Large-scale profiling of noncoding RNA function in yeast

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.

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

“…This GO enrichment is consistent with the known role of snR30 in ribosomal RNA processing [37]. SUT075 is required for normal transcript levels of its neighbouring essential gene PRP3 and can act in trans [10]. We note however, in our RNA-seq data, that the down-regulation of PRP3 was not significantly strong (fold change, FC, 0.7) in SUT075 Δ and instead a large global effect on the transcriptome was detected, including targets in common with the snR30 mutant.…”

“…We selected 18 haploid ncRNA deletion mutants from clusters 1, 2, 5 and 7-10 with different types of phenotypic changes ( i.e . growth defects, improvements and no changes) to study by RNA-seq, together with heterozygous deletions of 2 essential ncRNAs, namely SUT075 and snR30 (previously described in Parker et al [10]) (Table 1). As expected, we detected changes in the levels of at least one neighbouring transcript in 8 of the ncRNA deletion mutant strains analysed by RNA-seq.…”

“…More recently, RNA transcripts which are not translated into protein, have been described to have a prominent role as epigenetic modifiers [1,2]. There are an increasing number of examples of these non-coding RNA (ncRNA) transcripts regulating gene expression positively and negatively [3-10].…”

“…In many cases, ncRNAs appear to cause transcriptional interference [3,5,15,16,21-23] affecting the expression of neighbouring genes in cis . On the other hand, ncRNAs can be functional and regulate in trans the expression of genes located both nearby or at distant loci [4,6,10]. Although only a small number of functional ncRNAs have been well characterized to date, they have been shown to control gene expression at the transcriptional level.…”

“…Although only a small number of functional ncRNAs have been well characterized to date, they have been shown to control gene expression at the transcriptional level. For instance, SUT075 has recently been reported to regulate the expression of PRP3 when overexpressed remotely on a plasmid [10]. Another example is SUT457, which is involved in telomere organization.…”