Nuclear, Cytosolic, and Surface-Localized Poly(A)-Binding Proteins of Plasmodium yoelii.
Nuclear, Cytosolic, and Surface-Localized Poly(A)-Binding Proteins of Plasmodium yoelii.
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DOI：
10.1128/msphere.00435-17
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发表时间：
2018-01
期刊：
影响因子：
4.8
通讯作者：
Lindner SE
中科院分区：
文献类型：
--
作者：
Minns AM;Hart KJ;Subramanian S;Hafenstein S;Lindner SE
疟疾仍然是全球最大的健康问题之一。引起疟疾的寄生虫（疟原虫属）依赖于对其在脊椎动物宿主和蚊子之间传播的精确控制。它做到这一点的一个关键方法是主动产生建立新感染所需的 mRNA，但将它们沉默并储存起来直到需要它们为止。模型真核生物翻译抑制过程中的一个关键蛋白是多聚腺苷酸结合蛋白 (PABP)。在这里，我们证明约氏疟原虫利用核 PABP 和胞浆 PABP，两者都特异性结合聚腺苷酸化 RNA 序列。此外，我们发现胞质 PABP 在体外形成链，这与其在包裹 mRNA 聚腺苷酸尾方面的作用一致。最后，我们还令人惊讶地证实，胞质 PABP 存在于疟原虫子孢子的表面。将这些数据综合起来，我们建议 Plasmodium 采用一种更像后生动物的策略，使用专门的 PABP 进行 RNA 代谢。疟疾是一种毁灭性的疾病，每年导致约 50 万人死亡。引起疟疾的寄生虫（疟原虫属）利用翻译抑制过程来调节其生长、发育和传播。由于多聚腺苷酸结合蛋白 (PABP) 已被确定为模型真核生物和疟原虫中 RNA 代谢和翻译抑制的关键成分，因此我们在约氏疟原虫中鉴定并研究了两种 PABP：PyPABP1 和 PyPABP2。与大多数单细胞真核生物相比，疟原虫与后生动物非常相似，并且编码核 PABP 和胞浆 PABP；在这里，我们提供了多种证据来支持这一观察结果。 PyPABP1 和 PyPABP2 的保守结构域结构与酵母和后生动物的保守结构域结构相似，而多个独立的结合测定表明它们能够非常强烈且特异性地与 Poly(A) 序列结合。有趣的是，我们还观察到，尽管在体外进行了详尽的 RNase 处理，纯化的 PyPABP1 仍形成均聚物链。最后，我们通过间接免疫荧光测定（IFA）表明，PyPABP1 和 PyPABP2 在生命周期的血液阶段是细胞质和细胞核相关的 PABP。然而，令人惊讶的是，PyPABP1 也被观察到位于传播的唾液腺子孢子的表面上，并且当寄生虫在基质上滑行时沉积在痕迹中。这是第三个被证实在子孢子表面发现的 RNA 结合蛋白，这些数据可能表明宿主和寄生虫之间存在一个未被重视的以 RNA 为中心的界面。重要性 疟疾仍然是全球最大的健康问题之一。引起疟疾的寄生虫（疟原虫属）依赖于对其在脊椎动物宿主和蚊子之间传播的精确控制。它做到这一点的一个关键方法是主动产生建立新感染所需的 mRNA，但将它们沉默并储存起来直到需要它们为止。模型真核生物翻译抑制过程中的一个关键蛋白是多聚腺苷酸结合蛋白 (PABP)。在这里，我们证明约氏疟原虫利用核 PABP 和胞浆 PABP，两者都特异性结合聚腺苷酸化 RNA 序列。此外，我们发现胞质 PABP 在体外形成链，这与其在包裹 mRNA 聚腺苷酸尾方面的作用一致。最后，我们还令人惊讶地证实，胞质 PABP 存在于疟原虫子孢子的表面。将这些数据综合起来，我们建议 Plasmodium 采用一种更像后生动物的策略，使用专门的 PABP 进行 RNA 代谢。
Malaria remains one of the great global health problems. The parasite that causes malaria (Plasmodium genus) relies upon exquisite control of its transmission between vertebrate hosts and mosquitoes. One crucial way that it does so is by proactively producing mRNAs needed to establish the new infection but by silencing and storing them until they are needed. One key protein in this process of translational repression in model eukaryotes is poly(A)-binding protein (PABP). Here we have shown that Plasmodium yoelii utilizes both a nuclear PABP and a cytosolic PABP, both of which bind specifically to polyadenylated RNA sequences. Moreover, we find that the cytosolic PABP forms chains in vitro, consistent with its appreciated role in coating the poly(A) tails of mRNA. Finally, we have also verified that, surprisingly, the cytosolic PABP is found on the surface of Plasmodium sporozoites. Taking the data together, we propose that Plasmodium utilizes a more metazoan-like strategy for RNA metabolism using specialized PABPs. Malaria is a devastating illness that causes approximately 500,000 deaths annually. The malaria-causing parasite (Plasmodium genus) uses the process of translational repression to regulate its growth, development, and transmission. As poly(A)-binding proteins (PABP) have been identified as critical components of RNA metabolism and translational repression in model eukaryotes and in Plasmodium, we have identified and investigated two PABPs in Plasmodium yoelii, PyPABP1 and PyPABP2. In contrast to most single-celled eukaryotes, Plasmodium closely resembles metazoans and encodes both a nuclear PABP and a cytosolic PABP; here, we provide multiple lines of evidence in support of this observation. The conserved domain architectures of PyPABP1 and PyPABP2 resemble those of yeast and metazoans, while multiple independent binding assays demonstrated their ability to bind very strongly and specifically to poly(A) sequences. Interestingly, we also observed that purified PyPABP1 forms homopolymeric chains despite exhaustive RNase treatment in vitro. Finally, we show by indirect immunofluorescence assays (IFAs) that PyPABP1 and PyPABP2 are cytoplasm- and nucleus-associated PABPs during the blood stages of the life cycle. Surprisingly, however, PyPABP1 was instead observed to also be localized on the surface of transmitted salivary gland sporozoites and to be deposited in trails when parasites glide on a substrate. This is the third RNA-binding protein verified to be found on the sporozoite surface, and the data may point to an unappreciated RNA-centered interface between the host and parasite. IMPORTANCE Malaria remains one of the great global health problems. The parasite that causes malaria (Plasmodium genus) relies upon exquisite control of its transmission between vertebrate hosts and mosquitoes. One crucial way that it does so is by proactively producing mRNAs needed to establish the new infection but by silencing and storing them until they are needed. One key protein in this process of translational repression in model eukaryotes is poly(A)-binding protein (PABP). Here we have shown that Plasmodium yoelii utilizes both a nuclear PABP and a cytosolic PABP, both of which bind specifically to polyadenylated RNA sequences. Moreover, we find that the cytosolic PABP forms chains in vitro, consistent with its appreciated role in coating the poly(A) tails of mRNA. Finally, we have also verified that, surprisingly, the cytosolic PABP is found on the surface of Plasmodium sporozoites. Taking the data together, we propose that Plasmodium utilizes a more metazoan-like strategy for RNA metabolism using specialized PABPs.