Apparent non-canonical trans-splicing is generated by reverse transcriptase in vitro.
Apparent non-canonical trans-splicing is generated by reverse transcriptase in vitro.
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DOI：
10.1371/journal.pone.0012271
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发表时间：
2010-08-18
期刊：
影响因子：
3.7
通讯作者：
Tollervey D
中科院分区：
文献类型：
--
作者：
Houseley J;Tollervey D
反式剪接是两个独立转录的RNA分子在体内的连接，在低等真核生物中很有特点，但长期以来被认为在后生动物中缺失。然而，最近对EST序列的生物信息学分析表明，哺乳动物中普遍存在反式剪接。这些明显剪接的转录本通常缺乏规范的剪接位点，这让我们质疑它们的真实性。特别是，逆转录酶在转录过程中对模板切换的天然能力可以产生明显的反式剪接序列。在这里，我们报告了一个体外系统，用于分析反转录中的模板切换。使用高纯度的RNA底物，我们展示了两个RNA分子之间明显的反式剪接的可重复性发生。其他已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，据报道，人类中也存在大量的反义非编码RNA。我们认为，大多数已报道的后生动物中非规范剪接的例子都是在cDNA制备过程中通过逆转录酶进行模板切换而产生的。我们进一步证明，模板切换的产物可以在不同的逆转录酶之间变化，为识别许多这些实验伪像提供了一个简单的诊断方法。
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.
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