Murphy W J, Watkins K P, Agabian N
Cell. 1986 Nov 21;47(4):517-25. doi: 10.1016/0092-8674(86)90616-1.
We present evidence that addition of the 35 nucleotide spliced leader (SL) to the 5' end of T. brucei mRNAs occurs via trans RNA splicing. A 100 nucleotide fragment of the 135 base SL RNA (100-mer) is revealed by S1 nuclease analysis of total and poly(A)+ RNA. This 100-mer is not detected by Northern hybridization analysis, indicating that it does not exist free in the cell. The 5' end of the 100-mer maps precisely to the conserved splice junction sequence of the SL RNA. Purified debranching enzyme releases this 100-mer RNA as a free, 100 nucleotide species. This indicates that the 100-mer is covalently linked to poly(A)+ RNA by a 2'-5' phosphodiester bond, that the branched intermediate has a discontinuous intron or Y structure (rather than a lariat), which is expected of a trans-spliced mRNA, and that the SL RNA is indeed the donor of the SL sequence to trypanosome mRNAs.
我们提供的证据表明，布氏锥虫mRNA的5'端添加35个核苷酸的剪接前导序列（SL）是通过反式RNA剪接发生的。通过对总RNA和聚腺苷酸加尾RNA（poly(A)+ RNA）进行S1核酸酶分析，揭示了135个碱基的SL RNA的一个100个核苷酸的片段（100聚体）。Northern杂交分析未检测到该100聚体，表明它在细胞中不是游离存在的。100聚体的5'端精确地定位到SL RNA的保守剪接连接序列。纯化的去分支酶释放出作为游离的100个核苷酸物种的该100聚体RNA。这表明该100聚体通过2'-5'磷酸二酯键与聚腺苷酸加尾RNA共价连接，分支中间体具有不连续的内含子或Y结构（而不是套索结构），这是反式剪接mRNA所预期的，并且SL RNA确实是锥虫mRNA的SL序列的供体。
