Transcription termination and chimeric RNA formation controlled by Arabidopsis thaliana FPA.
Transcription termination and chimeric RNA formation controlled by Arabidopsis thaliana FPA.
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DOI：
10.1371/journal.pgen.1003867
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发表时间：
2013-10
期刊：
影响因子：
4.5
通讯作者：
Simpson GG
中科院分区：
文献类型：
--
作者：
Duc C;Sherstnev A;Cole C;Barton GJ;Simpson GG
选择性切割和聚腺苷酸化会影响 mRNA 的编码和调控潜力以及转录终止发生的位置。尽管这一过程很普遍，但很少有人知道该过程的调节者。拟南芥蛋白 FPA 是聚腺苷酸位点选择反式作用调节因子的罕见例子。因此，分析 fpa 突变体提供了一个揭示破坏这一过程的一般后果的机会。我们使用直接 RNA 测序来量化 fpa 突变体中 RNA 3' 形成的变化。在这里，我们表明，在其他独立基因的外显子之间形成的特定嵌合 RNA 是 FPA 功能丧失的一个显着后果。我们定义了 fpa 突变体中 RNA 3' 末端形成缺陷产生的基因间通读转录本，并详细介绍了与这些通读 RNA 相关的隐秘剪接和反义转录。我们鉴定了对 FPA 敏感的内含子内的替代多聚腺苷酸化，并在 IBM1 poly(A) 位点选择中显示出 FPA 依赖性转变，这与最近在基因内异染色质和 DNA 甲基化缺陷的突变体中定义的转变不同。最后，我们发现 fpa 突变体中特定位点的缺陷终止与 dicer-like 1 (dcl1) 或 dcl4 突变体共有，这使我们能够对这些蛋白质的某些沉默作用提出替代解释。我们将我们的发现与 3' 末端形成模式的改变可能对基因和基因组组织产生的影响联系起来。几乎所有真核蛋白质编码基因的末端都是由聚腺苷酸信号定义的。当基因被 RNA 聚合酶 II 转录为 mRNA 时，poly(A) 信号会引导前体 mRNA 在特定位点进行切割；这伴随着在 mRNA 上添加聚腺苷酸尾并终止转录。许多基因具有多个多聚腺苷酸信号，选择哪个信号的调节选择可以有效地确定该基因将编码什么、如何调节该基因以及转录终止发生在何处。我们在研究模式植物拟南芥花发育时发现了一个罕见的多聚腺苷酸位点选择调节因子 FPA。因此，研究 FPA 不仅提供了了解其在植物生物学中的作用，而且还了解破坏替代性聚腺苷酸化的一般后果的机会。在这项研究中，我们使用一种称为直接 RNA 测序的技术来量化缺乏 FPA 功能的植物中 Poly(A) 位点选择的全基因组变化。我们最引人注目的发现之一是，在没有 FPA 的情况下，我们检测到两个单独且特征明确的基因之间形成的嵌合 RNA。
Alternative cleavage and polyadenylation influence the coding and regulatory potential of mRNAs and where transcription termination occurs. Although widespread, few regulators of this process are known. The Arabidopsis thaliana protein FPA is a rare example of a trans-acting regulator of poly(A) site choice. Analysing fpa mutants therefore provides an opportunity to reveal generic consequences of disrupting this process. We used direct RNA sequencing to quantify shifts in RNA 3′ formation in fpa mutants. Here we show that specific chimeric RNAs formed between the exons of otherwise separate genes are a striking consequence of loss of FPA function. We define intergenic read-through transcripts resulting from defective RNA 3′ end formation in fpa mutants and detail cryptic splicing and antisense transcription associated with these read-through RNAs. We identify alternative polyadenylation within introns that is sensitive to FPA and show FPA-dependent shifts in IBM1 poly(A) site selection that differ from those recently defined in mutants defective in intragenic heterochromatin and DNA methylation. Finally, we show that defective termination at specific loci in fpa mutants is shared with dicer-like 1 (dcl1) or dcl4 mutants, leading us to develop alternative explanations for some silencing roles of these proteins. We relate our findings to the impact that altered patterns of 3′ end formation can have on gene and genome organisation. The ends of almost all eukaryotic protein-coding genes are defined by a poly(A) signal. When genes are transcribed into mRNA by RNA polymerase II, the poly(A) signal guides cleavage of the precursor mRNA at a particular site; this is accompanied by the addition of a poly(A) tail to the mRNA and termination of transcription. Many genes have more than one poly(A) signal and the regulated choice of which to select can effectively determine what the gene will code for, how the gene can be regulated and where transcription termination occurs. We discovered a rare example of a regulator of poly(A) site choice, called FPA, while studying flower development in the model plant Arabidopsis thaliana. Studying FPA therefore provides an opportunity to understand not only its roles in plant biology but also the generic consequences of disrupting alternative polyadenylation. In this study, we use a technique called direct RNA sequencing to quantify genome-wide shifts in poly(A) site selection in plants that lack FPA function. One of our most striking findings is that in the absence of FPA we detect chimeric RNAs formed between two otherwise separate and well-characterised genes.
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