Ultrashort and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution.
Ultrashort and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution.
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DOI：
10.1101/gr.131847.111
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发表时间：
2012-10
期刊：
影响因子：
7
通讯作者：
Dölken L
中科院分区：
文献类型：
--
作者：
Windhager L;Bonfert T;Burger K;Ruzsics Z;Krebs S;Kaufmann S;Malterer G;L'Hernault A;Schilhabel M;Schreiber S;Rosenstiel P;Zimmer R;Eick D;Friedel CC;Dölken L
RNA的合成和衰变速率决定了细胞RNA的稳态水平。4-硫脲（4sU）对新转录RNA的代谢标记可以揭示RNA合成和衰变速率的相对贡献。然而，RNA加工的动力学至今仍未得到解决。在这里，我们展示了超短4su标记不仅提供了真核生物基因表达的快照图片，而且当与渐进式4su标记和RNA-seq相结合时，可以在核苷酸分辨率上揭示全局RNA加工动力学。使用这种方法，我们鉴定了快速和缓慢剪接/降解的内含子类别。有趣的是，每一类剪接动力学的特点是与内含子长度、基因长度和剪接位点强度有明显的关联。对于一大组内含子，我们还观察到在初级转录本中长时间保留，但在稍后的时间点上有效的二次剪接或降解。最后，我们发现大多数（但不是全部）含小核仁（sno） rna内含子的加工效率非常低，大多数内含子被剪接和降解，而不是加工成成熟的snorna。总之，我们的研究对RNA加工动力学产生了无与伦比的见解，并为研究RNA加工的分子机制及其对基因表达调控的贡献提供了工具。
RNA synthesis and decay rates determine the steady-state levels of cellular RNAs. Metabolic tagging of newly transcribed RNA by 4-thiouridine (4sU) can reveal the relative contributions of RNA synthesis and decay rates. The kinetics of RNA processing, however, had so far remained unresolved. Here, we show that ultrashort 4sU-tagging not only provides snapshot pictures of eukaryotic gene expression but, when combined with progressive 4sU-tagging and RNA-seq, reveals global RNA processing kinetics at nucleotide resolution. Using this method, we identified classes of rapidly and slowly spliced/degraded introns. Interestingly, each class of splicing kinetics was characterized by a distinct association with intron length, gene length, and splice site strength. For a large group of introns, we also observed long lasting retention in the primary transcript, but efficient secondary splicing or degradation at later time points. Finally, we show that processing of most, but not all small nucleolar (sno)RNA-containing introns is remarkably inefficient with the majority of introns being spliced and degraded rather than processed into mature snoRNAs. In summary, our study yields unparalleled insights into the kinetics of RNA processing and provides the tools to study molecular mechanisms of RNA processing and their contribution to the regulation of gene expression.
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影响因子：
64.8
作者：
通讯作者：
--
影响因子：
64.5
作者：
Hafner M;Landthaler M;Burger L;Khorshid M;Hausser J;Berninger P;Rothballer A;Ascano M Jr;Jungkamp AC;Munschauer M;Ulrich A;Wardle GS;Dewell S;Zavolan M;Tuschl T
通讯作者：
Tuschl T
影响因子：
12.3
作者：
Langmead B;Schatz MC;Lin J;Pop M;Salzberg SL
通讯作者：
Salzberg SL
影响因子：
16
作者：
Hirose, T;Shu, MD;Steitz, JA
通讯作者：
Steitz, JA
DOI：
10.1111/j.2517-6161.1995.tb02031.x
发表时间：
1995-01-01
影响因子：
5.8
作者：
BENJAMINI, Y;HOCHBERG, Y
通讯作者：
HOCHBERG, Y