Details
| Original language | English |
|---|---|
| Pages (from-to) | 1614-1634 |
| Number of pages | 21 |
| Journal | Plant Journal |
| Volume | 117 |
| Issue number | 5 |
| Publication status | Published - 26 Feb 2024 |
Abstract
Ribosome profiling (Ribo-seq) is a powerful method for the deep analysis of translation mechanisms and regulatory circuits during gene expression. Extraction and sequencing of ribosome-protected fragments (RPFs) and parallel RNA-seq yields genome-wide insight into translational dynamics and post-transcriptional control of gene expression. Here, we provide details on the Ribo-seq method and the subsequent analysis with the unicellular model alga Chlamydomonas reinhardtii (Chlamydomonas) for generating high-resolution data covering more than 10 000 different transcripts. Detailed analysis of the ribosomal offsets on transcripts uncovers presumable transition states during translocation of elongating ribosomes within the 5′ and 3′ sections of transcripts and characteristics of eukaryotic translation termination, which are fundamentally distinct for chloroplast translation. In chloroplasts, a heterogeneous RPF size distribution along the coding sequence indicates specific regulatory phases during protein synthesis. For example, local accumulation of small RPFs correlates with local slowdown of psbA translation, possibly uncovering an uncharacterized regulatory step during PsbA/D1 synthesis. Further analyses of RPF distribution along specific cytosolic transcripts revealed characteristic patterns of translation elongation exemplified for the major light-harvesting complex proteins, LHCs. By providing high-quality datasets for all subcellular genomes and attaching our data to the Chlamydomonas reference genome, we aim to make ribosome profiles easily accessible for the broad research community. The data can be browsed without advanced bioinformatic background knowledge for translation output levels of specific genes and their splice variants and for monitoring genome annotation.
Keywords
- Chlamydomonas reinhardtii, next-generation sequencing, protein synthesis, ribosome profiling, ribosomes, transcript accumulation, translation
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Genetics
- Agricultural and Biological Sciences(all)
- Plant Science
- Biochemistry, Genetics and Molecular Biology(all)
- Cell Biology
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In: Plant Journal, Vol. 117, No. 5, 26.02.2024, p. 1614-1634.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Utilizing high-resolution ribosome profiling for the global investigation of gene expression in Chlamydomonas
AU - Gotsmann, Vincent Leon
AU - Ting, Michael Kien Yin
AU - Haase, Nadin
AU - Rudorf, Sophia
AU - Zoschke, Reimo
AU - Willmund, Felix
PY - 2024/2/26
Y1 - 2024/2/26
N2 - Ribosome profiling (Ribo-seq) is a powerful method for the deep analysis of translation mechanisms and regulatory circuits during gene expression. Extraction and sequencing of ribosome-protected fragments (RPFs) and parallel RNA-seq yields genome-wide insight into translational dynamics and post-transcriptional control of gene expression. Here, we provide details on the Ribo-seq method and the subsequent analysis with the unicellular model alga Chlamydomonas reinhardtii (Chlamydomonas) for generating high-resolution data covering more than 10 000 different transcripts. Detailed analysis of the ribosomal offsets on transcripts uncovers presumable transition states during translocation of elongating ribosomes within the 5′ and 3′ sections of transcripts and characteristics of eukaryotic translation termination, which are fundamentally distinct for chloroplast translation. In chloroplasts, a heterogeneous RPF size distribution along the coding sequence indicates specific regulatory phases during protein synthesis. For example, local accumulation of small RPFs correlates with local slowdown of psbA translation, possibly uncovering an uncharacterized regulatory step during PsbA/D1 synthesis. Further analyses of RPF distribution along specific cytosolic transcripts revealed characteristic patterns of translation elongation exemplified for the major light-harvesting complex proteins, LHCs. By providing high-quality datasets for all subcellular genomes and attaching our data to the Chlamydomonas reference genome, we aim to make ribosome profiles easily accessible for the broad research community. The data can be browsed without advanced bioinformatic background knowledge for translation output levels of specific genes and their splice variants and for monitoring genome annotation.
AB - Ribosome profiling (Ribo-seq) is a powerful method for the deep analysis of translation mechanisms and regulatory circuits during gene expression. Extraction and sequencing of ribosome-protected fragments (RPFs) and parallel RNA-seq yields genome-wide insight into translational dynamics and post-transcriptional control of gene expression. Here, we provide details on the Ribo-seq method and the subsequent analysis with the unicellular model alga Chlamydomonas reinhardtii (Chlamydomonas) for generating high-resolution data covering more than 10 000 different transcripts. Detailed analysis of the ribosomal offsets on transcripts uncovers presumable transition states during translocation of elongating ribosomes within the 5′ and 3′ sections of transcripts and characteristics of eukaryotic translation termination, which are fundamentally distinct for chloroplast translation. In chloroplasts, a heterogeneous RPF size distribution along the coding sequence indicates specific regulatory phases during protein synthesis. For example, local accumulation of small RPFs correlates with local slowdown of psbA translation, possibly uncovering an uncharacterized regulatory step during PsbA/D1 synthesis. Further analyses of RPF distribution along specific cytosolic transcripts revealed characteristic patterns of translation elongation exemplified for the major light-harvesting complex proteins, LHCs. By providing high-quality datasets for all subcellular genomes and attaching our data to the Chlamydomonas reference genome, we aim to make ribosome profiles easily accessible for the broad research community. The data can be browsed without advanced bioinformatic background knowledge for translation output levels of specific genes and their splice variants and for monitoring genome annotation.
KW - Chlamydomonas reinhardtii
KW - next-generation sequencing
KW - protein synthesis
KW - ribosome profiling
KW - ribosomes
KW - transcript accumulation
KW - translation
UR - http://www.scopus.com/inward/record.url?scp=85178433527&partnerID=8YFLogxK
U2 - 10.1111/tpj.16577
DO - 10.1111/tpj.16577
M3 - Article
AN - SCOPUS:85178433527
VL - 117
SP - 1614
EP - 1634
JO - Plant Journal
JF - Plant Journal
SN - 0960-7412
IS - 5
ER -