Quantitative analysis of C. elegans transcripts by Nanopore direct-cDNA sequencing reveals terminal hairpins in non trans-spliced mRNAs

Abstract

Nematode mRNA processing involves a trans-splicing step through which a 21nt sequence from a snRNP replaces the original 5' end of the primary transcript. It has long been held that 70% of C. elegans mRNAs are submitted to trans-splicing. Our recent work suggested that the mechanism is more pervasive but not fully captured by mainstream transcriptome sequencing methods. In this study, we used Oxford Nanopore's long-read amplification-free sequencing technology to perform a comprehensive analysis of trans-splicing in worms. We demonstrated that spliced leader (SL) sequences presence at the 5' end of the messengers affected library preparation and generated sequencing artefacts due to their self-complementarity. Consistent with our previous observations, we found evidence of trans-splicing for most genes. However, a subset of genes appears to be only marginally trans-spliced. These messengers all share the capacity to generate a 5' terminal hairpin structure mimicking the SL structure providing a mechanistic explanation for their non conformity. Altogether, our data provides the most comprehensive quantitative analysis of SL usage to date in C. elegans.