O'Connor J P, Peebles C L
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260.
Mol Cell Biol. 1991 Jan;11(1):425-39. doi: 10.1128/mcb.11.1.425-439.1991.
We have surveyed intron-containing RNAs of the yeast Saccharomyces cerevisiae by filter hybridization with pre-tRNA intron-specific oligonucleotide probes. We have classified various RNAs as pre-tRNAs, splicing intermediates, or excised intron products according to apparent size and structure. Linear, excised intron products were detected, and one example was isolated and sequenced directly. Additional probes designed to detect other precursor sequences were used to verify the identification of several intermediates. Pre-tRNA species with both 5' leader and 3' extension, with 3' extension only, and with mature ends were distinguished. From these results, we conclude that the processing reactions used to remove the 5' leader and 3' extension from the transcript are ordered 5' end trimming before 3' end trimming. Splicing intermediates containing the 5' exon plus the intron were detected. The splice site cleavage reactions are probably ordered 3' splice site cleavage before 5' splice site cleavage. Surprisingly, we also detected a splicing intermediate with the 5' leader and a spliced product with both 5' leader and 3' extension. Evidently, splicing and end trimming are not ordered relative to each other, splicing occurring either before or after end trimming.
我们通过与前体tRNA内含子特异性寡核苷酸探针进行滤膜杂交，对酿酒酵母中含内含子的RNA进行了研究。我们根据明显的大小和结构，将各种RNA分类为前体tRNA、剪接中间体或切除的内含子产物。检测到了线性的、切除的内含子产物，并直接分离出一个例子进行测序。设计用于检测其他前体序列的额外探针，被用于验证几种中间体的鉴定。区分了具有5'前导序列和3'延伸序列、仅具有3'延伸序列以及具有成熟末端的前体tRNA种类。从这些结果中，我们得出结论，用于从转录本中去除5'前导序列和3'延伸序列的加工反应是有序的，即先进行5'末端修剪，然后进行3'末端修剪。检测到了包含5'外显子加内含子的剪接中间体。剪接位点的切割反应可能是先进行3'剪接位点切割，然后进行5'剪接位点切割。令人惊讶的是，我们还检测到了带有5'前导序列的剪接中间体以及带有5'前导序列和3'延伸序列的剪接产物。显然，剪接和末端修剪在顺序上并不相互关联，剪接可以在末端修剪之前或之后发生。
