Functional and transcriptional profiling of non-coding RNAs in yeast reveal context-dependent phenotypes and in trans effects on the protein regulatory network

Author summary A quarter of the yeast genome comprises non-coding RNA molecules (ncRNAs), which do not translate into proteins but are involved in regulation of gene expression. ncRNAs can affect nearby genes by physically interfering with their transcription (cis mode of action), or they interact with DNA, proteins or other RNAs to regulate the expression of distant genes (trans mode of action). Examples of cis-acting ncRNAs have been broadly described, however genome-wide studies to identify functional trans-acting ncRNAs involved in global gene regulation are still lacking. Here, we used a ncRNA yeast deletion collection to score ncRNA impact on cellular function in different environmental conditions. A group of 20 ncRNA deletion mutants with broad fitness diversity were selected to investigate the ncRNA effect on the protein and ncRNA expression network. We show a high correlation between altered phenotypes and global transcriptional changes, in an environmental dependent manner. We confirmed the trans acting regulation of ncRNAs in the genome and their role in altering the expression of transcription factors. These findings support the notion of the involvement of ncRNAs in fine tuning cellular expression via regulation of transcription factors, as an advantageous RNA-mediated mechanism that can be fast and cost-effective for the cells. Non-coding RNAs (ncRNAs), including the more recently identified Stable Unannotated Transcripts (SUTs) and Cryptic Unstable Transcripts (CUTs), are increasingly being shown to play pivotal roles in the transcriptional and post-transcriptional regulation of genes in eukaryotes. Here, we carried out a large-scale screening of ncRNAs in Saccharomyces cerevisiae, and provide evidence for SUT and CUT function. Phenotypic data on 372 ncRNA deletion strains in 23 different growth conditions were collected, identifying ncRNAs responsible for significant cellular fitness changes. Transcriptome profiles were assembled for 18 haploid ncRNA deletion mutants and 2 essential ncRNA heterozygous deletants. Guided by the resulting RNA-seq data we analysed the genome-wide dysregulation of protein coding genes and non-coding transcripts. Novel functional ncRNAs, SUT125, SUT126, SUT035 and SUT532 that act in trans by modulating transcription factors were identified. Furthermore, we described the impact of SUTs and CUTs in modulating coding gene expression in response of different environmental conditions, regulating important biological process such as respiration (SUT125, SUT126, SUT035, SUT432), steroid biosynthesis (CUT494, SUT053, SUT468) or rRNA processing (SUT075 and snR30). Overall, these data captures and integrates the regulatory and phenotypic network of ncRNAs and protein coding genes, providing genome-wide evidence of the impact of ncRNAs on cellular homeostasis.