The dynamic landscape of fission yeast meiosis alternative-splice isoforms

Abstract

Alternative splicing increases the diversity of transcriptomes and proteomes in metazoans. The extent to which alternative splicing is active and functional in unicellular organisms is less understood. Here, we exploit a single-molecule long-read sequencing technique and develop an open-source software program called SpliceHunter to characterize the transcriptome in the meiosis of fission yeast. We reveal 14,353 alternative splicing events in 17,669 novel isoforms at different stages of meiosis, including antisense and read-through transcripts. Intron retention is the major type of alternative splicing, followed by alternate "intron in exon." Seven hundred seventy novel transcription units are detected; 53 of the predicted proteins show homology in other species and form theoretical stable structures. We report the complexity of alternative splicing along isoforms, including 683 intra-molecularly co-associated intron pairs. We compare the dynamics of novel isoforms based on the number of supporting full-length reads with those of annotated isoforms and explore the translational capacity and quality of novel isoforms. The evaluation of these factors indicates that the majority of novel isoforms are unlikely to be both condition-specific and translatable but consistent with the possibility of biologically functional novel isoforms. Moreover, the co-option of these unusual transcripts into newly born genes seems likely. Together, the results of this study highlight the diversity and dynamics at the isoform level in the sexual development of fission yeast.

Long-read sequencing of nascent RNA reveals coupling among RNA processing events

“…Comparison of our nRNA LRS and published mRNA LRS data showed a decrease of partially spliced transcripts to 43% from nRNA to mRNA (Supplemental Fig. S5C), indicating that a substantial fraction of partially spliced transcripts will be stable, polyadenylated, cytoplasmic mRNAs that likely represent alternative mRNA isoforms in S. pombe (Kuang et al 2017). In conclusion, LRS of nascent transcripts provides a snapshot of splicing states for multi-B A Figure 3.…”

“…Analysis of splicing frequency on a per intron basis from nascent RNA-seq showed that most S. pombe introns are spliced cotranscriptionally (median 0.59), which is lower than in S. cerevisiae (median 0.74), Drosophila, or human cells (Brugiolo et al 2013). Notably, 5% of mRNAs contain introns; recent studies suggest that translation of retained introns contributes to protein diversity in S. pombe (Duncan and Mata 2014;Kuang et al 2017). Intron length is often associated with reduced cotranscriptional splicing (Brugiolo et al 2013).…”

“…Full-length nascent transcript sequences reveal the life history of individual (pre-)mRNAs that cannot be inferred from conventional short-read sequencing. Long-read sequencing has been previously used to characterize full-length mRNA splicing products (Sharon et al 2013;Kuang et al 2017). Here, we used it to analyze full-length nascent transcripts-splicing substrates and intermediates-relative to the progression of transcription which is marked by nascent RNA 3 ′ ends.…”

“…Middle panel: Proportion of the three splicing categories from nRNA LRS as in Figure 3B. Right panel: Proportion of the three splicing categories from total mRNA LRS (data from Kuang et al 2017). (B) Predominant "all or none" splicing in multi-intron genes.…”

Veni, vidi, vici: the success of wtf meiotic drivers in fission yeast

“…It is likely that the other identified driving wtf genes also act via a similar mechanism. Two transcripts are common amongst wtf genes and the other three bona fide wtf drive genes also contain a potential alternative translational start site near the beginning of exon 2 (Hu et al, 2017;Kuang, Boeke, & Canzar, 2016;Nuckolls et al, 2017). In addition, Hu et al (2017) found that deletions of the regions upstream of exon 1 of cw9 and cw27 generated poison-only separation of function alleles.…”