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A fluorescent assay for cryptic transcription in Saccharomyces cerevisiae [MNase-seq]

ABSTRACT: A fluorescent assay for cryptic transcription in Saccharomyces cerevisiae [MNase-seq]

PROVIDER: PRJNA1049345 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR27109002_1.fastq.gz	Fastqsanger.gz
	SRR27109002_2.fastq.gz	Fastqsanger.gz
	SRR27109003_1.fastq.gz	Fastqsanger.gz
	SRR27109003_2.fastq.gz	Fastqsanger.gz
	SRR27109004_1.fastq.gz	Fastqsanger.gz

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A fluorescent assay for cryptic transcription in Saccharomyces cerevisiae

Project description:A fluorescent assay for cryptic transcription in Saccharomyces cerevisiae

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A fluorescent assay for cryptic transcription in Saccharomyces cerevisiae [MNase-seq]

Project description:The disruption of chromatin structure can result in transcription initiation from cryptic promoters within gene bodies. While the passage of RNA polymerase II is a well-characterized chromatin-disrupting force, numerous factors, including histone chaperones, normally stabilize chromatin on transcribed genes, thereby repressing cryptic transcription. DNA replication, which requires a partially overlapping set of histone chaperones, is also inherently disruptive to chromatin, but a role for DNA replication in cryptic transcription has never been examined. In this study, we tested the hypothesis that, in the absence of chromatin-stabilizing factors, DNA replication can promote cryptic transcription in S. cerevisiae. Using a novel fluorescent reporter assay, we show that multiple factors, including Asf1, CAF-1, Rtt106, Spt6, and FACT, block transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Collectively, these results demonstrate that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA.

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Project description:We investigated cryptic transcription start site usage, chromatin organization and post-transcriptional consequences in Saccharomyces cerevisiae. We used 5PSeq approach, which measures ribosome dynamics by sequencing the presence of co-translation mRNA degradation intermediates, to assess if cryptic transcripts are engaged in active translation. We show that chromatin-dependent cryptic transcripts can be recognized by ribosomes and have the potential to produce truncated polypeptides by using downs-stream, in-frame start codons. Our work suggests that a significant fraction of chromatin-dependent internal cryptic promoters are in fact alternative truncated mRNA isoforms.

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Project description:In cells lacking the histone methyltransferase Set2, initiation of RNA polymerase II transcription occurs inappropriately within the protein-coding regions of genes, rather than being restricted to the proximal promoter. Here, we mapped the transcripts produced in an S. cerevisiae strain lacking Set2, and applied rigorous statistical methods to identify sites of cryptic transcription at high resolution. Wild type (BY4741) and set2â (BY4741) strains were grown at 30Â°C in YPD (1% yeast extract, 2% peptone, 2% dextrose) to an OD600 of 0.6-0.8. For each of the three replicates, Total RNA was extracted by acid-phenol method (Xiao et al. 2003). Double-stranded cDNA was prepared using an Invitrogen SuperScriptâ¢ (Cat No. 11917-010) primed with Oligo(dt) and random hexamers. For each replicate, the wt and set2â cDNA were independetly fluorescently labeled and comparatively hybridized to high-resolution 385K Saccharomyces cerevisiae CGH arrays (2005-08-16_SCER_WG_CGH) with Tm-normalized probes. In one of the replicates, assignment of the fluorescent label was reversed.

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