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Updated: Mar 26, 2026
Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling
Published on: December 21, 2017
Genome-wide quantification of 5'-phosphorylated mRNA degradation intermediates for analysis of ribosome dynamics
Vicent Pelechano1, Wu Wei2,3, Lars M Steinmetz1,2,3
1European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany.
Abstract:
Co-translational mRNA degradation is a widespread process in which 5'-3' exonucleolytic degradation follows the last translating ribosome, thus producing an in vivo ribosomal footprint that delimits the 5' position of the mRNA molecule within the ribosome. To study this degradation process and ribosome dynamics, we developed 5PSeq, which is a method that profiles the genome-wide abundance of mRNA degradation intermediates by virtue of their 5'-phosphorylated (5'P) ends. The approach involves targeted ligation of an oligonucleotide to the 5'P end of mRNA degradation intermediates, followed by depletion of rRNA molecules, reverse transcription of 5'P mRNAs and Illumina high-throughput sequencing. 5PSeq can identify translational pauses at rare codons that are often masked when using alternative methods. This approach can be applied to previously extracted RNA samples, and it is straightforward and does not require polyribosome purification or in vitro RNA footprinting. The protocol we describe here can be applied to Saccharomyces cerevisiae and potentially to other eukaryotic organisms. Three days are required to generate 5PSeq libraries.
Insights
We developed 5PSeq, a new method to study mRNA degradation and ribosome dynamics by profiling 5-phosphorylated mRNA ends. This technique reveals translational pauses at rare codons, offering insights into gene expression regulation.
Area of Science:
- Molecular Biology
- Genomics
- RNA Biology
Background:
- Co-translational mRNA degradation involves 5'-3' exonucleolytic decay following the ribosome.
- This process leaves a ribosomal footprint, indicating the mRNA's 5' position within the ribosome.
Purpose of the Study:
- To develop a novel method for profiling genome-wide mRNA degradation intermediates.
- To study co-translational mRNA degradation and ribosome dynamics.
- To identify translational pauses, particularly at rare codons.
Main Methods:
- Developed 5PSeq (5'-phosphorylated sequencing) to profile mRNA degradation intermediates.
- Utilized targeted oligonucleotide ligation to 5'-phosphorylated mRNA ends.
- Employed rRNA depletion, reverse transcription, and Illumina high-throughput sequencing.
Main Results:
- 5PSeq successfully profiles genome-wide mRNA degradation intermediates.
- The method identifies translational pauses at rare codons, often missed by other techniques.
- The protocol is applicable to previously extracted RNA and does not require polyribosome purification.
Conclusions:
- 5PSeq is a straightforward and effective method for studying mRNA degradation and ribosome dynamics.
- This technique provides valuable insights into translational regulation and gene expression.
- The protocol is adaptable for use in Saccharomyces cerevisiae and potentially other eukaryotes.
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