A resource for functional profiling of noncoding RNA in the yeast Saccharomyces cerevisiae

Abstract

Eukaryotic genomes are extensively transcribed, generating many different RNAs with no known function. We have constructed 1502 molecular barcoded ncRNA gene deletion strains encompassing 443 ncRNAs in the yeast Saccharomyces cerevisiae as tools for ncRNA functional analysis. This resource includes deletions of small nuclear RNAs (snRNAs), transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and other annotated ncRNAs as well as the more recently identified stable unannotated transcripts (SUTs) and cryptic unstable transcripts (CUTs) whose functions are largely unknown. Specifically, deletions have been constructed for ncRNAs found in the intergenic regions, not overlapping genes or their promoters (i.e., at least 200 bp minimum distance from the closest gene start codon). The deletion strains carry molecular barcodes designed to be complementary with the protein gene deletion collection enabling parallel analysis experiments. These strains will be useful for the numerous genomic and molecular techniques that utilize deletion strains, including genome-wide phenotypic screens under different growth conditions, pooled chemogenomic screens with drugs or chemicals, synthetic genetic array analysis to uncover novel genetic interactions, and synthetic dosage lethality screens to analyze gene dosage. Overall, we created a valuable resource for the RNA community and for future ncRNA research.

Keywords: noncoding RNA; snRNA; snoRNA; tRNA; yeast.

FIGURE 1.

The ncRNA deletion strategy and composition of the ncRNA deletion collection. (A) A schematic of the PCR-based ncRNA deletion strategy. The kanMX4 ncRNA deletion cassette was amplified from the pFA6a-kanMX4 plasmid using primers “Up primer (101mer)” and “Down primer (101mer).” The primers contain two barcodes (Tag), unique for each ncRNA and genome target homologous region (HR) sequences (upstream of and downstream from the ncRNA) to allow ncRNA disruption by homologous recombination. The kanMX4 ncRNA deletion cassettes were integrated into the genome at least 200 bp away from the closest open reading frame (ORF) start codon. (B) Composition of the ncRNA deletion collection. The “other ncRNA” are NME1, RPR1, RUF21, and TLC1.