Abstract
Eukaryotic genomes express numerous long intergenic non-coding RNAs (lincRNAs) that do not overlap any coding genes. Some lincRNAs function in various aspects of gene regulation, but it is not clear in general to what extent lincRNAs contribute to the information flow from genotype to phenotype. To explore this question, we systematically analysed cellular roles of lincRNAs in Schizosaccharomyces pombe. Using seamless CRISPR/Cas9-based genome editing, we deleted 141 lincRNA genes to broadly phenotype these mutants, together with 238 diverse coding-gene mutants for functional context. We applied high-throughput colony-based assays to determine mutant growth and viability in benign conditions and in response to 145 different nutrient, drug, and stress conditions. These analyses uncovered phenotypes for 47.5% of the lincRNAs and 96% of the protein-coding genes. For 110 lincRNA mutants, we also performed high-throughput microscopy and flow cytometry assays, linking 37% of these lincRNAs with cell-size and/or cell-cycle control. With all assays combined, we detected phenotypes for 84 (59.6%) of all lincRNA deletion mutants tested. For complementary functional inference, we analysed colony growth of strains ectopically overexpressing 113 lincRNA genes under 47 different conditions. Of these overexpression strains, 102 (90.3%) showed altered growth under certain conditions. Clustering analyses provided further functional clues and relationships for some of the lincRNAs. These rich phenomics datasets associate lincRNA mutants with hundreds of phenotypes, indicating that most of the lincRNAs analysed exert cellular functions in specific environmental or physiological contexts. This study provides groundwork to further dissect the roles of these lincRNAs in the relevant conditions.
Highlights
Genomes produce pervasive and diverse non-coding RNAs
Gene overexpression (‘gain of function’) provides complementary phenotype information to gene deletion (Prelich, 2012); any phenotype caused by a long intergenic non-coding RNA (lincRNA) that is ectopically expressed from a plasmid points to a function that is exerted over a distance via the lincRNA itself rather than via its transcription or other local effects
We applied a phenomics approach to explore the functional importance of S. pombe lincRNAs, including colony-based and cellular assays of deletion mutants and colony-based assays of overexpression strains
Summary
Genomes produce pervasive and diverse non-coding RNAs. How much genetic information is transacted by this non-coding ‘dark matter’ remains a matter of debate. A substantial but poorly understood portion of transcriptomes consists of long intergenic non-coding RNAs (lincRNAs). LincRNAs are longer than 200 nucleotides, lack long open reading frames, and do not overlap any neighbouring coding regions. While not all lincRNAs may be functional, several have well-d efined roles in gene regulation and some other cellular processes. Different lincRNAs can control gene expression at different levels, from transcription to translation, and either in cis (acting on neighbouring genes) or in trans (acting on distant genes) (Fauquenoy et al, 2018; Popadin et al, 2013; Rinn and Chang, 2012; Schlackow et al, 2017; Ulitsky and Bartel, 2013; Yamashita et al, 2016). LincRNAs show little sequence conservation between species, functional principles seem to be conserved which
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