The conserved SEN1 DNA/RNA helicase has multiple functions during yeast meiosis
DNA:RNA hybrids are unusual structures found throughout the genomes of many species, including yeast and mammals. While DNA:RNA hybrids may promote various cellular functions, persistent hybrids lead to double strand breaks, resulting in genomic instability. DNA:RNA hybrid formation and removal are therefore highly regulated, including by enzymes that either degrade or unwind RNA from the hybrid. Meiosis is the specialized cell division that creates haploid gametes for sexual reproduction. Previous work in yeast and mammals showed that elimination of DNA:RNA hybrids by RNase H facilitates meiotic recombination. This work demonstrates that the conserved Sen1 DNA/RNA helicase functions during three temporally distinct processes during yeast meiosis. First, SEN1 allows meiosis-specific genes to be expressed at the proper time to allow entry into meiosis. Second, SEN1 prevents the accumulation of hybrids during premeiotic DNA replication. Third, SEN1 promotes the repair of programmed meiotic double strand breaks that are necessary to form crossovers between homologous chromosomes to allow their proper segregation at the first meiotic division. Given the evolutionary conservation of Sen1 with its mammalian counterpart, Senataxin, studies of Sen1 function in yeast are likely to be informative about the regulation of DNA:RNA hybrids during humans as well. Overall design: To investigate how SEN1 contributes to the regulation of IME1, nanopore direct RNA sequencing (dRNA-seq) was performed on coding and non-coding transcripts isolated from SEN1 and sen1-md cells in Spo medium. dRNA-seq requires that each transcript has a 3' poly(A) tail to which a sequencing adapter and motor protein that threads the RNA through a pore can be ligated. Non-coding RNAs—particularly those that fail to terminate properly in the absence of the Nrd1-Nab3-Sen1 (NNS) complex lack poly(A) tails and therefore would be underrepresented. To capture these transcripts, poly(A) tails are added in vitro. However the high abundance of the four ribosomal RNAs (rRNAs) (25S, 18S, 5.8S and 5S) in total RNA preparations would swamp out this reaction and so must be removed prior to polyadenylation. The four rRNAs were depleted from total RNA isolated from SEN1 and sen1-md cells by hybridization to the combined biotinylated anti-sense transcripts for each rRNA, followed by streptavidin-based removal. The resulting rRNA-depleted RNA was then polyadenylated in vitro using E. coli poly(A) polymerase prior to nanopore library preparation. Two biological replicates were performed for SEN1 and sen1-md strains after two hours in Spo medium, yielding ~6-7 million uniquely aligned reads per replicate to the S. cerevisiae SK1 genome.