Kamina Anyango D, Jaremko Daniel, Christen Linda, Williams Noreen
Department of Microbiology and Immunology and Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, Buffalo, New York, USA.
mSphere. 2017 Oct 18;2(5). doi: 10.1128/mSphere.00394-17. eCollection 2017 Sep-Oct.
Ribosome biogenesis is a highly complex and conserved cellular process that is responsible for making ribosomes. During this process, there are several assembly steps that function as regulators to ensure proper ribosome formation. One of these steps is the assembly of the 5S ribonucleoprotein particle (5S RNP) in the central protuberance of the 60S ribosomal subunit. In eukaryotes, the 5S RNP is composed of 5S rRNA, ribosomal proteins L5 and L11, and assembly factors Rpf2 and Rrs1. Our laboratory previously showed that in , the 5S RNP is composed of 5S rRNA, L5, and trypanosome-specific RNA binding proteins P34 and P37. In this study, we characterize an additional component of the 5S RNP, the homolog of Rpf2. This is the first study to functionally characterize interactions mediated by Rpf2 in an organism other than fungi. . Rpf2 (TbRpf2) was identified from tandem affinity purification using extracts prepared from protein A-tobacco etch virus (TEV)-protein C (PTP)-tagged L5, P34, and P37 cell lines, followed by mass spectrometry analysis. We characterized the binding interactions between TbRpf2 and the previously characterized members of the 5S RNP. Our studies show that TbRpf2 mediates conserved binding interactions with 5S rRNA and L5 and that TbRpf2 also interacts with trypanosome-specific proteins P34 and P37. We performed RNA interference (RNAi) knockdown of TbRpf2 and showed that this protein is essential for the survival of the parasites and is critical for proper ribosome formation. These studies provide new insights into a critical checkpoint in the ribosome biogenesis pathway in . is the parasitic protozoan that causes African sleeping sickness. Ribosome assembly is essential for the survival of this parasite through the different host environments it encounters during its life cycle. The assembly of the 5S ribonucleoprotein particle (5S RNP) functions as one of the regulatory checkpoints during ribosome biogenesis. We have previously characterized the 5S RNP in and showed that trypanosome-specific proteins P34 and P37 are part of this complex. In this study, we characterize for the first time the interactions of the homolog of the assembly factor Rpf2 with members of the 5S RNP in another organism besides fungi. Our studies show that Rpf2 is essential in and that it forms unique interactions within the 5S RNP, particularly with P34 and P37. These studies have identified parasite-specific interactions that can potentially function as new therapeutic targets against sleeping sickness.
核糖体生物合成是一个高度复杂且保守的细胞过程，负责核糖体的生成。在此过程中，有几个组装步骤起到调节作用，以确保核糖体的正确形成。其中一个步骤是在60S核糖体亚基的中央突起处组装5S核糖核蛋白颗粒（5S RNP）。在真核生物中，5S RNP由5S rRNA、核糖体蛋白L5和L11以及组装因子Rpf2和Rrs1组成。我们实验室先前表明，在[此处原文缺失相关信息]中，5S RNP由5S rRNA、L5以及锥虫特异性RNA结合蛋白P34和P37组成。在本研究中，我们鉴定了5S RNP的另一个组分，即Rpf2的同源物。这是首次在真菌以外的生物体中对Rpf2介导的相互作用进行功能表征的研究。Rpf2（TbRpf2）是通过串联亲和纯化从用蛋白A - 烟草蚀纹病毒（TEV） - 蛋白C（PTP）标记的L5、P34和P37细胞系制备的提取物中鉴定出来的，随后进行质谱分析。我们表征了TbRpf2与先前鉴定的5S RNP成员之间的结合相互作用。我们的研究表明，TbRpf2介导与5S rRNA和L5的保守结合相互作用，并且TbRpf2还与锥虫特异性蛋白P34和P37相互作用。我们对TbRpf2进行了RNA干扰（RNAi）敲低实验，结果表明该蛋白对寄生虫的存活至关重要，并且对核糖体的正确形成至关重要。这些研究为[此处原文缺失相关信息]核糖体生物合成途径中的一个关键检查点提供了新的见解。[此处原文缺失相关信息]是引起非洲昏睡病的寄生原生动物。核糖体组装对于这种寄生虫在其生命周期中遇到的不同宿主环境中的存活至关重要。5S核糖核蛋白颗粒（5S RNP）的组装是核糖体生物合成过程中的调节检查点之一。我们先前已对[此处原文缺失相关信息]中的5S RNP进行了表征，并表明锥虫特异性蛋白P34和P37是该复合物的一部分。在本研究中，我们首次表征了除真菌外另一种生物体中组装因子Rpf2的同源物与5S RNP成员之间的相互作用。我们的研究表明，Rpf2在[此处原文缺失相关信息]中至关重要，并且它在5S RNP内形成独特的相互作用，特别是与P34和P37。这些研究确定了寄生虫特异性相互作用，这些相互作用有可能作为针对昏睡病的新治疗靶点。
