Large-scale profiling of noncoding RNA function in yeast

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Abstract
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. Genomes from different organisms produce noncoding RNAs that are not translated to make proteins and whose functions are largely unknown. There are approximately 2,000 noncoding RNAs that make up almost 25% of the yeast genome compared to the approximately 6,000 protein coding genes that make up 70% of the yeast genome. With this large number of ncRNAs there is a need for large-scale studies to determine the functional roles of ncRNAs. We take advantage of a recently developed resource of 532 yeast strains in which individual noncoding RNA genes have been deleted. We grow these yeast noncoding RNA deletion strains in different conditions and catalogue how each noncoding RNA contributes to cell growth. Improvement or inhibition of cell growth under particular conditions implicates the deleted RNA in cellular responses to those conditions. We have also investigated in more detail the function of specific noncoding RNAs, revealing examples of how a deletion influences nearby genes, and other examples of noncoding RNAs that regulate genes at distant genomic locations. We have made our extensive data on the fitness of noncoding RNA deletion mutants publically available for searching and bulk download in a new online resource, called Yeast ncRNA Analysis (YNCA). This large-scale analysis of noncoding RNA deletion mutants reveals the importance of many noncoding RNAs in cellular function.
Funding Information
  • Wellcome (094225)
  • Wellcome (104981)

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