Genome‐wide screen of fission yeast mutants for sensitivity to 6‐azauracil, an inhibitor of transcriptional elongation

Abstract

Abstract6-Azauracil (6 AU) inhibits enzymes in nucleoside synthesis and depletes the intracellular GTP/UTP pool. Mutations in transcriptional elongation machinery, as well as mutations in a variety of other pathways, exaggerate the growth defect of cells in the presence of 6 AU. Thus, identification of mutations that render cells sensitive to 6 AU will benefit study on the basis of 6 AU-sensitive phenotype. Here we performed a genome-wide screen of a fission yeast deletion library. Of 3235 single-gene deletions, 66 mutants displayed at least 50% drop of fitness in the presence of 6 AU and 60 mutants were reported for the first time; five deletions showed synthetic decrease of fitness when combined with deletion of set3 + , which encodes a transcriptional regulator. Genes conferring tolerance to 6 AU were enriched in various processes, especially in chromosome segregation. Accordingly, genes encoding subunits of CLRC complex and spindle pole body were over-represented. Mutants were subjected to an in vivo transcript length-dependent reporter assay to assess the potential roles of deleted genes in transcriptional elongation. As with the deletions known to affect elongation, nab2Δ, nxt1Δ, rhp18Δ, SPAC24C9.08Δ, clr3Δ and ncs1Δset3Δ mutants exhibited defects in expressing long transcripts. New 6 AU-sensitive mutants identified here will help to elucidate the mechanism of action of 6 AU in the cells. Meanwhile, our study revealed novel genes potentially involved in transcriptional elongation and provided valuable targets for transcription study.

Expanding the Substrate Specificity of Macro Domains toward 3″-Isomer of O-Acetyl-ADP-ribose

“…From our qualitative sensitivity analysis, the poa1 null strain was sensitive to 6-azauracil, which was presumed to affect elongation in yeast cells by depleting the pools of UTP and GTP and affecting induction of IMD2, which encodes IMP dehydrogenase . Nonetheless, we could not find poa1 from the genome-wide analysis of 6-azauracil-sensitive mutants, but we did find some known poa1 -interacting partners such as nuclear components ccr4 and nab2 . With the use of STRING analysis, Poa1p shows high correlation with the predicted functional partners of HTA1, HTA2, and HTB1 proteins, which are core histone proteins required for chromatin assembly and chromosome function in the nucleus .…”

“…55 Nonetheless, we could not find poa1 from the genome-wide analysis of 6-azauracilsensitive mutants, but we did find some known poa1-interacting partners such as nuclear components ccr4 and nab2. 56 With the use of STRING analysis, Poa1p shows high correlation with the predicted functional partners of HTA1, HTA2, and HTB1 proteins, which are core histone proteins required for chromatin assembly and chromosome function in the nucleus. 57 Accordingly, we used the DeepLoc server 58 for predicting the subcellular localization of Poa1p and found more than 90% probability of Poa1p localized in cytoplasm, with only a few in the nucleus and mitochondria.…”

Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe

“…To explore further whether Upf1 has a role in Pol II transcription, we examined whether upf1 Δ cells are hypersensitive to 6-azauracil (6AU). It has previously been reported that strains carrying mutations in components of the RNA polymerase II transcription elongation machinery are hypersensitive to 6AU ( 43 , 44 ). 6AU is an inhibitor of enzymes that are involved in nucleotide biosynthesis; 6AU treatment leads to nucleotide depletion and hence can diminish transcription elongation ( 45 ).…”

“…In view of these changes in Pol II loading and Ser2 phosphorylation, the possibility that Pol II elongation is altered in upf1 Δ cannot be ruled out. Specifically, upf1 Δ shows apparent 6AU hypersensitivity (Figure 7 ), which is a characteristic of transcription elongation S. pombe mutant strains ( 43 ). Additionally, an S. pombe strain carrying a mutant Pol II with reduced elongation rate is also hypersensitive to 6AU ( 47 ).…”

A Bifunctional Nucleoside Probe for the Inhibition of the Human Immunodeficiency Virus-Type 1 Reverse Transcriptase

“…Early studies of 6-azauracil demonstrated that it inhibited the growth of yeast and determined that this was achieved through the inhibition of transcriptional elongation by affecting GTP synthesis and depleting UTP pools . While the literature does not suggest a direct interaction of 6-azauracil with polymerases, studies have demonstrated that uracil can be deleterious if incorporated into DNA .…”