Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.
PLoS One. 2010 Aug 18;5(8):e12271. doi: 10.1371/journal.pone.0012271.
Trans-splicing, the in vivo joining of two independently transcribed RNA molecules, is well characterized in lower eukaryotes, but was long thought absent from metazoans. However, recent bioinformatic analyses of EST sequences suggested widespread trans-splicing in mammals. These apparently spliced transcripts generally lacked canonical splice sites, leading us to question their authenticity. Particularly, the native ability of reverse transcriptase enzymes to template switch during transcription could produce apparently trans-spliced sequences.
Here we report an in vitro system for the analysis of template switching in reverse transcription. Using highly purified RNA substrates, we show the reproducible occurrence of apparent trans-splicing between two RNA molecules. Other reported non-canonical splicing events such as exon shuffling and sense-antisense fusions were also readily detected. The latter caused the production of apparent antisense non-coding RNAs, which are also reported to be abundant in humans.
We propose that most reported examples of non-canonical splicing in metazoans arise through template switching by reverse transcriptase during cDNA preparation. We further show that the products of template switching can vary between reverse transcriptases, providing a simple diagnostic for identifying many of these experimental artifacts.
转译拼接，即两个独立转录的 RNA 分子在体内的连接，在低等真核生物中已有很好的研究，但长期以来被认为不存在于后生动物中。然而，最近对 EST 序列的生物信息学分析表明，哺乳动物中广泛存在转译拼接。这些明显拼接的转录本通常缺乏规范的剪接位点，这使我们对其真实性产生了质疑。特别是，逆转录酶在转录过程中模板转换的固有能力可能产生明显的转译拼接序列。
本文报道了一种体外分析逆转录中模板转换的系统。使用高度纯化的 RNA 底物，我们证明了两个 RNA 分子之间明显的转译拼接的重现发生。其他报道的非规范剪接事件，如外显子改组和正义-反义融合，也很容易被检测到。后者导致了明显的反义非编码 RNA 的产生，这些 RNA 在人类中也被报道是丰富的。
我们提出，后生动物中大多数报道的非规范剪接实例都是通过 cDNA 制备过程中逆转录酶的模板转换产生的。我们进一步表明，模板转换的产物在逆转录酶之间可能不同，这为识别许多这些实验假象提供了一个简单的诊断方法。
