Current approaches to profiling tissue-specific gene expression in C. elegans require delicate manipulation and are difficult under certain conditions, e.g. from dauer or aging worms. Researchers at Tsinghua University have developed an easy and robust method for tissue-specific RNA-seq by taking advantage of the endogenous trans-splicing process. In this method, transgenic worms are generated in which a spliced leader (SL) RNA gene is fused with a sequence tag and driven by a tissue-specific promoter. Only in the tissue of interest, the tagged SL RNA gene is transcribed and then trans-spliced onto mRNAs. The tag allows enrichment and sequencing of mRNAs from that tissue only.
As a proof of principle, the researchers profiled the muscle transcriptome, which showed high coverage and efficient enrichment of muscle specific genes, with low background noise. To demonstrate the robustness of the method, they profiled muscle gene expression in dauer larvae and aging worms, revealing gene expression changes consistent with the physiology of these stages. The resulting muscle transcriptome also revealed 461 novel RNA transcripts, likely muscle-expressed long non-coding RNAs.
Schematic representation of the SRT method
(A) The worm strain carries a sequence-tagged SL1 transgene driven by a tissue-specific promoter. Only in this tissue, the transgenic tag::SL1 will express and trans-splice onto pre-mRNA, resulting in the tag::SL1 fused to the 5′ end of mRNAs. The green bar represents the G16C point mutation introduced to SL1 to distinguish from endogenous wild-type SL1. (B) Flowchart of sequencing library construction. After RNAs were purified from a large number of transgenic worms, those from the tissue of interest were pulled down using the tag sequence as bait. These enriched RNAs are reverse transcribed using polyT primer and polymerase chain reaction (PCR) amplified using the tag as a primer. The tag-based PCR further enriches mRNAs from the target tissue. PCR products are used to construct Illumina sequencing libraries (Supplementary Figure S2A shows more detail).
In summary, the splicing-based RNA tagging (SRT) method provides a convenient and robust tool to profile trans-spliced genes and identify novel transcripts in a tissue-specific manner, with a low false positive rate.