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Development of High-throughput 5Pseq and detection of ribosome stalls at termination level in Saccharomyces cerevisiae

ABSTRACT: Development of High-throughput 5Pseq and detection of ribosome stalls at termination level in Saccharomyces cerevisiae

PROVIDER: PRJNA639129 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12004671_1.fastq.gz	Fastqsanger.gz
	SRR12004671_2.fastq.gz	Fastqsanger.gz
	SRR12004672_1.fastq.gz	Fastqsanger.gz
	SRR12004672_2.fastq.gz	Fastqsanger.gz
	SRR12004673_1.fastq.gz	Fastqsanger.gz

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Development of High-throughput 5Pseq and detection of ribosome stalls at termination level in Saccharomyces cerevisiae

Project description:We developed a high throughput 5’P sequencing approach (HT-5PSeq) to investigate 5'P mRNA degradome in relation with translation process. This approach is easy and scalable; and uses an affordable rRNA depletion based on duplex-specific nuclease treatment. We use it to investigate in vivo ribosome stalls in S. cerevisiae and S. pombe at single nucleotide resolution. We investigate the 5’P degradation profiles associated to ribosome pausing, its regulation in stress conditions and the relative poly(A) length of mRNA degradation intermediates.

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Project description:RNA degradation is critical for gene expression and mRNA quality control. mRNA degradation is connected to the translation process up to the degree that 5'-3' mRNA degradation follows the last translating ribosome. Here, we present an improved high-throughput 5'P degradome RNA-sequencing method (HT-5Pseq). HT-5Pseq is easy, scalable, and uses affordable duplex-specific nuclease-based rRNA depletion. We investigate in vivo ribosome stalls focusing on translation termination. By comparing ribosome stalls identified by ribosome profiling, disome-seq and HT-5Pseq, we find that degradation-associated ribosome stalls are often enriched in Arg preceding the stop codon. On the contrary, mRNAs depleted for those stalls use more frequently a TAA stop codon preceded by hydrophobic amino acids. Finally, we show that termination stalls found by HT-5Pseq, and not by other approaches, are associated with decreased mRNA stability. Our work suggests that ribosome stalls associated with mRNA decay can be easily captured by investigating the 5'P degradome.

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Project description:Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here, we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but employs duplex-specific nuclease as a convenient, bias-free, species-independent way to reduce rRNA contamination. Our protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream and overlapping open reading frames (ORFs). This feature also allows the identification of an alternative ribosome conformation evident during termination of protein synthesis. To test the effectiveness of DSN in ribosome profiling, we generated Ribo-Seq libraries from mouse tissue culture cells and from the green alga Chlamydomonas reinhardtii.

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