Control of Cap-independent Translation by a 3' Untranslated Region

摘要MCB 9974590PI：W.Allen Miller翻译最初被认为是线性的：核糖体及其相关因子识别其5‘m7GpppG帽结构的信使RNA，然后沿3’方向扫描到蛋白质合成开始的第一个8月密码子。两个重大发现改变了这一观点：(I)某些RNA序列促进帽非依赖性翻译的能力，使核糖体内部与帽部位结合；(Ii)3‘非翻译区(UTR)，包括Poly(A)尾巴，在调节翻译中的重要作用。这表明，在起始过程中，mRNAs必须被蛋白质因素所循环。这些发现仍然需要核糖体与起始密码子上游的mRNA结合，所有3‘UTR控制模型都依赖于对5’帽结构的识别来使mRNA循环。与所有已知的模型不同的是，研究人员在植物病毒基因组的3‘UTR中发现了一个序列，该序列在缺少5’帽和PolyA尾巴的mRNA的5‘端促进了非常有效的翻译起始。这项研究的目的是了解在没有5‘帽的情况下，3’非编码区如何招募核糖体，并将其传递到遥远上游的特定起始密码子。本工作要检验的假设是，3‘UTR中的特定RNA序列和结构模仿5’端的帽和聚(A)尾，并通过特定的蛋白质相互作用与5‘端的UTR相互作用，将翻译机制招募到5’-近端的AUG密码子。将在体外和体内翻译的报告基因mRNA的UTRs的缺失和定点突变将被用来确定UTRs中核糖体招募和与5‘端通讯所需的特定序列。酶和化学探查也将用于确定RNA的二级结构。最后，将利用酵母3-杂交系统和RNA亲和结合柱开始寻找介导这一过程的细胞蛋白。这将阐明翻译机器可以通过令人惊讶的多样化的方式组装在一个活跃的mRNA上。储存为核酸序列的遗传信息转化为执行细胞功能的蛋白质的过程称为翻译。这需要一个被称为核糖体的复杂的大分子机器，以及相关的蛋白质因子，来识别信使RNA(信使RNA)，并以受控的方式阅读它。除了编码蛋白质序列的部分信使核糖核酸外，位于信使核糖核酸末端的非编码序列经常调节其与核糖体的相互作用，以控制蛋白质的合成。关于这一复杂的过程，还有很多需要了解。病毒通常使用非常规方法绕过正常的主机转换机制，这使它们能够避开主机防御。它们也是理解翻译的有用工具。这个项目的重点是一种不寻常的植物病毒mRNAs，它在“错误的”未翻译末端(编码区的“下游”)附近有一个序列，它以某种方式将核糖体招募到mRNA的另一端，在翻译开始的地方。因此，信使核糖核酸必须循环。正常的mRNAs也通过与蛋白质因子复杂组合相互作用的非翻译区中的特殊修饰或序列进行循环。该项目中的病毒RNA缺乏这些修饰和序列，推测有不同的序列模仿传统的RNA，它们可能至少由一些不同的蛋白质相互作用，而不是用来翻译正常的mRNAs。目标是识别参与这一过程的RNA序列和蛋白质。翻译机制在所有动植物中基本上是相同的。因此，这项工作对我们理解两个王国的翻译都是至关重要的。首先在病毒中发现的新的基因表达机制通常后来被发现存在于细胞基因中。这项工作可能揭示通过抑制病毒特有的基因表达机制来控制病毒的新方法，并可能为构建绕过调控和限制基因表达的细胞机制的基因提供基因工程工具。

AbstractMCB 9974590PI: W. Allen Miller Translation was originally thought to be linear: ribosomes, with associated factors, recognize the messenger RNA at its 5' m7GpppG cap structure, then scan in a 3' direction to the first AUG codon at which protein synthesis begins. Two major discoveries have changed this view: (i) the ability of certain RNA sequences to facilitate cap-independent translation, allowing the ribosome to bind internally to the cap site; and (ii) the important role of the 3' untranslated region (UTR), including the poly(A) tail, in regulating translation. This revealed that mRNAs must be circularized by protein factors in the initiation process. These discoveries still require that the ribosome binds the mRNA upstream of the start codon, and all 3' UTR control models rely on recognition of the 5' cap structure to circularize the mRNA. In contrast to all known models, the investigator discovered a sequence in the 3' UTR of a plant viral genome that facilitates very efficient translation initiation at the 5' end of mRNA lacking a 5' cap and a poly(A) tail. The goal of this research is to understand how a 3'UTR can recruit the ribosome and deliver it to a specific start codon far upstream, in the absence of a 5' cap. The hypothesis to be tested in this work is that specific RNA sequences and structures in the 3' UTR mimic a 5' cap and a poly(A) tail and, via specific protein interactions, interact with the 5' UTR to recruit the translational machinery to the 5'-proximal AUG codon. Deletion and site-directed mutagenesis of UTRs of a reporter gene mRNA that will be translated in vitro and in vivo will be used to identify the specific sequences in the UTRs that are required for ribosome recruitment and communication with the 5' end. Enzymatic and chemical probing will also be used to determine the RNA secondary structure. Finally, a search for cellular proteins that mediate this process by using the yeast 3-hybrid system, and by RNA affinity binding columns will be initiated. This will shed light on the surprisingly diverse ways by which the translational machinery can be assembled on an active mRNA. The process by which genetic information that is stored as a nucleic acid sequence is converted to a protein that carries out the functions of the cell is called translation. This requires a complex macromolecular machine called the ribosome, along with associated protein factors, to recognize the messenger RNA (mRNA), and "read" it in a controlled fashion. In addition to the portion of the mRNA that codes for a protein sequence, non-coding sequences at the ends of the mRNA often regulate its interaction with the ribosome, to control protein synthesis. Much remains to be learned about this complex process. Viruses often employ unconventional means of bypassing normal host translation mechanisms, that allow them to avoid host defenses. They also serve as useful tools for understanding translation in general. This project focuses on an unusual plant viral mRNA that has a sequence near the "wrong" untranslated end ("downstream" of the coding region), that somehow recruits the ribosome to the other end of the mRNA where translation begins. Thus, the mRNA must be circularized. Normal mRNAs are also circularized, via special modifications or sequences in the untranslated regions that interact with a complex assemblage of protein factors. The viral RNA in this project lacks these modifications and sequences, and presumably has different sequences that mimic the conventional ones, and they likely interact by at least some different proteins than are used in translation of normal mRNAs. The goal is to identify both the RNA sequences and the proteins that are involved in this process. Translation mechanisms are fundamentally the same in all plants and animals. Thus, this work is of fundamental relevance to our understanding of translation in both kingdoms. Novel gene expression mechanisms first discovered in viruses often are found later to exist in cellular genes. This work may reveal new means of controlling viruses by inhibiting gene expression mechanisms that are unique to viruses, and it may provide genetic engineering tools for constructing genes that bypass cellular mechanisms of regulating and limiting gene expression.