MFB: RNA modifications of frameshifting stimulators: cellular platforms to engineer gene expression by computational mutation predictions and functional experiments

在这个生物技术分子基础（MFB）项目中，纽约大学的Tamar Schlick博士和北卡罗来纳大学的Alain Laederach博士将开发先进的计算工具来预测和控制当细胞的机制（核糖体）发生变化时病毒蛋白合成是如何受到影响的，从而改变了信使RNA （mRNA）中三个字母代码的读取方式。在病毒和人类细胞中，这种翻译mRNA三重码的移框被发现是预先编程的，以修改基因产物的表达并调节生化过程。本研究旨在通过计算预测和实验测试将突变引入mRNA如何影响其三维结构，从而影响原型病毒基因组中的程序化帧移。揭示原型病毒中移帧的特定结构和序列要求将有助于设计新的高效移帧元件，具有潜在的应用于病毒基因包装。该项目将为学生提供数学、计算机科学、生物、物理、化学和工程方面的跨学科培训，特别强调加强少数民族参与STEM活动。将开展公共宣传工作，以接触普通受众，并强调对人类健康有影响的数学、生物学、计算和生物技术的交叉。程序性核糖体移框（PRF）是一种通过改变mRNA三联体核苷酸转录物来产生替代基因产物来修饰基因表达的广泛机制。PRF对于包括HIV和sars相关冠状病毒在内的许多病毒来说是翻译重叠的mRNA阅读框必不可少的，它也是内源性人类、真核和原核基因中的一种机制。由于PRF已被证明能显著影响病毒活力或人类过程的生化调节，因此对帧移的调节定义了一个工程基因表达的平台。然而，在工程和治疗策略取得成功之前，必须了解移框的复杂方面和RNA移框元件（FSE）的结构可塑性。在这个协同的生物、化学、数学和计算研究项目中，将开发基于图论的工具来预测原型病毒系统的FSE突变，旨在大幅降低帧移效率，作为一种新的生物技术策略，对抗病毒感染和与PRF相关的人类疾病。这些突变的影响将通过荧光素酶测定来评估，并通过适用于具有多种构象的rna的技术来分析产生的FSE结构景观。除了改进对帧移机制的理解和用于预测fse景观改变突变的计算工具外，该项目还将产生新的生物技术，RNA修饰工具，作为针对RNA病毒的潜在治疗药物，或适用于使用帧移的人类和其他基因。由于帧移位是一种存储基因编码信息的紧凑机制，可以用来克服基因组大小的限制，因此也出现了病毒包装/药物递送的应用。该项目由数学和物理科学理事会（MPS）化学部（CHE）、数学科学部（DMS）和物理部（PHY）共同资助。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

In this Molecular Foundations for Biotechnology (MFB) project, Dr. Tamar Schlick from New York University and Dr. Alain Laederach from the University of North Carolina will develop advanced computational tools to predict and control how viral protein synthesis is affected when the cell’s machinery (the ribosome) shifts and thus changes how the three-letter code in messenger RNA (mRNA) is read. This frameshifting in translating the mRNA triplet code has been found to be preprogrammed in viruses and human cells to modify the expression of gene products and to regulate biochemical processes. This study aims to computationally predict and experimentally test how introducing mutations to mRNA affects its three-dimensional structure and, consequently, programmed frameshifting in prototypical viral genomes. Revealing the specific structural and sequence requirements for frameshifting in prototype viruses will facilitate the design of novel efficient frameshifting elements, with potential applications to viral packaging of genes. This project will provide interdisciplinary training to students in mathematics, computer science, biology, physics, chemistry, and engineering, with particular emphasis on enhancing minority participation in STEM activities. Public outreach efforts will be included to reach general audiences and highlight the intersection of mathematics, biology, computing, and biotechnologies that have implications in human health. Programmed ribosomal frameshifting (PRF) is a widespread mechanism for modifying the gene expressed by altering the mRNA triplet-nucleotide transcript to generate an alternate gene product. Indispensable to many viruses including HIV and SARS-associated coronaviruses for translating overlapping mRNA reading frames, PRF is also a mechanism in endogenous human, eukaryotic and prokaryotic genes. Because PRF has been shown to dramatically influence viral viability or the biochemical regulation of human processes, the modulation of frameshifting defines a platform for engineering gene expression. However, the complex aspects of frameshifting and the structural plasticity of the RNA frameshifting element (FSE) must be understood before engineering and therapeutic strategies can succeed. In this synergistic biological, chemical, mathematical, and computational research program, graph-theory-based tools will be developed to predict FSE mutations for prototype viral systems aimed at substantially lowering frameshifting efficiency as a novel biotechnological strategy against viral infections and related human diseases associated with PRF. The effect of these mutations will be assessed by Luciferase assay measurements, and the resulting FSE structural landscapes analyzed by techniques suitable for RNAs with multiple conformations. Besides an improved understanding of the mechanisms of frameshifting and computational tools for predicting FSE-landscape-altering mutations, this project will produce new biotechnological, RNA modifying tools as potential therapeutic agents against RNA viruses or applicable to human and other genes that employ frameshifting. Applications to viral packaging/drug delivery also arise, as frameshifting is a compact mechanism to store gene coding information and can be exploited to overcome genomic size limitations.This project is jointly funded by the Division of Chemistry (CHE), the Division of Mathematical Sciences (DMS), and the Division of Physics (PHY) in the Directorate for Mathematical and Physical Sciences (MPS).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.