Massive functional mapping of a 5'-UTR by saturation mutagenesis, phenotypic sorting and deep sequencing.
Massive functional mapping of a 5'-UTR by saturation mutagenesis, phenotypic sorting and deep sequencing.
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DOI：
10.1093/nar/gkt267
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发表时间：
2013-07
影响因子：
14.9
通讯作者：
Wagner EG
中科院分区：
文献类型：
--
作者：
Holmqvist E;Reimegård J;Wagner EG
我们在这里提出了一种方法，通过结合随机突变、表型细胞分类和高通量测序，在单个实验中能够对大量突变进行功能筛选。作为测试案例，我们研究了细菌csgD信使RNA的转录后基因调控，它由一个小RNA(SRNA)调控。CsgD 5‘-非编码区内的109个碱基的序列，包含了表达和sRNA依赖调控的所有元件，突变接近饱和。我们监测了翻译GFP融合的表达，并通过荧光激活细胞分选收集了不同表达水平的细胞片段。对来自不同活性排序组分的细胞的突变质粒进行深度测序，确定了信使RNA中影响内在(翻译活性本身)和外在(基于sRNA)基因调控的重要功能位置。所获得的结果证实了先前公布的数据。除了精确定位改变表达水平的核苷酸位置外，我们的方法还揭示了在基因表达和/或调节方面沉默的突变。这种方法为研究RNA中的调控序列提供了一个简单而信息丰富的工具，特别是解决了RNA结构-功能关系(例如，sRNA介导的调控，核糖开关元件)。然而，轻微的方案修改也允许绘制蛋白质中功能DNA元件和功能重要区域的图谱。
We present here a method that enables functional screening of large number of mutations in a single experiment through the combination of random mutagenesis, phenotypic cell sorting and high-throughput sequencing. As a test case, we studied post-transcriptional gene regulation of the bacterial csgD messenger RNA, which is regulated by a small RNA (sRNA). A 109 bp sequence within the csgD 5′-UTR, containing all elements for expression and sRNA-dependent control, was mutagenized close to saturation. We monitored expression from a translational gfp fusion and collected fractions of cells with distinct expression levels by fluorescence-activated cell sorting. Deep sequencing of mutant plasmids from cells in different activity-sorted fractions identified functionally important positions in the messenger RNA that impact on intrinsic (translational activity per se) and extrinsic (sRNA-based) gene regulation. The results obtained corroborate previously published data. In addition to pinpointing nucleotide positions that change expression levels, our approach also reveals mutations that are silent in terms of gene expression and/or regulation. This method provides a simple and informative tool for studies of regulatory sequences in RNA, in particular addressing RNA structure–function relationships (e.g. sRNA-mediated control, riboswitch elements). However, slight protocol modifications also permit mapping of functional DNA elements and functionally important regions in proteins.
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影响因子：
46.9
作者：
通讯作者：
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影响因子：
3.6
作者：
Jorgensen, Mikkel Girke;Nielsen, Jesper S.;Valentin-Hansen, Poul
通讯作者：
Valentin-Hansen, Poul
影响因子：
3.6
作者：
Mika F;Busse S;Possling A;Berkholz J;Tschowri N;Sommerfeldt N;Pruteanu M;Hengge R
通讯作者：
Hengge R
影响因子：
3.6
作者：
Gerstel, U;Park, C;Römling, U
通讯作者：
Römling, U
影响因子：
3.5
作者：
Cormack, BP;Valdivia, RH;Falkow, S
通讯作者：
Falkow, S