The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing.
The in vivo kinetics of RNA polymerase II elongation during co-transcriptional splicing.
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DOI：
10.1371/journal.pbio.1000573
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发表时间：
2011-01-11
期刊：
影响因子：
9.8
通讯作者：
Shav-Tal Y
中科院分区：
文献类型：
--
作者：
Brody Y;Neufeld N;Bieberstein N;Causse SZ;Böhnlein EM;Neugebauer KM;Darzacq X;Shav-Tal Y
动力学分析表明，RNA聚合酶的延伸动力学不受共转录剪接的调节，并且转录后剪接可在转录位点进行，无需聚合酶存在。
在转录的前体mRNA上发生的RNA加工事件包括加帽、剪接、编辑、3′加工和多聚腺苷酸化。这些过程大多在RNA聚合酶II（Pol II）参与转录延伸时共转录发生。Pol II延伸速率如何受剪接影响尚不清楚。我们构建了一系列可诱导的基因构建体，其中包含数量逐渐增加的内含子和外显子，它们稳定整合在人类细胞中，作为活跃转录的基因位点。通过在体内监测这些基因上转录和剪接机制的关联，我们发现只有U1小核核糖核蛋白（snRNP）定位于无内含子基因，这与U1 snRNP在转录中不依赖剪接的作用一致。相反，所有snRNP都聚集在含内含子的基因上，并且内含子数量的增加会使招募的剪接体成分增多。这表明新生RNA可同时组装多个剪接体。对体内Pol II延伸的动力学测量、Pol II染色质免疫沉淀（ChIP）以及使用剪接抑制素和美阿霉素剪接抑制剂的实验表明，聚合酶延伸速率与正在进行的剪接无关。这项研究表明，在本文所研究的模型基因中，转录延伸动力学独立于剪接进行。令人惊讶的是，在转录终止后，在转录位点检测到多聚腺苷酸化mRNA的滞留。这表明聚合酶在剪接完成之前从染色质上释放，并且前体mRNA在转录后进行加工时仍与基因末端附近的染色质相连。
在真核生物转录过程中，从RNA聚合酶II产生的前体mRNA会经历一系列加工事件。这些包括5′加帽、剪接过程中的内含子切除和外显子连接、3′末端加工以及多聚腺苷酸化。加工事件共转录发生，这意味着在聚合酶仍在进行转录时，多种酶会在前体mRNA上组装。共转录mRNA加工的概念引发了关于转录聚合酶和加工机制之间可能存在的耦合的问题。在这里，我们研究了剪接机制（剪接体）的共转录组装如何影响RNA聚合酶的延伸动力学。通过活细胞显微镜技术，我们追踪了包含数量逐渐增加的内含子的基因的转录动力学，并测量了转录和剪接因子的招募情况。令人惊讶的是，一部分剪接因子被招募到无内含子基因，这意味着存在一种针对内含子序列的与聚合酶耦合的扫描机制。有内含子和无内含子基因上的聚合酶延伸速率没有差异，这表明剪接体不调节延伸动力学。包括抑制剪接或转录的实验，以及随机计算模拟，都表明当聚合酶终止先于剪接完成时，前体mRNA可保留在基因上。总之，我们表明聚合酶延伸动力学不受新生前体mRNA上的剪接事件影响，增加剪接会导致更多剪接因子被招募到mRNA上，并且在没有聚合酶的情况下，转录后剪接可在转录位点进行。
Kinetic analysis shows that RNA polymerase elongation kinetics are not modulated by co-transcriptional splicing and that post-transcriptional splicing can proceed at the site of transcription without the presence of the polymerase. RNA processing events that take place on the transcribed pre-mRNA include capping, splicing, editing, 3′ processing, and polyadenylation. Most of these processes occur co-transcriptionally while the RNA polymerase II (Pol II) enzyme is engaged in transcriptional elongation. How Pol II elongation rates are influenced by splicing is not well understood. We generated a family of inducible gene constructs containing increasing numbers of introns and exons, which were stably integrated in human cells to serve as actively transcribing gene loci. By monitoring the association of the transcription and splicing machineries on these genes in vivo, we showed that only U1 snRNP localized to the intronless gene, consistent with a splicing-independent role for U1 snRNP in transcription. In contrast, all snRNPs accumulated on intron-containing genes, and increasing the number of introns increased the amount of spliceosome components recruited. This indicates that nascent RNA can assemble multiple spliceosomes simultaneously. Kinetic measurements of Pol II elongation in vivo, Pol II ChIP, as well as use of Spliceostatin and Meayamycin splicing inhibitors showed that polymerase elongation rates were uncoupled from ongoing splicing. This study shows that transcription elongation kinetics proceed independently of splicing at the model genes studied here. Surprisingly, retention of polyadenylated mRNA was detected at the transcription site after transcription termination. This suggests that the polymerase is released from chromatin prior to the completion of splicing, and the pre-mRNA is post-transcriptionally processed while still tethered to chromatin near the gene end. The pre-mRNA emerging from RNA polymerase II during eukaryotic transcription undergoes a series of processing events. These include 5′-capping, intron excision and exon ligation during splicing, 3′-end processing, and polyadenylation. Processing events occur co-transcriptionally, meaning that a variety of enzymes assemble on the pre-mRNA while the polymerase is still engaged in transcription. The concept of co-transcriptional mRNA processing raises questions about the possible coupling between the transcribing polymerase and the processing machineries. Here we examine how the co-transcriptional assembly of the splicing machinery (the spliceosome) might affect the elongation kinetics of the RNA polymerase. Using live-cell microscopy, we followed the kinetics of transcription of genes containing increasing numbers of introns and measured the recruitment of transcription and splicing factors. Surprisingly, a sub-set of splicing factors was recruited to an intronless gene, implying that there is a polymerase-coupled scanning mechanism for intronic sequences. There was no difference in polymerase elongation rates on genes with or without introns, suggesting that the spliceosome does not modulate elongation kinetics. Experiments including inhibition of splicing or transcription, together with stochastic computational simulation, demonstrated that pre-mRNAs can be retained on the gene when polymerase termination precedes completion of splicing. Altogether we show that polymerase elongation kinetics are not affected by splicing events on the emerging pre-mRNA, that increased splicing leads to more splicing factors being recruited to the mRNA, and that post-transcriptional splicing can proceed at the site of transcription in the absence of the polymerase.
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DOI：
10.1083/jcb.200601105
发表时间：
2006-04-24
期刊：
The Journal of cell biology
影响因子：
--
作者：
Darzacq X;Kittur N;Roy S;Shav-Tal Y;Singer RH;Meier UT
通讯作者：
Meier UT
影响因子：
14.9
作者：
KOLE, R;WEISSMAN, SM
通讯作者：
WEISSMAN, SM
影响因子：
5.7
作者：
Albert BJ;McPherson PA;O'Brien K;Czaicki NL;Destefino V;Osman S;Li M;Day BW;Grabowski PJ;Moore MJ;Vogt A;Koide K
通讯作者：
Koide K
影响因子：
4
作者：
Ben-Ari, Ya'ara;Brody, Yehuda;Shav-Tal, Yaron
通讯作者：
Shav-Tal, Yaron
影响因子：
5.3
作者：
KESSLER, O;JIANG, YJ;CHASIN, LA
通讯作者：
CHASIN, LA