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

Journal Article · 15 November 2019 · Nucleic Acids Research

DOI: https://doi.org/10.1093/nar/gkz1059 · OSTI ID:1606070

Castandet, Benoît ^[1]; Germain, Arnaud ^[2]; Hotto, Amber M. ^[2]; Stern, David B.  ^[2]

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1. Boyce Thompson Institute, Ithaca, NY 14853, USA, Institut des Sciences des Plantes de Paris Saclay (IPS2), UEVE, INRA, CNRS, Univ. Paris Sud, Université Paris-Saclay, F-91192 Gif sur Yvette, France, Université de Paris, IPS2, F-91192 Gif sur Yvette, France
2. Boyce Thompson Institute, Ithaca, NY 14853, USA

Abstract 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.

• Sponsoring Organization: USDOE
• Grant/Contract Number: FG02-90ER20015
• OSTI ID: 1606070
• Journal Information: Nucleic Acids Research, Journal Name: Nucleic Acids Research; ISSN 0305-1048
• Publisher: Oxford University PressCopyright Statement
• Country of Publication: United Kingdom
• Language: English