Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom.
Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, United Kingdom.
PLoS Genet. 2018 Mar 12;14(3):e1007253. doi: 10.1371/journal.pgen.1007253. eCollection 2018 Mar.
Noncoding RNAs (ncRNAs) are emerging as key regulators of cellular function. We have exploited the recently developed barcoded ncRNA gene deletion strain collections in the yeast Saccharomyces cerevisiae to investigate the numerous ncRNAs in yeast with no known function. The ncRNA deletion collection contains deletions of tRNAs, snoRNAs, snRNAs, stable unannotated transcripts (SUTs), cryptic unstable transcripts (CUTs) and other annotated ncRNAs encompassing 532 different individual ncRNA deletions. We have profiled the fitness of the diploid heterozygous ncRNA deletion strain collection in six conditions using batch and continuous liquid culture, as well as the haploid ncRNA deletion strain collections arrayed individually onto solid rich media. These analyses revealed many novel environmental-specific haplo-insufficient and haplo-proficient phenotypes providing key information on the importance of each specific ncRNA in every condition. Co-fitness analysis using fitness data from the heterozygous ncRNA deletion strain collection identified two ncRNA groups required for growth during heat stress and nutrient deprivation. The extensive fitness data for each ncRNA deletion strain has been compiled into an easy to navigate database called Yeast ncRNA Analysis (YNCA). By expanding the original ncRNA deletion strain collection we identified four novel essential ncRNAs; SUT527, SUT075, SUT367 and SUT259/691. We defined the effects of each new essential ncRNA on adjacent gene expression in the heterozygote background identifying both repression and induction of nearby genes. Additionally, we discovered a function for SUT527 in the expression, 3' end formation and localization of SEC4, an essential protein coding mRNA. Finally, using plasmid complementation we rescued the SUT075 lethal phenotype revealing that this ncRNA acts in trans. Overall, our findings provide important new insights into the function of ncRNAs.
非编码 RNA（ncRNAs）正在成为细胞功能的关键调节因子。我们利用最近开发的酵母酿酒酵母中带有条形码的 ncRNA 基因缺失菌株文库，研究了酵母中许多具有未知功能的 ncRNAs。ncRNA 缺失文库包含 tRNA、snoRNA、snRNA、稳定未注释转录本（SUT）、隐蔽不稳定转录本（CUT）和其他注释 ncRNA 的缺失，涵盖了 532 个不同的单个 ncRNA 缺失。我们使用批量和连续液体培养，以及单独排列在丰富固体培养基上的单倍体 ncRNA 缺失菌株文库，在六种条件下对二倍体杂合 ncRNA 缺失菌株文库的适应性进行了分析。这些分析揭示了许多新的环境特异性单倍体不足和单倍体有效表型，为每个特定 ncRNA 在每种条件下的重要性提供了关键信息。使用杂合 ncRNA 缺失菌株文库的适应性数据进行的共适应性分析确定了在热应激和营养剥夺期间生长所需的两个 ncRNA 组。每个 ncRNA 缺失菌株的广泛适应性数据已被编译到一个名为 Yeast ncRNA Analysis（YNCA）的易于导航的数据库中。通过扩展原始的 ncRNA 缺失菌株文库，我们鉴定了四个新的必需 ncRNA：SUT527、SUT075、SUT367 和 SUT259/691。我们定义了每个新必需 ncRNA 在杂合子背景下对相邻基因表达的影响，确定了附近基因的抑制和诱导。此外，我们发现 SUT527 在 SEC4（一种必需的蛋白质编码 mRNA）的表达、3' 末端形成和定位中的功能。最后，我们使用质粒互补拯救了 SUT075 的致死表型，表明该 ncRNA 是反式作用的。总的来说，我们的研究结果为 ncRNA 的功能提供了重要的新见解。
