Systematic sequencing of chloroplast transcript termini from Arabidopsis thaliana reveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures

Chloroplast transcription requires numerous quality control steps to generate the complex but selective mixture of accumulating RNAs. To gain insight into how this RNA diversity is achieved and regulated, we systematically mapped transcript ends by developing a protocol called Terminome-seq. Using A...

Descripción completa

Detalles Bibliográficos
Autores principales: Castandet, Benoît, Germain, Arnaud, Hotto, Amber M, Stern, David B
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145512/
https://www.ncbi.nlm.nih.gov/pubmed/31732725
http://dx.doi.org/10.1093/nar/gkz1059
_version_ 1783520004335992832
author Castandet, Benoît
Germain, Arnaud
Hotto, Amber M
Stern, David B
author_facet Castandet, Benoît
Germain, Arnaud
Hotto, Amber M
Stern, David B
author_sort Castandet, Benoît
collection PubMed
description Chloroplast transcription requires numerous quality control steps to generate the complex but selective mixture of accumulating RNAs. To gain insight into how this RNA diversity is achieved and regulated, we systematically mapped transcript ends by developing a protocol called Terminome-seq. Using Arabidopsis thaliana as a model, we catalogued >215 primary 5′ ends corresponding to transcription start sites (TSS), as well as 1628 processed 5′ ends and 1299 3′ ends. While most termini were found in intergenic regions, numerous abundant termini were also found within coding regions and introns, including several major TSS at unexpected locations. A consistent feature was the clustering of both 5′ and 3′ ends, contrasting with the prevailing description of discrete 5′ termini, suggesting an imprecision of the transcription and/or RNA processing machinery. Numerous termini correlated with the extremities of small RNA footprints or predicted stem-loop structures, in agreement with the model of passive RNA protection. Terminome-seq was also implemented for pnp1–1, a mutant lacking the processing enzyme polynucleotide phosphorylase. Nearly 2000 termini were altered in pnp1–1, revealing a dominant role in shaping the transcriptome. In summary, Terminome-seq permits precise delineation of the roles and regulation of the many factors involved in organellar transcriptome quality control.
format Online
Article
Text
id pubmed-7145512
institution National Center for Biotechnology Information
language English
publishDate 2019
publisher Oxford University Press
record_format MEDLINE/PubMed
spelling pubmed-71455122020-04-13 Systematic sequencing of chloroplast transcript termini from Arabidopsis thaliana reveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures Castandet, Benoît Germain, Arnaud Hotto, Amber M Stern, David B Nucleic Acids Res RNA and RNA-Protein Complexes Chloroplast transcription requires numerous quality control steps to generate the complex but selective mixture of accumulating RNAs. To gain insight into how this RNA diversity is achieved and regulated, we systematically mapped transcript ends by developing a protocol called Terminome-seq. Using Arabidopsis thaliana as a model, we catalogued >215 primary 5′ ends corresponding to transcription start sites (TSS), as well as 1628 processed 5′ ends and 1299 3′ ends. While most termini were found in intergenic regions, numerous abundant termini were also found within coding regions and introns, including several major TSS at unexpected locations. A consistent feature was the clustering of both 5′ and 3′ ends, contrasting with the prevailing description of discrete 5′ termini, suggesting an imprecision of the transcription and/or RNA processing machinery. Numerous termini correlated with the extremities of small RNA footprints or predicted stem-loop structures, in agreement with the model of passive RNA protection. Terminome-seq was also implemented for pnp1–1, a mutant lacking the processing enzyme polynucleotide phosphorylase. Nearly 2000 termini were altered in pnp1–1, revealing a dominant role in shaping the transcriptome. In summary, Terminome-seq permits precise delineation of the roles and regulation of the many factors involved in organellar transcriptome quality control. Oxford University Press 2019-12-16 2019-11-16 /pmc/articles/PMC7145512/ /pubmed/31732725 http://dx.doi.org/10.1093/nar/gkz1059 Text en © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle RNA and RNA-Protein Complexes
Castandet, Benoît
Germain, Arnaud
Hotto, Amber M
Stern, David B
Systematic sequencing of chloroplast transcript termini from Arabidopsis thaliana reveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures
title Systematic sequencing of chloroplast transcript termini from Arabidopsis thaliana reveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures
title_full Systematic sequencing of chloroplast transcript termini from Arabidopsis thaliana reveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures
title_fullStr Systematic sequencing of chloroplast transcript termini from Arabidopsis thaliana reveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures
title_full_unstemmed Systematic sequencing of chloroplast transcript termini from Arabidopsis thaliana reveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures
title_short Systematic sequencing of chloroplast transcript termini from Arabidopsis thaliana reveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures
title_sort systematic sequencing of chloroplast transcript termini from arabidopsis thaliana reveals >200 transcription initiation sites and the extensive imprints of rna-binding proteins and secondary structures
topic RNA and RNA-Protein Complexes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145512/
https://www.ncbi.nlm.nih.gov/pubmed/31732725
http://dx.doi.org/10.1093/nar/gkz1059
work_keys_str_mv AT castandetbenoit systematicsequencingofchloroplasttranscriptterminifromarabidopsisthalianareveals200transcriptioninitiationsitesandtheextensiveimprintsofrnabindingproteinsandsecondarystructures
AT germainarnaud systematicsequencingofchloroplasttranscriptterminifromarabidopsisthalianareveals200transcriptioninitiationsitesandtheextensiveimprintsofrnabindingproteinsandsecondarystructures
AT hottoamberm systematicsequencingofchloroplasttranscriptterminifromarabidopsisthalianareveals200transcriptioninitiationsitesandtheextensiveimprintsofrnabindingproteinsandsecondarystructures
AT sterndavidb systematicsequencingofchloroplasttranscriptterminifromarabidopsisthalianareveals200transcriptioninitiationsitesandtheextensiveimprintsofrnabindingproteinsandsecondarystructures