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
2019
Castandet, Benoît | Germain, Arnaud | Hotto, Amber | Stern, David | Boyce Thompson Institute [Ithaca] | Institut des Sciences des Plantes de Paris-Saclay (IPS2 (UMR_9213 / UMR_1403)) ; Institut National de la Recherche Agronomique (INRA)-Université Paris-Sud - Paris 11 (UP11)-Université Paris Diderot - Paris 7 (UPD7)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS) | United States Department of Energy (DOE) DE-FG02-10ER20015LabEx Saclay Plant Sciences-SPS ANR-10-LABX-0040SPS | ANR-10-LABX-0040,SPS,Saclay Plant Sciences(2010)
International audience
显示更多 [+] 显示较少 [-]英语. 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 Ara-bidopsis 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, Terminomes-eq permits precise delineation of the roles and regulation of the many factors involved in organellar transcriptome quality control.
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