DataSheet_1_Deep mutational scanning of the RNase III-like domain in Trypanosoma brucei RNA editing protein KREPB4.docx

Kinetoplastid pathogens including Trypanosoma brucei, T. cruzi, and Leishmania species, are early diverged, eukaryotic, unicellular parasites. Functional understanding of many proteins from these pathogens has been hampered by limited sequence homology to proteins from other model organisms. Here we describe the development of a high-throughput deep mutational scanning approach in T. brucei that facilitates rapid and unbiased assessment of the impacts of many possible amino acid substitutions within a protein on cell fitness, as measured by relative cell growth. The approach leverages several molecular technologies: cells with conditional expression of a wild-type gene of interest and constitutive expression of a library of mutant variants, degron-controlled stabilization of I-SceI meganuclease to mediate highly efficient transfection of a mutant allele library, and a high-throughput sequencing readout for cell growth upon conditional knockdown of wild-type gene expression and exclusive expression of mutant variants. Using this method, we queried the effects of amino acid substitutions in the apparently non-catalytic RNase III-like domain of KREPB4 (B4), which is an essential component of the RNA Editing Catalytic Complexes (RECCs) that carry out mitochondrial RNA editing in T. brucei. We measured the impacts of thousands of B4 variants on bloodstream form cell growth and validated the most deleterious variants containing single amino acid substitutions. Crucially, there was no correlation between phenotypes and amino acid conservation, demonstrating the greater power of this method over traditional sequence homology searching to identify functional residues. The bloodstream form cell growth phenotypes were combined with structural modeling, RECC protein proximity data, and analysis of selected substitutions in procyclic form T. brucei. These analyses revealed that the B4 RNaseIII-like domain is essential for maintenance of RECC integrity and RECC protein abundances and is also involved in changes in RECCs that occur between bloodstream and procyclic form life cycle stages.

动质体病原体（Kinetoplastid pathogens）包括布氏锥虫（Trypanosoma brucei）、克氏锥虫（T. cruzi）以及利什曼原虫（Leishmania）物种，均为早期分化的真核单细胞寄生虫。由于此类病原体的蛋白质与其他模式生物的蛋白质序列同源性有限，学界对其多数蛋白质的功能认知长期存在瓶颈。在此，我们介绍了一种在布氏锥虫中建立的高通量深度突变扫描（deep mutational scanning）方法，该方法可通过相对细胞生长水平，快速且无偏地评估蛋白质内任意可能的氨基酸替换对细胞适应性的影响。该方法依托多项分子技术体系：包括携带目标野生型基因条件性表达与突变变体库组成型表达的工程细胞、通过降解标签（degron）调控稳定的I-SceI大范围核酸酶（I-SceI meganuclease）以实现突变等位基因库的高效转染，以及在野生型基因条件性敲低且仅表达突变变体的条件下，用于检测细胞生长水平的高通量测序读值方案。借助该方法，我们探究了KREPB4（简称B4）的看似非催化性的RNase III样结构域（RNase III-like domain）内的氨基酸替换所产生的功能效应；KREPB4是布氏锥虫中负责线粒体RNA编辑的RNA编辑催化复合物（RNA Editing Catalytic Complexes, RECCs）的核心组成元件。我们检测了数千个B4变体对布氏锥虫血流型（bloodstream form）细胞生长的影响，并验证了其中携带单氨基酸替换的危害性最显著的变体。尤为关键的是，细胞表型与氨基酸保守性之间并无显著关联，这表明相较于传统的序列同源性搜索方法，本方法在识别功能残基（functional residues）方面具有更优异的效能。我们将布氏锥虫血流型细胞生长表型数据与结构建模结果、RECC蛋白邻近性分析数据，以及对布氏锥虫前循环型（procyclic form）中选定替换变体的功能分析相结合。上述分析结果表明，B4的RNase III样结构域对于维持RECC的完整性与RECC蛋白的丰度至关重要，同时也参与了布氏锥虫在血流型与前循环型生命周期阶段之间RECC复合物所发生的动态调控变化。