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. 2010 Aug 18;5(8):e12271.
doi: 10.1371/journal.pone.0012271.

Apparent non-canonical trans-splicing is generated by reverse transcriptase in vitro

Jonathan Houseley  1 , David Tollervey
Affiliations

Apparent non-canonical trans-splicing is generated by reverse transcriptase in vitro

Jonathan Houseley et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. An apparent non-canonical intron in the IGS1 R non-coding RNA.
A: 35 cycle RT-PCR across the apparent intron on cDNA synthesized with Superscript II and genomic DNA. cDNA was produced from a trf4Δ strain where this non-coding RNA is stabilized. B: Hairpin structure of IGS1 R, short homologous repeats are underlined in grey. C: 35 cycle RT-PCR across the apparent intron on trf4Δ cDNA synthesized using Superscript II at 42°C or Superscript III at 55°C. D: 35 cycle RT-PCR across the apparent intron on trf4Δ cDNA synthesized using Superscipt II or AMV. Control shows 30 cycle RT-PCR reaction across the ASC1 mRNA intron on the same cDNA samples.
Figure 2
Figure 2. An in vitro system for the analysis of apparent trans-splicing.
A: HXK1 and KRE29 substrate RNAs showing primer locations. Template switching events produced by Superscript II and AMV are indicated. B: Purified substrate RNAs. C: RT-PCR using primers complementary to each RNA on three independent RT reactions (lanes 1–3), and a no RT control (Lanes 4–5). The template for the DNA control (lanes 6–7) was HeLa cDNA with restriction fragments encompassing the entire sequence of the substrate RNAs. Upper panel 35 cycles, other panels 25 cycles. D: PCR reactions performed as in C on cDNA produced with AMV reverse transcriptase. Upper panel 35 cycles; lower panels 25 cycles.
Figure 3
Figure 3. In vitro formation of sense-antisense fusions.
A: Proposed mechanism of sense-antisense fusion formation. B: Schematic of SPT7 RNA showing primer binding sites and observed sense-antisense fusions. C: Purified SPT7 substrate. D: RT-PCR experiments performed on SPT7 substrate performed as in Fig. 2C. Upper panel shows a 32 cycle PCR reaction, other panels show 25 cycles. E: Schematic of SPT7 RNA showing primer binding sites and observed exon shuffling events. F: RT-PCR experiments performed as in d. Sequenced bands are indicated by *.
Figure 4
Figure 4. Generality of trans-splicing artifacts.
A: PCR reactions for detecting sense-antisense fusion were performed on SPT7 substrate RNA reverse transcribed with Superscript II or III at the given temperatures. Upper panel 30 cycle PCR reactions; lower panel 25 cycles. B: PCR reactions for detecting sense-antisense fusion (upper panel) and exon shuffling (middle panel) were performed on SPT7 substrate RNA reverse transcribed with Superscript II or AMV. Upper panels 32 cycle PCR reactions; lower panel 25 cycles. C: RT-PCR using primers to detect sense-antisense fusions on poly(A) tailed RNA. Upper to lower panels show 32, 25 and 30 cycle PCRs. D: PCR reactions performed as in B using Superscript II, products from two RT reactions with and two RT reactions without 6 µg ml−1 Actinomycin D are shown.
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