Université de Bordeaux, Inserm U1212, CNRS UMR5320, Institut Européen de Chimie et Biologie (IECB), 2, rue Robert Escarpit, 33607, Pessac, France.
Department of Neurobiology, Wise Faculty of Life Sciences & Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel.
Nat Commun. 2023 Mar 3;14(1):1229. doi: 10.1038/s41467-023-36915-0.
In nematodes and kinetoplastids, mRNA processing involves a trans-splicing step through which a short 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. Here we use Oxford Nanopore's long-read amplification-free sequencing technology to perform a comprehensive analysis of trans-splicing in worms. We demonstrate that spliced leader (SL) sequences at the 5' end of the mRNAs affect library preparation and generate sequencing artefacts due to their self-complementarity. Consistent with our previous observations, we find evidence of trans-splicing for most genes. However, a subset of genes appears to be only marginally trans-spliced. These mRNAs all share the capacity to generate a 5' terminal hairpin structure mimicking the SL structure and offering a mechanistic explanation for their non conformity. Altogether, our data provide a comprehensive quantitative analysis of SL usage in C. elegans.
在线虫和动基体目生物中,mRNA 处理涉及一个转剪接步骤,通过该步骤,snRNP 中的一小段序列取代了初级转录本的原始 5' 端。长期以来,人们一直认为 70%的秀丽隐杆线虫的 mRNA 经历了转剪接。我们最近的工作表明,这种机制比主流转录组测序方法更普遍,但并没有完全捕获到。在这里,我们使用 Oxford Nanopore 的长读长扩增免费测序技术对线虫中转剪接进行了全面分析。我们证明,mRNA 5' 端的拼接先导 (SL) 序列会影响文库制备,并由于其自我互补性而产生测序伪影。与我们之前的观察结果一致,我们发现大多数基因都有转剪接的证据。然而,一部分基因似乎只有轻微的转剪接。这些 mRNA 都具有产生类似于 SL 结构的 5' 端发夹结构的能力,为它们的非一致性提供了一种机制解释。总的来说,我们的数据提供了秀丽隐杆线虫中 SL 使用的全面定量分析。