The unicellular yeast Schizosaccharomyces pombe (fission yeast) retains many of the splicing features observed in humans and is thus an excellent model to study the basic mechanisms of splicing. Nearly half the genes contain introns, but the impact of alternative splicing in gene regulation and proteome diversification remains largely unexplored. Here we leverage Oxford Nanopore Technologies native RNA sequencing (dRNA), as well as ribosome profiling data, to uncover the full range of polyadenylated transcripts and translated open reading frames. We identify 332 alternative isoforms affecting the coding sequences of 262 different genes, 97 of which occur at frequencies >20%, indicating that functional alternative splicing in S. pombe is more prevalent than previously suspected. Intron retention events make ∼80% of the cases; these events may be involved in the regulation of gene expression and, in some cases, generate novel protein isoforms, as supported by ribosome profiling data in 18 of the intron retention isoforms. One example is the rpl22 gene, in which intron retention is associated with the translation of a protein of only 13 amino acids. We also find that lowly expressed transcripts tend to have longer poly(A) tails than highly expressed transcripts, highlighting an interdependence between poly(A) tail length and transcript expression level. Finally, we discover 214 novel transcripts that are not annotated, including 158 antisense transcripts, some of which also show translation evidence. The methodologies described in this work open new opportunities to study the regulation of splicing in a simple eukaryotic model.

中文翻译：

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裂殖酵母中的天然 RNA 测序揭示了频繁的可变剪接亚型

单细胞酵母Schizosaccharomyces pombe（裂殖酵母）保留了许多在人类中观察到的剪接特征，因此是研究剪接基本机制的绝佳模型。将近一半的基因含有内含子，但可变剪接对基因调控和蛋白质组多样化的影响在很大程度上仍未得到探索。在这里，我们利用 Oxford Nanopore Technologies 天然 RNA 测序 (dRNA) 以及核糖体分析数据，来揭示全范围的聚腺苷酸化转录本和翻译的开放阅读框。我们鉴定了影响 262 个不同基因编码序列的 332 种替代亚型，其中 97 种出现频率 > 20%，表明裂殖酵母中的功能性替代剪接比以前怀疑的更普遍。内含子保留事件占案例的 80%；这些事件可能参与基因表达的调节，并且在某些情况下，产生新的蛋白质亚型，18 种内含子保留亚型的核糖体分析数据支持这一点。一个例子是rpl22基因，其中内含子保留与仅 13 个氨基酸的蛋白质的翻译有关。我们还发现低表达的转录物往往比高表达的转录物具有更长的 poly(A) 尾巴，突出了 poly(A) 尾巴长度和转录物表达水平之间的相互依赖性。最后，我们发现了 214 个未注释的新转录本，包括 158 个反义转录本，其中一些还显示了翻译证据。这项工作中描述的方法为研究简单真核模型中剪接的调控开辟了新的机会。