Departamento de Biologia Celular e Molecular, Universidade Federal do Paraná, Curitiba, Brazil.
PLoS One. 2011;6(6):e20730. doi: 10.1371/journal.pone.0020730. Epub 2011 Jun 8.
In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX) multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei -except the fibrillar center of nucleolus- and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II), but not RNA polymerase I (RNA pol I) or Spliced Leader (SL) transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and decrease of translation levels, reinforcing that Trypanosoma-Sub2 (Tryp-Sub2) is a component of mRNA transcription/export pathway in trypanosomes.
在真核细胞中，不同的 RNA 物种通过专门的途径从细胞核输出。mRNA 输出机制与 mRNA 加工高度整合，包括不同的核转运适配器以及其他 mRNA 结合蛋白、RNA 解旋酶和 NPC 相关蛋白。原生动物寄生虫克氏锥虫是恰加斯病的病原体，恰加斯病是一种广泛存在且被忽视的人类疾病，流行于拉丁美洲。克氏锥虫的基因表达具有独特的特征，例如蛋白质编码基因的组成性多顺反子转录和通过反式剪接进行的 mRNA 加工。一般来说，转录后事件是这些寄生虫基因表达调控的主要点。然而，mRNA 从细胞核的输出途径知之甚少。本研究调查了 TcSub2 的功能，它是 Sub2/UAP56 的高度保守蛋白同源物，Sub2/UAP56 是连接酵母/人类转录与 mRNA 输出的转录/输出 (TREX) 多蛋白复合物的一个组成部分。与它的同源物相似，TcSub2 是一种核蛋白，定位于核内弥散的焦点-除了核仁纤维中心-和致密和非致密染色质区域之间的界面，表明 TcSub2 与转录/加工位点相关联。通过 BrUTP 掺入测定进一步分析了这些发现，并证实 TcSub2 与活性 RNA 聚合酶 II（RNA pol II）物理相关，但与 RNA 聚合酶 I（RNA pol I）或拼接前导（SL）转录无关，表明其特别参与 T. cruzi 的核 mRNA 代谢。TcSub2 基因的双敲除在克氏锥虫中是致命的，表明它具有必需的功能。或者，在布氏锥虫中进行了 RNA 干扰测定。这表明，除了作为必需蛋白外，其敲低还会导致核内 mRNA 积累和翻译水平降低，这强化了锥虫 Sub2（Tryp-Sub2）是锥虫 mRNA 转录/输出途径的一个组成部分。
