The Mina & Everard Goodman Faculty of Life Sciences, and Advanced Materials and Nanotechnology Institute Bar-Ilan University, Ramat-Gan 52900, Israel.
Nucleic Acids Res. 2010 Jun;38(10):e114. doi: 10.1093/nar/gkq065. Epub 2010 Feb 16.
In trypanosomes a 39 nucleotide exon, the spliced leader (SL) is donated to all mRNAs from a small RNA, the SL RNA, by trans-splicing. Since the discovery of trans-splicing in trypanosomes two decades ago, numerous attempts failed to reconstitute the reaction in vitro. In this study, a crude whole-cell extract utilizing the endogenous SL RNA and synthetic tubulin pre-mRNA were used to reconstitute the trans-splicing reaction. An RNase protection assay was used to detect the trans-spliced product. The reaction was optimized and shown to depend on ATP and intact U2 and U6 snRNPs. Mutations introduced at the polypyrimidine tract and the AG splice site reduced the reaction efficiency. To simplify the assay, RT-PCR and quantitative real-time PCR assays were established. The system was used to examine the structural requirements for SL RNA as a substrate in the reaction. Interestingly, synthetic SL RNA assembled poorly to its cognate particle and was not utilized in the reaction. However, SL RNA synthesized in cells lacking Sm proteins, which is defective in cap-4 modification, was active in the reaction. This study is the first step towards further elucidating the mechanism of trans-splicing, an essential reaction which determines the trypanosome transcriptome.
在锥虫中，通过反式剪接，39 个核苷酸的外显子——拼接 leader（SL）由一个小 RNA——SL RNA 提供给所有的 mRNA。自从二十年前在锥虫中发现反式剪接以来，许多试图在体外重新构建该反应的尝试都失败了。在这项研究中，使用内源性 SL RNA 和合成的微管蛋白前体 mRNA 的粗制全细胞提取物来重新构建反式剪接反应。使用 RNase 保护测定法来检测反式剪接产物。优化了该反应并表明它依赖于 ATP 和完整的 U2 和 U6 snRNP。在多嘧啶区和 AG 剪接位点引入的突变降低了反应效率。为了简化测定法，建立了 RT-PCR 和定量实时 PCR 测定法。该系统用于检查 SL RNA 作为反应中底物的结构要求。有趣的是，合成的 SL RNA 与其同源颗粒组装不良，并且未在反应中使用。然而，在缺乏 Sm 蛋白的细胞中合成的 SL RNA（其在帽 4 修饰中存在缺陷）在反应中是活跃的。这项研究是进一步阐明反式剪接机制的第一步，反式剪接是决定锥虫转录组的必要反应。
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