Roles for transcript leaders in translation and mRNA decay revealed by transcript leader sequencing.
Roles for transcript leaders in translation and mRNA decay revealed by transcript leader sequencing.
复制标题
DOI：
10.1101/gr.150342.112
复制
发表时间：
2013-06
期刊：
影响因子：
7
通讯作者：
Gilbert WV
中科院分区：
文献类型：
--
作者：
Arribere JA;Gilbert WV
转录前导序列（TL）对mRNA的翻译和稳定性有着深远的影响。为了在全基因组范围内定位TL边界，我们开发了TL测序（TL-seq），这是一种结合酶捕获m7 G加帽mRNA 5′端与高通量测序的技术。TL-seq鉴定了大多数酵母基因的mRNA起始位点，并揭示了许多基因内TL异质性的例子。令人惊讶的是，TL-seq在6%的蛋白质编码区内鉴定了转录起始位点，并且这些位点集中在ORF的5′端附近。此外，核糖体密度分析显示这些截短的mRNA被翻译。翻译相关的TL-seq（TATL-seq）将TL-seq与多核糖体分级分离相结合，使得能够注释TL，并同时测定其在翻译中的功能。使用TATL-seq来解决TL特征与下游ORF翻译之间的关系，我们观察到上游AUG（uAUG），而没有其他上游密码子，与翻译不良和无义介导的mRNA衰变（NMD）相关。我们还鉴定了数百个具有非常短的TL的基因，并证明短TL与注释起始密码子处的翻译起始差和下游AUG处的起始增加相关。这经常导致框外翻译和随后在提前终止密码子处终止，最终导致转录物的NMD。与以前的方法不同，我们的技术能够观察到数百个基因的替代TL变体，并揭示了具有不同TL异构体的基因在翻译中的显着差异。TL-seq和TATL-seq是用于注释和功能表征TL的有用工具，并且可以应用于任何真核系统以研究TL介导的基因表达调控。
Transcript leaders (TLs) can have profound effects on mRNA translation and stability. To map TL boundaries genome-wide, we developed TL-sequencing (TL-seq), a technique combining enzymatic capture of m7G-capped mRNA 5′ ends with high-throughput sequencing. TL-seq identified mRNA start sites for the majority of yeast genes and revealed many examples of intragenic TL heterogeneity. Surprisingly, TL-seq identified transcription initiation sites within 6% of protein-coding regions, and these sites were concentrated near the 5′ ends of ORFs. Furthermore, ribosome density analysis showed these truncated mRNAs are translated. Translation-associated TL-seq (TATL-seq), which combines TL-seq with polysome fractionation, enabled annotation of TLs, and simultaneously assayed their function in translation. Using TATL-seq to address relationships between TL features and translation of the downstream ORF, we observed that upstream AUGs (uAUGs), and no other upstream codons, were associated with poor translation and nonsense-mediated mRNA decay (NMD). We also identified hundreds of genes with very short TLs, and demonstrated that short TLs were associated with poor translation initiation at the annotated start codon and increased initiation at downstream AUGs. This frequently resulted in out-of-frame translation and subsequent termination at premature termination codons, culminating in NMD of the transcript. Unlike previous approaches, our technique enabled observation of alternative TL variants for hundreds of genes and revealed significant differences in translation in genes with distinct TL isoforms. TL-seq and TATL-seq are useful tools for annotation and functional characterization of TLs, and can be applied to any eukaryotic system to investigate TL-mediated regulation of gene expression.
登录
查看更多内容
影响因子：
4.8
作者：
Chang, KJ;Wang, CC
通讯作者：
Wang, CC
影响因子：
56.9
作者：
Gilbert, Wendy V.;Zhou, Kaihong;Doudna, Jennifer A.
通讯作者：
Doudna, Jennifer A.
使用启发式和进化保护鉴定酵母基因组中推定的调节性上游ORF。
影响因子：
3
作者：
通讯作者：
--
影响因子：
16
作者：
He, F;Li, XR;Jacobson, A
通讯作者：
Jacobson, A
影响因子：
9.8
作者：
Hendrickson DG;Hogan DJ;McCullough HL;Myers JW;Herschlag D;Ferrell JE;Brown PO
通讯作者：
Brown PO