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 is highly promiscuous, requiring numerous quality control steps to generate the ultimately complex but selective mixture of accumulating RNAs. To gain insight into how this RNA diversity is achieved and regulated, we focused on mapping transcript ends (the terminome) using Terminome-Seq, a protocol to systematically sequence RNA termini. Using Arabidopsis thaliana as a model, we generated a catalogue of >215 primary 5′ ends corresponding to transcription start sites (TSS), as well as 1,628 processed 5′ ends and 1,299 3′ ends. While most termini were found in intergenic regions, as expected, numerous abundant termini were also found within coding regions and introns, including a major TSS within the first clpP intron, and other TSS internal to gene clusters. A consistent feature was that both 5′ and 3′ ends tended to occur in clusters, 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 current model of passive RNA protection. Terminome-Seq was also implemented for pnp1-1, a mutant lacking the well-characterized 3′ to 5′ exoribonuclease polynucleotide phosphorylase. Most 3′ ends, and many 5′ ends differed in pnp1-1 compared to wild-type plants, underscoring its near-ubiquitous role in shaping the transcriptome. Both examples establish the potential of Terminome-Seq to decipher precisely the roles and regulation of ribonucleases, RNA-binding proteins and transcription initiation factors in organellar transcriptome quality control.