Kinetic analysis demonstrates a requirement for the Rat1 exonuclease in cotranscriptional pre-rRNA cleavage.
Kinetic analysis demonstrates a requirement for the Rat1 exonuclease in cotranscriptional pre-rRNA cleavage.
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DOI：
10.1371/journal.pone.0085703
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Tollervey D
中科院分区：
文献类型：
--
作者：
Axt K;French SL;Beyer AL;Tollervey D
在酵母核糖体合成过程中，三次早期裂解产生40S亚基18S rRNA组分的20S前体。这些切割既可以发生在新生转录物上（新生转录物切割；NTC），也可以发生在已经完全转录并从rDNA上释放的35S pre-rRNA上（释放转录物切割；RTC）。由于NTC和RTC的前rrna产物是相同的，这些替代途径无法通过传统的RNA分析进行评估。然而，它们可以通过代谢标记进行动力学区分，并通过动力学数据建模进行量化。这项工作的目的是利用这些方法作为一种实用的工具，以确定调解NTC和RTC利用之间的决策因素。40S和60S核糖体亚基的成熟途径在很大程度上是不同的。然而，一些早期作用的60S合成因子，包括5 ' -核酸外切酶Rat1的缺失，导致35S pre-rRNA的积累和20S pre-rRNA的合成延迟。我们推测这可能反映了NTC的损失。Rat1在5.8S和25S rRNA加工过程中起催化作用，但在这些活性之前与pre-rRNA结合。缺乏Rat1菌株的动力学数据与强还原NTC的模型效应吻合较好。新生前rrna转录物的“米勒”染色质扩散的EM可视化证实了这一点。模型进一步表明，NTC发生在有限的时间窗口内，当聚合酶转录过A2切割位点约1.5Kb时。我们推测，在NTC之前，早期作用的60S合成因子的组装是一个质量控制系统。
During yeast ribosome synthesis, three early cleavages generate the 20S precursor to the 18S rRNA component of the 40S subunits. These cleavages can occur either on the nascent transcript (nascent transcript cleavage; NTC) or on the 35S pre-rRNA that has been fully transcribed and released from the rDNA (released transcript cleavage; RTC). These alternative pathways cannot be assessed by conventional RNA analyses, since the pre-rRNA products of NTC and RTC are identical. They can, however, be distinguished kinetically by metabolic labeling and quantified by modeling of the kinetic data. The aim of this work was to use these approaches as a practical tool to identify factors that mediate the decision between utilization of NTC and RTC. The maturation pathways of the 40S and 60S ribosomal subunits are largely distinct. However, depletion of some early-acting 60S synthesis factors, including the 5′-exonuclease Rat1, leads to accumulation of the 35S pre-rRNA and delayed 20S pre-rRNA synthesis. We speculated that this might reflect the loss of NTC. Rat1 acts catalytically in 5.8S and 25S rRNA processing but binds to the pre-rRNA prior to these activities. Kinetic data for strains depleted of Rat1 match well with the modeled effects of strongly reduced NTC. This was confirmed by EM visualization of “Miller” chromatin spreads of nascent pre-rRNA transcripts. Modeling further indicates that NTC takes place in a limited time window, when the polymerase has transcribed ∼1.5Kb past the A2 cleavage site. We speculate that assembly of early-acting 60S synthesis factors is monitored as a quality control system prior to NTC.
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影响因子：
10.5
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