Kister K P, Eckert W A
Nucleic Acids Res. 1987 Mar 11;15(5):1905-20. doi: 10.1093/nar/15.5.1905.
We have characterized a 1.5 kb RNA species in T. thermophila macronuclei previously found in vivo and including intron sequences linked to the 3' exon. This IVS-3' exon RNA could be detected in gels as a discrete molecule only after denaturation of nuclear RNA. After addition of 32P-GTP, as splicing cofactor in a nuclear in vitro system, the IVS-3' exon RNA was labeled at its 5' terminus, as was the by-product of splicing, the excised IVS RNA. The time course of labeling indicates that the IVS-3' exon RNA acts like a reaction intermediate and specifically a kinetic precursor to IVS RNA. Partial nuclease digestions showed that the IVS-3' exon RNA and the IVS RNA have the same 5' terminal sequence. In addition the IVS-3' exon RNA can release the 15-mer oligonucleotide cleaved off during circularization of IVS RNA under conditions of high temperature. Taken together, the structural, functional, and kinetic properties of the IVS-3' exon RNA strongly suggest that it represents a previously postulated in vivo intermediate in the splicing pathway.
我们已经对嗜热栖热菌大核中一种1.5 kb的RNA物种进行了表征，该物种先前在体内被发现，包含与3'外显子相连的内含子序列。只有在核RNA变性后，这种IVS - 3'外显子RNA才能在凝胶中作为离散分子被检测到。在核体外系统中加入32P - GTP作为剪接辅因子后，IVS - 3'外显子RNA在其5'末端被标记，剪接副产物即切除的IVS RNA也是如此。标记的时间进程表明，IVS - 3'外显子RNA的作用类似于反应中间体，特别是IVS RNA的动力学前体。部分核酸酶消化表明，IVS - 3'外显子RNA和IVS RNA具有相同的5'末端序列。此外，在高温条件下，IVS - 3'外显子RNA可以释放出在IVS RNA环化过程中被切割下来的15聚体寡核苷酸。综上所述，IVS - 3'外显子RNA的结构、功能和动力学特性强烈表明，它代表了剪接途径中先前假定的体内中间体。
