Single-Molecule Analysis of the Influenza Virus Replication Initiation Mechanism
Single-Molecule Analysis of the Influenza Virus Replication Initiation Mechanism
复制标题
流感病毒复制启动机制的单分子分析
DOI：
10.1016/j.bpj.2017.11.1370
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发表时间：
2018
影响因子：
3.4
通讯作者：
Robb N
中科院分区：
文献类型：
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作者：
Robb N
1240-核散斑联合会对B149转录的增强作用Jiah Kim，Nimish卡纳，Andrew S.贝尔蒙特。伊利诺伊大学厄巴纳-香槟分校，厄巴纳，伊利诺伊州，美国。教科书上对核斑点的看法是，它们主要是作为RNA加工因子的储存位点。一个长期存在的竞争模型是，核斑点作为一个转录枢纽的一个子集的活性基因;然而，这个枢纽模型是基于分析只有几个基因。通过TSA-Seq将Hi-CA 1活性亚区室分配为对应于位于靠近核斑点的基因组区域，现在极大地支持了核斑点作为转录中心的这种模型，然而这种TSA-Seq作图仍然是相关的。在这里，我们证明了斑点协会和热休克诱导的转录激活使用BAC转基因系统，密切概括的内源性热休克蛋白70基因座的转录诱导动力学之间的紧密的时间关系。smRNA FISH显示，热休克后3-5分钟，Hsp 70 BAC转基因和内源性Hsp 70基因座同步激活转录。然而，斑点相关的转基因有4，12，和50倍增加的新生转录水平相比，非相关的热休克后30分钟，1小时，2小时，分别为13倍的差异，在2小时观察到内源性Hsp 70位点。此外，活细胞成像揭示了稳健的转录激活中的几分钟延迟与BAC转基因移动到核斑点所需的时间相关。活细胞数据的进一步分析揭示了BAC转基因远离斑点后转录降低的例子，甚至在BAC转基因形成新的斑点关联后再次增加或重新开始。斑点关联和基因表达变化之间的这种强时间相关性表明，基因表达的随机变化可能与活性基因子集相对于核斑点的空间定位有关。
1240-Pos Board B149 Transcription Enhancement by Nuclear Speckle Association Jiah Kim, Nimish Khanna, Andrew S. Belmont. University of Illinois at Urbana-Champaign, Urbana, IL, USA. The textbook view of nuclear speckles is that they serve primarily as storage sites for RNA processing factors. A long-standing competing model has been that nuclear speckles act as a transcriptional hub for a subset of active genes; however, this hub model was based on analysis of just a few genes. The assignment by TSA-Seq of the Hi-C A1 active subcompartment as corresponding to genomic regions positioned close to nuclear speckles, now greatly bolsters this model of nuclear speckles as a transcriptional hub, yet this TSA-Seq mapping remains correlative. Here we demonstrate a tight temporal relationship between speckle association and heat-shock induced transcriptional activation using a BAC transgene system that closely recapitulates the transcriptional induction kinetics of the endogenous Hsp70 locus. smRNA FISH shows that both Hsp70 BAC transgenes and the endogenous Hsp70 locus synchronously activate transcription between 3-5 minutes after heat shock. However, speckleassociated transgenes have 4, 12, and 50-fold increased levels of nascent transcripts compared to non-associated ones at 30 mins, 1 hr, and 2 hrs after heat shock, respectively; a 13-fold difference at 2 hrs is observed for the endogenous Hsp70 loci. Moreover, live-cell imaging reveals several minute delays in robust transcriptional activation correlate with the time required for a BAC transgene to move to a nuclear speckle. Further analysis of live-cell data reveals examples where transcription decreases after BAC transgenes move away from speckles, and even increase again or restart after the BAC transgene forms a new speckle association. This strong temporal correlation between speckle association and changes in gene expression suggests that stochastic variations in gene expression may be related to spatial positioning relative to nuclear speckles for a subset of active genes.