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RNAseq analysis of mutants in coding and non-coding transcription of phosphate genes in the yeast Saccharomyces cerevisiae.

ABSTRACT: This study addresses the effect of mutations related to sense and antisense transcription of Saccharomyces cerevisiae phosphate genes using strand-specific RNAseq. Replacement of the transcriptional terminator of PHO84 by that of CYC1 resulted, unexpectedly, in an increased antisense transcription and a strongly reduced sense transcription of PHO84. Also RNA levels of SPL2 were strongly reduced. Deletion of the PHO84 coding region did not markedly affect the expression of SPL2, suggesting that antisense transcription of PHO84 and not the Pho84 transporter affects the expression of SPL2. Deletion of the two putative binding sites for the transcriptional regulator Ume6 in the SPL2 promoter resulted in a slightly increased level of SPL2 RNA, whereas deletion of the UME6 gene resulted in a decreased level of SPL2 and PHO84 RNA. These results suggests that Ume6 is involved in the regulation of SPL2 by a mechanism different from a simple binding to the putative Ume6 binding sites.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE226971 | GEO | 2024/03/06

REPOSITORIES: GEO

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Project description:Non-coding sense and antisense germline transcription within the immunoglobulin heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germline transcription throughout the Igh locus has been done. Therefore, we performed directional RNAseq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two PAIR elements in the distal IghV region. Importantly, long-distance loops measured by 3C are observed between these two active PAIR promoters and Eμ, the start site of Iμ germline transcription, in a lineage- and stage-specific manner, even though this antisense transcription is Eμ-independent. YY1-/- pro-B cells are greatly impaired in distal VH gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eμ interactions. ChIP-seq shows high level YY1 binding only at Eμ, but low levels near some antisense promoters. PAIR-Eμ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal VH genes close to the DJH rearrangement, which is adjacent to Eμ. Therefore, we hypothesize that one key role of non-coding germline transcription is to facilitate locus compaction, allowing distal VH genes to undergo efficient rearrangement. In order to determine the amount and location of sense and antisense non-coding RNA in the Igh locus, we prepared total RNA from CD19+ RAG1-/- pro-B cells. Samples were either pre-enriched in custom Agilent arrays, or directly sequenced. Data from one sample for each condition is included as reflected in the publication.

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RNAseq analysis of mutants in coding and non-coding transcription of phosphate genes in the yeast Saccharomyces cerevisiae.

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