Assessing long-distance RNA sequence connectivity via RNA-templated DNA–DNA ligation

Abstract

Many RNAs, including pre-mRNAs and long non-coding RNAs, can be thousands of nucleotides long and undergo complex post-transcriptional processing. Multiple sites of alternative splicing within a single gene exponentially increase the number of possible spliced isoforms, with most human genes currently estimated to express at least ten. To understand the mechanisms underlying these complex isoform expression patterns, methods are needed that faithfully maintain long-range exon connectivity information in individual RNA molecules. In this study, we describe SeqZip, a methodology that uses RNA-templated DNA–DNA ligation to retain and compress connectivity between distant sequences within single RNA molecules. Using this assay, we test proposed coordination between distant sites of alternative exon utilization in mouse Fn1, and we characterize the extraordinary exon diversity of Drosophila melanogaster Dscam1.DOI: http://dx.doi.org/10.7554/eLife.03700.001

Intron-targeted mutagenesis reveals roles forDscam1RNA pairing-mediated splicing bias in neuronal wiring

“…This may explain why the docking site deletion markedly reduced inclusion of exon 9.9 but not exon 9.6, even though exon 9.9 resided farther from the docking site than exon 9.6. These observations together with disrupting and compensatory mutations on RNA secondary structures in exon 9 cluster of silkworm Consistent with previous studies 6,21,34 , comparative analyses did not reveal any significant difference between wild type and DscamΔ4D −/− flies in the inclusion frequency of most alternative exons 6 or 9, showing largely independent splicing of exon clusters 4, 6, and 9. ( Fig.…”

Circular RNAs: analysis, expression and potential functions

“…Template switching during the RT step ( Fig. 2A) can lead to a variety of artifactual templates that could give rise to PCR products even with inverse primers (Cocquet et al, 2006;Luo and Taylor, 1990;Roy et al, 2015). Thus, it is imperative that the PCR products are sequenced to verify the diagnostic scrambled junction.…”

Detecting circular RNAs: bioinformatic and experimental challenges

“…In fact, such artefacts can dominate the results for some genes, with specific examples suggesting that these artefacts can account for 34–55% of the isoforms detected, sometimes with specific artefactual isoforms detected at higher levels than any of the truly expressed isoforms 44 . As such artefacts can be generated at high levels, filtering out the circRNAs that are supported by only a few reads is not sufficient to eliminate the false positives generated due to template switching, and can result in true isoforms being overlooked.…”