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Disome-seq data in yeast cells

ABSTRACT: Disome-seq data in yeast cells

PROVIDER: PRJNA665694 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12712400_1.fastq.gz	Fastqsanger.gz
	SRR12712400_2.fastq.gz	Fastqsanger.gz
	SRR12712401_1.fastq.gz	Fastqsanger.gz
	SRR12712401_2.fastq.gz	Fastqsanger.gz
	SRR12712402_1.fastq.gz	Fastqsanger.gz

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Disome-seq data in yeast cells

Project description:Genome-wide detection of monosome and disome distributions in yeast cells

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Disome-seq reveals sequence-mediated coupling of translational pauses and protein structures

Project description:The regulation of translation elongation plays a vital role in protein folding; an adequate translational pause provides time and cellular environments for the co-translational folding of nascent peptides. However, the genomic landscape, sequence determinants, and molecular consequences of translational pausing remain mostly unknown. In this study, we performed disome-seq that sequenced mRNA fragments protected by two consecutive ribosomes – a product of severe translational pauses during which the upstream ribosome collides into the paused one. We detected severe translational pauses on ~75% of yeast genes. These pauses were often explained by one of the three mechanisms: 1) slow ribosome releasing at stop codons, 2) slow peptide formation from proline, glycine, asparagine, and cysteine, and 3) slow leaving of polylysine from the exit tunnel of ribosomes. Notably, these amino acids also terminate the α-helical conformation. Such dual roles of amino acids establish an inborn coupling between the synthetic completion of a structural motif and a translational pause. Furthermore, paused ribosomes often recruit chaperones to assist protein folding. As a consequence, emergent protein structures during evolution should be ready to be correctly folded. Collectively, our study shows widespread translational pauses and sheds lights on a better understanding of the regulation of co-translational protein folding.

2020-12-10 | PXD015271 | Pride

Project description:Disome-seq data in yeast cells

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Disome and trisome profiling reveal targets of ribosome quality control

Project description:The ribosome-associated protein quality control (RQC) system that resolves stalled translation events is activated when ribosomes collide and form disome, trisome or higher order complexes. However, it is unclear whether this system distinguishes collision complexes formed on defective mRNAs from those with functional roles on endogenous transcripts. Here, we performed disome and trisome footprint profiling in yeast and found collisions were enriched on diverse sequence motifs known to slow translation. When 60S recycling was inhibited, disomes accumulated at stop codons and could move into the 3’UTR to reinitiate translation. The ubiquitin ligase and RQC factor Hel2/ZNF598 generally recognized collisions but did not trigger degradation of endogenous transcripts. However, loss of Hel2 triggered the integrated stress response, via phosphorylation of eIF2alpha, thus linking these pathways. Our results suggest that Hel2 has a role in sensing ribosome collisions on endogenous mRNAs and such events may be important for cellular homeostasis.

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Monosome and disome profiling of Flag-STAU1 Flp-In 293 T-REx cells

Project description:Monosome and disome profiling was performed on Flag-STAU1 Flp-In 293 T-REx to study the causes of ribosomal collisions, and whether this may be modulated by the presence/absence of Staufen-1. Cells were treated with either an siRNA targeting STAU1 transcript (4x samples) or a control siRNA (2x samples). Two of the four samples treated with the STAU1 siRNA had siRNA-resistant STAU1 mRNA expression induced by doxycycline (rescue). Sequencing libraries from monosome and disome fractions were generated in parallel from the same samples. Note that unique molecular identifiers/random barcodes (UMIs/RBCs) were included in the sequencing experiment. Each UMI has been moved to the fastq read name of each read. For example \\"xxxxxxrbc:AGCCAAT\\" in the read name signifies that the given read had a UMI of \\"AGCCAAT\\". Using these UMIs, PCR duplicates can be removed with UMI-Tools following read alignment.

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