Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706
Genetics. 2019 May;212(1):111-124. doi: 10.1534/genetics.119.301922. Epub 2019 Mar 21.
Selection of suppressor mutations that correct growth defects caused by substitutions in an RNA or protein can reveal functionally important molecular structures and interactions in living cells. This approach is particularly useful for the study of complex biological pathways involving many macromolecules, such as premessenger RNA (pre-mRNA) splicing. When a sufficiently large number of suppressor mutations is obtained and structural information is available, it is possible to generate detailed models of molecular function. However, the laborious and expensive task of identifying suppressor mutations in whole-genome selections limits the utility of this approach. Here I show that a custom targeted sequencing panel can greatly accelerate the identification of suppressor mutations in the genome. Using a panel that targets 112 genes encoding pre-mRNA splicing factors, I identified 27 unique mutations in six protein-coding genes that each overcome the cold-sensitive block to spliceosome activation caused by a substitution in U4 small nuclear RNA. When mapped to existing structures of spliceosomal complexes, the identified suppressors implicate specific molecular contacts between the proteins Brr2, Prp6, Prp8, Prp31, Sad1, and Snu114 as functionally important in an early step of catalytic activation of the spliceosome. This approach shows great promise for elucidating the allosteric cascade of molecular interactions that direct accurate and efficient pre-mRNA splicing and should be broadly useful for understanding the dynamics of other complex biological assemblies or pathways.
选择能够纠正 RNA 或蛋白质中取代引起的生长缺陷的抑制突变，可以揭示活细胞中功能重要的分子结构和相互作用。这种方法特别适用于研究涉及许多大分子的复杂生物途径，例如前信使 RNA (pre-mRNA) 剪接。当获得足够数量的抑制突变并且有结构信息时，就有可能生成分子功能的详细模型。然而，全基因组选择中识别抑制突变的繁琐和昂贵的任务限制了这种方法的实用性。在这里，我表明定制的靶向测序面板可以大大加速基因组中抑制突变的鉴定。使用靶向编码 pre-mRNA 剪接因子的 112 个基因的面板，我在六个编码蛋白质的基因中鉴定了 27 个独特的突变，这些突变都克服了 U4 小核 RNA 取代引起的剪接体激活的冷敏感阻断。当映射到剪接体复合物的现有结构时，鉴定出的抑制子暗示 Brr2、Prp6、Prp8、Prp31、Sad1 和 Snu114 之间的特定分子接触在剪接体催化激活的早期步骤中具有功能重要性。这种方法有望阐明指导准确和有效 pre-mRNA 剪接的变构级联分子相互作用，并应广泛用于理解其他复杂生物组装或途径的动态。
