FLAM-seq: full-length mRNA sequencing reveals principles of poly(A) tail length control
FLAM-seq: full-length mRNA sequencing reveals principles of poly(A) tail length control
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DOI：
10.1038/s41592-019-0503-y
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发表时间：
2019-09-01
期刊：
影响因子：
48
通讯作者：
Rajewsky, Nikolaus
中科院分区：
文献类型：
--
作者：
Legnini, Ivano;Alles, Jonathan;Rajewsky, Nikolaus
虽然信使RNA是理解生命的关键分子，但到目前为止，还没有方法确定内源RNA的全长序列，包括它们的Poly(A)尾巴。此外，尽管非A核苷酸可以被掺入到聚(A)尾巴中，但也没有方法对它们进行准确的测序。在这里，我们提出了全长聚(A)和mRNA测序(FLAM-SEQ)，一种快速和简单的方法，高质量的整个mRNAs测序。我们报道了一种结合单分子测序的互补DNA文库制备方法来进行FLAM-SEQ。使用人类细胞系、脑器官和秀丽线虫，我们发现FLAM-SEQ为每个样本提供了数千个不同基因的高质量全长mRNA序列。我们发现，3‘非翻译区长度与Poly(A)尾长相关，同一基因的不同聚腺苷酸化位点和不同启动子与不同的尾长相关，尾巴含有大量的胞嘧啶。
Although messenger RNAs are key molecules for understanding life, until now, no method has existed to determine the fulllength sequence of endogenous mRNAs including their poly(A) tails. Moreover, although non-A nucleotides can be incorporated in poly(A) tails, there also exists no method to accurately sequence them. Here, we present full-length poly(A) and mRNA sequencing (FLAM-seq), a rapid and simple method for high-quality sequencing of entire mRNAs. We report a complementary DNA library preparation method coupled to single-molecule sequencing to perform FLAM-seq. Using human cell lines, brain organoids and Caenorhabditis elegans we show that FLAM-seq delivers high-quality full-length mRNA sequences for thousands of different genes per sample. We find that 3' untranslated region length is correlated with poly(A) tail length, that alternative polyadenylation sites and alternative promoters for the same gene are linked to different tail lengths, and that tails contain a substantial number of cytosines.