Newman A J, Lin R J, Cheng S C, Abelson J
Cell. 1985 Aug;42(1):335-44. doi: 10.1016/s0092-8674(85)80129-x.
We have altered the TACTAAC sequence in the yeast CYH2m gene intron to TACTACC. This mutation changes the nucleotide at the normal position of the branch in intron RNA lariats produced during pre-mRNA splicing, and it prevents splicing in vivo. In a yeast pre-mRNA splicing system, CYH2m pre-mRNA carrying the TACTACC mutation is not specifically cut or rearranged in any way. Substitution of an A for the first G of the CYH2m intron, converting the highly conserved GTATGT 5' splice site sequence to ATATGT, also blocks intron excision in vivo and in vitro: pre-mRNA carrying this mutation was still cut normally at the mutant 5' splice site in vitro, to give authentic exon 1 and an intron-exon 2 lariat RNA with an A-A 2'-5' phosphodiester linkage at the branch point. This lariat RNA is a dead-end product. The subsequent cleavage at the 3' splice site is therefore sensitive to the sequence of the 5' end of the intron attached at the branch point.
我们已将酵母CYH2m基因内含子中的TACTAAC序列改变为TACTACC。这种突变改变了前体mRNA剪接过程中产生的内含子RNA套索状结构分支正常位置处的核苷酸，并且阻止了体内剪接。在酵母前体mRNA剪接系统中，携带TACTACC突变的CYH2m前体mRNA不会以任何方式被特异性切割或重排。将CYH2m内含子的第一个G替换为A，将高度保守的GTATGT 5'剪接位点序列转换为ATATGT，同样会在体内和体外阻断内含子切除：携带这种突变的前体mRNA在体外仍在突变的5'剪接位点正常切割，产生真实的外显子1和一个内含子-外显子2套索状RNA，其在分支点处具有A-A 2'-5'磷酸二酯键。这种套索状RNA是一种无后续作用的产物。因此，3'剪接位点的后续切割对连接在分支点处的内含子5'末端序列敏感。
