50. doi: 10.1128/mcb.14.4.2640-2650.1994.
The mRNA encoding the soybean rbcS gene, SRS4, is degraded into a set of discrete lower-molecular-weight products in light-grown soybean seedlings and in transgenic petunia leaves. The 5'-proximal products have intact 5' ends, lack poly(A) tails, lack various amounts of 3'-end sequences, and are found at higher concentrations in the polysomal fraction. To study the mechanisms of SRS4 mRNA decay more closely, we developed a cell-free RNA degradation system based on a polysomal fraction isolated from soybean seedlings or mature petunia leaves. In the soybean in vitro degradation system, endogenous SRS4 mRNA and proximal product levels decreased over a 6-h time course. When full-length in vitro-synthesized SRS4 RNAs were added to either in vitro degradation system, the RNAs were degraded into the expected set of proximal products, such as those observed for total endogenous RNA samples. When exogenously added SRS4 RNAs already truncated at their 3' ends were added to either system, they too were degraded into the expected subset of proximal products. A set of distal fragments containing intact 3' ends and lacking various portions of 5'-end sequences were identified in vivo when the heterogeneous 3' ends of the SRS4 RNAs were removed by oligonucleotide-directed RNase H cleavage. Significant amounts of distal fragments which comigrated with the in vivo products were also observed when exogenous SRS4 RNAs were degraded in either in vitro system. These proximal and distal products lacking various portions of their 3' and 5' sequences, respectively, were generated in essentially a random order, a result supporting a nonprocessive mechanism. Tagging of the in vitro-synthesized RNAs on their 5' and 3' ends with plasmid vector sequences or truncation of the 3' end had no apparent effect on the degradation pattern. Therefore, RNA sequences and/or structures in the immediate vicinity of each 3' end point may be important in the degradation machinery. Together, these data suggest that SRS4 mRNA is degraded by a stochastic mechanism and that endonucleolytic cleavage may be the initial event. These plant in vitro systems should be useful in identifying the cis- and trans-acting factors involved in the degradation of mRNAs.
编码大豆rbcS基因SRS4的mRNA在光照下生长的大豆幼苗和转基因矮牵牛叶片中被降解为一组离散的低分子量产物。5'近端产物具有完整的5'端，缺乏多聚腺苷酸尾巴，缺乏不同长度的3'端序列，并且在多核糖体组分中浓度更高。为了更深入地研究SRS4 mRNA降解的机制，我们基于从大豆幼苗或成熟矮牵牛叶片中分离的多核糖体组分开发了一种无细胞RNA降解系统。在大豆体外降解系统中，内源性SRS4 mRNA和近端产物水平在6小时的时间进程中下降。当将体外合成的全长SRS4 RNA添加到任一体外降解系统中时，这些RNA被降解为预期的近端产物组，就像在总内源性RNA样品中观察到的那样。当将已经在其3'端截短的外源添加的SRS4 RNA添加到任一系统中时，它们也被降解为预期的近端产物子集。当通过寡核苷酸定向的RNase H切割去除SRS4 RNA的异质3'端时，在体内鉴定出一组含有完整3'端且缺乏5'端序列不同部分的远端片段。当外源SRS4 RNA在任一体外系统中降解时，也观察到大量与体内产物共迁移的远端片段。这些分别缺乏其3'和5'序列不同部分的近端和远端产物基本上以随机顺序产生，这一结果支持了非连续机制。用质粒载体序列在体外合成的RNA的5'和3'端进行标记或3'端截短对降解模式没有明显影响。因此，每个3'端点紧邻区域的RNA序列和/或结构可能在降解机制中很重要。总之，这些数据表明SRS4 mRNA通过随机机制降解，并且内切核酸酶切割可能是初始事件。这些植物体外系统在鉴定参与mRNA降解的顺式和反式作用因子方面应该是有用的。
