Kleinschmidt A M, Pederson T
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
Mol Cell Biol. 1987 Sep;7(9):3131-7. doi: 10.1128/mcb.7.9.3131-3137.1987.
The small nuclear RNAs U1, U2, U4, and U5 are cofactors in mRNA splicing and, like the pre-mRNAs with which they interact, are transcribed by RNA polymerase II. Also like mRNAs, mature U1 and U2 RNAs are generated by 3' processing of their primary transcripts. In this study we have investigated the in vitro processing of an SP6-transcribed human U2 RNA precursor, the 3' end of which matches that of authentic human U2 RNA precursor molecules. Although the SP6-U2 RNA precursor was efficiently processed in an ammonium sulfate-fractionated HeLa cytoplasmic S100 extract, the product RNA was unstable. Further purification of the processing activity on glycerol gradients resolved a 7S activity that nonspecifically cleaved all RNAs tested and a 15S activity that efficiently processed the 3' end of pre-U2 RNA. The 15S activity did not process the 3' end of a tRNA precursor molecule. As demonstrated by RNase protection, the processed 3' end of the SP6-U2 RNA maps to the same nucleotides as does mature HeLa U2 RNA.
小核RNA U1、U2、U4和U5是mRNA剪接的辅助因子，与它们相互作用的前体mRNA一样，由RNA聚合酶II转录。同样与mRNA相似，成熟的U1和U2 RNA是通过其初级转录本的3' 加工产生的。在本研究中，我们研究了SP6转录的人U2 RNA前体的体外加工过程，其3' 末端与真实的人U2 RNA前体分子的3' 末端相匹配。尽管SP6-U2 RNA前体在硫酸铵分级分离的HeLa细胞质S100提取物中被有效加工，但产物RNA不稳定。在甘油梯度上对加工活性进行进一步纯化，分离出一种7S活性，它能非特异性切割所有测试的RNA，还有一种15S活性，它能有效加工前体U2 RNA的3' 末端。15S活性不能加工tRNA前体分子的3' 末端。如核糖核酸酶保护实验所示，SP6-U2 RNA加工后的3' 末端与成熟的HeLa U2 RNA的核苷酸相同。
