The Trypanosomatid-Specific N Terminus of RPA2 Is Required for RNA Polymerase I Assembly, Localization, and Function
The Trypanosomatid-Specific N Terminus of RPA2 Is Required for RNA Polymerase I Assembly, Localization, and Function
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DOI：
10.1128/ec.00036-12
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发表时间：
2012-05-01
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通讯作者：
Wickstead, Bill
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作者：
Daniels, Jan-Peter;Gull, Keith;Wickstead, Bill
非洲锥虫是唯一已知使用RNA聚合酶I（pol I）转录蛋白质编码基因的生物。这些基因包括VSG，它是免疫逃避所必需的，并且是从核仁表达位点体（ESB）转录的。几个锥虫pol I亚基不同的同源物相比，在其他地方，和这些变化如何涉及到pol I功能的问题。一个明显的例子是在pol I的第二大亚基RPA 2上发现的N-末端延伸。在这里，我们确定了该区域的一个重要作用。RPA 2截短导致核排斥和表型模仿单等位基因敲除的生长缺陷。然而，N末端不是一般的核定位信号（NLS），并且它不能在核中积累不相关的蛋白质。异位NLS足以恢复截短的RPA 2的核定位，但不能恢复功能。此外，NLS标记的截短RPA 2具有与全长蛋白不同的亚核分布，并且不能构建稳定的pol I复合物。我们的结论是，RPA 2 N-末端的延伸没有专门的蛋白质编码基因的表达的作用，但它是必不可少的锥虫中的所有pol I功能，因为它指导锥虫特异性相互作用与RPA 1。
African trypanosomes are the only organisms known to use RNA polymerase I (pol I) to transcribe protein-coding genes. These genes include VSG, which is essential for immune evasion and is transcribed from an extranucleolar expression site body (ESB). Several trypanosome pol I subunits vary compared to their homologues elsewhere, and the question arises as to how these variations relate to pol I function. A clear example is the N-terminal extension found on the second-largest subunit of pol I, RPA2. Here, we identify an essential role for this region. RPA2 truncation leads to nuclear exclusion and a growth defect which phenocopies single-allele knockout. The N terminus is not a general nuclear localization signal (NLS), however, and it fails to accumulate unrelated proteins in the nucleus. An ectopic NLS is sufficient to reinstate nuclear localization of truncated RPA2, but it does not restore function. Moreover, NLS-tagged, truncated RPA2 has a different subnuclear distribution to full-length protein and is unable to build stable pol I complexes. We conclude that the RPA2 N-terminal extension does not have a role exclusive to the expression of protein-coding genes, but it is essential for all pol I functions in trypanosomes because it directs trypanosomatid-specific interactions with RPA1.