apparent non-canonical trans-splicing is generated by reverse transcriptase in vitro明显的非规范trans-splicing是由逆转录酶体外.pdfVIP

Apparent Non-Canonical Trans-Splicing Is Generated by Reverse Transcriptase In Vitro ¤ Jonathan Houseley* , David Tollervey* Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, United Kingdom Abstract Background: 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. Principal Findings: 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. Conclusions: 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. Citation: Houseley J, Tollervey D (2010) Apparent Non-Canonical Trans-Splicing Is Generated by Reverse Transcriptase In Vitro. PLoS ONE 5(8): e