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TRNA pool analysis of S. cerevisiae lacking Paf1 and Rtf1 using tRNA-HySeq

ABSTRACT: tRNA pool analysis of S. cerevisiae lacking Paf1 and Rtf1 using tRNA-HySeq

PROVIDER: PRJNA557037 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9850460.fastq.gz	Fastqsanger.gz
	SRR9850461.fastq.gz	Fastqsanger.gz
	SRR9850462.fastq.gz	Fastqsanger.gz
	SRR9850463.fastq.gz	Fastqsanger.gz
	SRR9850458.fastq.gz	Fastqsanger.gz

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tRNA pool analysis of S. cerevisiae lacking Paf1 and Rtf1 using tRNA-HySeq

Project description:tRNAs isolated from log phase yeast cells were hydrolyzed, followed by adapter ligation and sequencing.

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Comparative analysis of Rtf1- and PAF1C-regulated genes

Project description:Paf1 and Ski8 were selected as representative subunits of the Paf1 complex (PAF1C), and RNA-seq analysis was performed in triplicate to compare the genes affected by Paf1, Ski8, and Rtf1 knockdown in HeLa cells.

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tRNA pool analysis of S. cerevisiae using tRNA-HySeq

Project description:tRNA pool analysis of S. cerevisiae using tRNA-HySeq

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tRNA pool analysis of S. cerevisiae using tRNA-HySeq

Project description:For a typical RNA polymerase (pol) II transcribed budding yeast gene, the 5' -end is characterized by a nucleosome-free region (NFR) immediate upstream of the transcription start site (TSS), flanked by two well-positioned nucleosomes (-1 and +1) containing H2A.Z. A similar arrangement of nucleosomes containing H2A.Z is found on the genes transcribed by pol III, which reside in the NFR actively maintained by the chromatin remodeling complexes. We did genome-wide MNase-seq and ChIP-seq experiments to study the nucleosome arrangement near pol III transcribed genes. We also measured the levels of different tRNAs in the tRNA pool of the wild type and Spt16 mutant (*spt16-197*) cells using tRNA-HySeq method. Although, it is difficult to measure the primary transcripts of tRNA due to their quick processing and the sequence degeneracy of the tRNA isogenes; a comparison of the wild type and Spt16 mutant showed both increase or decrease of tRNA transcripts. The result suggest that Spt16 may not be necessary for the transcription per se of tRNA genes.

2023-12-31 | GSE109527 | GEO

RTF1 and SPT6 stimulate transcription elongation by two distinct mechanisms

Project description:We have previously shown that the RNA polymerase II (Pol II)-DSIF complex associates with the PAF1 complex (PAF), RTF1 and SPT6 to form an activated elongation complex in vitro. Here we investigate the mechanisms that these factors use to stimulate Pol II elongation in vivo. We combine rapid factor depletion from human cells with genome-wide analyses of changes in RNA synthesis and occupancy with engaged Pol II. Whereas depletion of PAF subunits has little effect on transcription in vivo, depletion of RTF1 or SPT6 strongly compromises RNA synthesis, albeit in fundamentally different ways. RTF1 depletion decreases Pol II velocity, whereas SPT6 depletion impairs Pol II progression through nucleosomes. These results show that distinct transcription elongation factors stimulate Pol II velocity and Pol II progression through chromatin in vivo. Our results also provide evidence for two distinct barriers to elongation at the beginning of genes, the promoter-proximal pause site and the +1 nucleosome.

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The Histone Modification Domain of Paf1 Complex Subunit Rtf1 Promotes H2B Ubiquitylation Through a Direct Interaction with the Ubiquitin Conjugase Rad6

Project description:Transcription by RNA polymerase II is regulated by epigenetic modifications to the chromatin template. The mono-ubiquitylation of H2B is established during transcription elongation and broadly impacts chromatin architecture and gene expression. The Polymerase Associated Factor 1 complex (Paf1C) is required for H2B ubiquitylation through an unknown mechanism. Here, we find that a 66-amino acid histone modification domain (HMD) within the Rtf1 subunit of Paf1C promotes H2B ubiquitylation in cells lacking all Paf1C members and present the crystal structure of this domain. Using site-specific in vivo crosslinking, we show that Rtf1 directly interacts with the ubiquitin conjugase Rad6 through a conserved surface on the HMD. Through ChIP-exo analysis, we observe that enrichment of Paf1C correlates with H2B ubiquitylation, and that the HMD, Rad6 and Bre1 localize to H2B. Finally, we demonstrate that the HMD directly stimulates H2B ubiquitylation in a reconstituted system, arguing that Paf1C functions as a cofactor for Rad6-Bre1 mediated catalysis.

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Project description:Genomic features of DSB re-landscaping in rtf1 mutants. Histone modification is a critical determinant of frequency and location of double-strand breaks (DSBs), which induce recombination during meiosis. The Set1-dependent histone H3K4 and Dot1-dependent H3K79 methylations play an important role in DSB formations in budding yeast. Both methylations are promoted by the RNA polymerase II associated factor 1 (Paf1) complex, Paf1C. This study addressed a role of the Paf1C component Rtf1, which is critical for H3K4 and H3K79 methylations, for the regulation of meiotic DSB formation. Similar to set1 mutation, rtf1 mutation decreased the occurrence of DSBs in the genome. The rtf1 set1 double mutant exhibited a larger reduction in the levels of DSBs than the frequency of DSBs detected in either of the single mutants; this indicates independent roles of Rtf1 and Set1 in DSB formation. Importantly, the distribution of DSBs along chromosomes in the rtf1 mutant changed in a different manner than the pattern observed in the set1 and set1 dot1 mutants; this was characterized by enhanced DSB formation at some DSB-cold regions. These observations suggest that Rtf1, and, possibly, the Paf1C, determine DSB landscape in the genome, independent of H3K4 methylation.

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Rtf1-dependent transcriptional pausing regulates cardiogenesis

Project description:During heart development, an evolutionarily conserved network of cardiac transcription factors collaborate to define the precise timing and location of cardiac progenitor specification. Accumulating evidence suggests that cardiac progenitor specification is subject to transcriptional control beyond the level of transcription initiation. The PAF1C component Rtf1 is a multifunctional transcription regulatory protein that modulates pausing and elongation of RNA Pol II, as well as histone epigenetic modifications. By transient knockdown and CRISPR mutagenesis, we found that Rtf1 is essential for cardiogenesis and that without Rtf1 activity, cardiac progenitors arrest in an immature state. This role in early cardiogenesis was evolutionarily conserved between fish and mammals. We also found that Rtf1's Plus3 domain, which confers interaction with the pausing/elongation factor Spt5, was required for Rtf1's ability to support cardiac progenitor formation, while other regions of the protein were dispensable. We examined the occupancy of RNA Pol II at cardiac genes in rtf1 morphants using ChIP-seq and found that Pol II signals at the TSS of genes was reduced, suggesting a reduction in transcriptional pausing. Intriguingly, pharmacological or morpholino antisense reduction of pause release in rtf1 morphants and mutants restored the formation of cardiac cells and improved Pol II occupancy at the TSS of key cardiac genes. Our findings highlight the crucial role that transcriptional pausing plays in promoting normal levels of gene expression in a cardiac developmental context.

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Manipulation of the human tRNA pool reveals distinct tRNA sets that act in cellular proliferation or cell cycle arrest [tRNA-seq]

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