An Integrative Approach to Elucidate RNA Replication and Systemic Trafficking

个体细胞中的基本过程如何在生物体水平上整合以控制发育和行为是当前生物学中的一个主要问题。RNA复制和运输正在成为一种新的范式，以发展对整合系统性基因调控、病毒感染、细胞抗病毒反应和细胞间通讯基本机制的生物学原理的理解。该项目解决了一个基本的但知之甚少的问题，即RNA是如何通过核机制复制的，并通过使用马铃薯纺锤体块茎类病毒（PSTVd）作为一个高度易处理的模型，进一步选择运输到植物的邻近细胞和远端器官。这种小的（359个核苷酸）感染性RNA具有在细胞核中复制并在细胞和器官之间运输以建立感染的能力。因为它没有蛋白质编码能力和辅助病毒，这种RNA必须进化出独特的结构基序，以利用预先存在的细胞机制来完成复制和运输。利用生物化学、细胞学、遗传学、分子学和病毒学方法的组合，该项目将（1）研究在复制中发挥特定作用的PSTVd RNA基序，（2）分离和表征细胞因子RNA连接酶在复制中的作用，以及（3）解剖介导特定细胞之间运输的RNA基序。该项目的结果有望为感染性RNA如何利用宿主细胞机制建立感染提供新的见解，并有助于为进一步研究许多生物过程中RNA复制和贩运的机制和功能建立概念框架。该项目还将培训本科生/研究生和博士后研究员，以开发尖端的多学科方法，深入和广泛地解决基本生物学问题。

How basic processes in individual cells are integrated at the organismal level to control development and behavior is a major question in current biology. RNA replication and trafficking are emerging as a new paradigm to develop an understanding of the biological principles that integrate systemic gene regulation, viral infection, cellular antiviral responses, and basic mechanisms of cell-to-cell communication. This project addresses the fundamental and yet poorly understood question of how an RNA is replicated by the nuclear machinery and is further selected for transport into neighboring cells and distant organs in a plant, by using Potato spindle tuber viroid (PSTVd) as a highly tractable model. This small (359 nucleotides) infectious RNA has the capacity to replicate in the nucleus and traffic between cells and organs to establish infection. Because it does not have protein-coding capacity and helper viruses, this RNA must have evolved distinct structural motifs to exploit preexisting cellular machineries to accomplish replication and trafficking. Using a combination of biochemical, cellular, genetic, molecular and virological approaches, this project will (1) investigate the PSTVd RNA motifs that play specific roles in replication, (2) isolate and characterize the role of a cellular factor, RNA ligase, in replication, and (3) dissect the RNA motifs that mediate trafficking between specific cells. Results from the project are expected to provide new insights about how an infectious RNA utilizes the host cellular machinery to establish infection, and to help develop a conceptual framework for further studies on the mechanisms and functions of RNA replication and trafficking underlying many biological processes. This project will also train undergraduate/graduate students and postdoctoral fellows in developing cutting-edge multidisciplinary approaches to address fundamental biological problems in great depth and breadth.