An improved MS2 system for accurate reporting of the mRNA life cycle.
An improved MS2 system for accurate reporting of the mRNA life cycle.
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DOI：
10.1038/nmeth.4502
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发表时间：
2018-01
期刊：
影响因子：
48
通讯作者：
Singer RH
中科院分区：
文献类型：
--
作者：
Tutucci E;Vera M;Biswas J;Garcia J;Parker R;Singer RH
MS 2-MCP系统允许以高时间和空间分辨率对mRNA生命周期的多个步骤进行成像。然而，对于短寿命的mRNA，MS 2外壳蛋白（MCP）与MS 2结合位点（MBS）的紧密结合保护RNA不被有效降解，混淆了mRNA调控的研究。在这里，我们描述了一个报告系统（MBSV 6）与MCP的亲和力降低，允许mRNA降解，同时保留单分子检测确定的smFISH或活成像。在S.酿酒酵母正常降解。因此，快速翻转的mRNA在其整个生命周期中被成像。MBSV 6在活哺乳动物细胞中提供单分子检测。MBSV 6报告显示，协调募集的mRNA在专门的结构，如P-体在应力过程中没有发生，降解是不均匀分布在细胞质中。
The MS2-MCP system allows imaging multiple steps of the mRNA life cycle with high temporal and spatial resolution. However for short-lived mRNAs, the tight binding of the MS2 coat protein (MCP) to the MS2 binding sites (MBS) protects the RNA from being efficiently degraded, confounding the study of mRNA regulation. Here, we describe a reporter system (MBSV6) with reduced affinity for the MCP, allowing mRNA degradation while preserving single molecule detection determined by smFISH or live imaging. Constitutive mRNAs (MDN1 and DOA1) or highly-regulated mRNAs (GAL1 and ASH1) endogenously tagged with MBSV6 in S. cerevisiae degrade normally. As a result, rapidly turning over mRNAs were imaged throughout their complete life cycle. MBSV6 provided single molecule detection in live mammalian cells. The MBSV6 reporter revealed that coordinated recruitment of mRNAs at specialized structures such as P-bodies during stress did not occur and that degradation was heterogeneously distributed in the cytoplasm.
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影响因子：
4.5
作者：
Brodsky, AS;Silver, PA
通讯作者：
Silver, PA
DOI：
10.1126/science.1202142
发表时间：
2011-04-22
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Larson DR;Zenklusen D;Wu B;Chao JA;Singer RH
通讯作者：
Singer RH
将菠菜标签的RNA和基因定位与活酵母中的基因表达相结合。
影响因子：
16.6
作者：
Guet D;Burns LT;Maji S;Boulanger J;Hersen P;Wente SR;Salamero J;Dargemont C
通讯作者：
Dargemont C
影响因子：
3.3
作者：
Kshirsagar, M;Parker, R
通讯作者：
Parker, R
影响因子：
56.9
作者：
Long, RM;Singer, RH;Jansen, RP
通讯作者：
Jansen, RP