Herzel Lydia, Neugebauer Karla M
Molecular Biophysics and Biochemistry, Yale University, 333 Cedar St, New Haven, CT 06520, United States.
Molecular Biophysics and Biochemistry, Yale University, 333 Cedar St, New Haven, CT 06520, United States.
Methods. 2015 Sep 1;85:36-43. doi: 10.1016/j.ymeth.2015.04.024. Epub 2015 Apr 27.
During gene expression, protein-coding transcripts are shaped by multiple processing events: 5' end capping, pre-mRNA splicing, RNA editing, and 3' end cleavage and polyadenylation. These events are required to produce mature mRNA, which can be subsequently translated. Nearly all of these RNA processing steps occur during transcription, while the nascent RNA is still attached to the DNA template by RNA polymerase II (i.e. co-transcriptionally). Polyadenylation occurs after 3' end cleavage or post-transcriptionally. Pre-mRNA splicing - the removal of introns and ligation of exons - can be initiated and concluded co-transcriptionally, although this is not strictly required. Recently, a number of studies using global methods have shown that the majority of splicing is co-transcriptional, yet not all published studies agree in their conclusions. Short read sequencing of RNA (RNA-Seq) is the prevailing approach to measuring splicing levels in nascent RNA, mRNA or total RNA. Here, we compare four different strategies for analyzing and quantifying co-transcriptional splicing. To do so, we reanalyze two nascent RNA-Seq datasets of the same species, but different cell type and RNA isolation procedure. Average co-transcriptional splicing values calculated on a per intron basis are similar, independent of the strategy used. We emphasize the technical requirements for identifying co-transcriptional splicing events with high confidence, e.g. how to calculate co-transcriptional splicing from nascent RNA- versus mRNA-Seq data, the number of biological replicates needed, depletion of polyA+RNA, and appropriate normalization. Finally, we present guidelines for planning a nascent RNA-Seq experiment.
在基因表达过程中，蛋白质编码转录本通过多种加工事件形成：5' 端加帽、前体 mRNA 剪接、RNA 编辑以及 3' 端切割和多聚腺苷酸化。这些事件是产生成熟 mRNA 所必需的，成熟 mRNA 随后可被翻译。几乎所有这些 RNA 加工步骤都发生在转录过程中，此时新生 RNA 仍通过 RNA 聚合酶 II 附着在 DNA 模板上（即共转录过程）。多聚腺苷酸化发生在 3' 端切割之后或转录后。前体 mRNA 剪接——去除内含子并连接外显子——可以在共转录过程中起始和完成，尽管这并非严格必需。最近，一些使用全局方法的研究表明，大多数剪接是共转录的，但并非所有已发表的研究都认同这一结论。RNA 的短读测序（RNA-Seq）是测量新生 RNA、mRNA 或总 RNA 中剪接水平的主流方法。在这里，我们比较了四种分析和量化共转录剪接的不同策略。为此，我们重新分析了同一物种但不同细胞类型和 RNA 分离程序的两个新生 RNA-Seq 数据集。基于每个内含子计算的平均共转录剪接值相似，与所使用的策略无关。我们强调了高置信度识别共转录剪接事件的技术要求，例如如何从新生 RNA 与 mRNA-Seq 数据计算共转录剪接、所需的生物学重复次数、去除 polyA+RNA 以及适当的标准化。最后，我们给出了规划新生 RNA-Seq 实验的指南。
