Systematic sequencing of chloroplast transcript termini fromArabidopsis thalianareveals >200 transcription initiation sites and the extensive imprints of RNA-binding proteins and secondary structures

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 1,628 processed 5' ends and 1,299 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 2,000 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.

The Arabidopsis mTERF‐repeat MDA1 protein plays a dual function in transcription and stabilization of specific chloroplast transcripts within the psbE and ndhH operons

“…These results argue that MDA1 contributes to the stabilization of the À67 ndhA 5 0 -end in vivo. Interestingly, the position of this processed 5 0 -end coincides with the existence of a recently identified TSS suggesting that the 5 0 -end processing of ndhA mRNA is concomitant with transcription (Castandet et al, 2019).…”

“…The RNA blotting revealed that the abundance of distinct mRNAs containing ndhA and ndhI was specifically reduced in mda1 whereas mRNAs containing genes upstream or downstream were barely affected. We were able to assign positions for these disturbed transcripts based on their size, the probes to which they hybridized and the positions of chloroplast RNA termini in Arabidopsis that were mapped in this work or in a recent study (Castandet et al, 2019). Transcripts whose abundance is diminished in mda1 start with an ndhA 5 0 -end.…”

Organellar and Secretory Ribonucleases: Major Players in Plant RNA Homeostasis

“…Under phosphorus deprivation, Arabidopsis mutants fail to initiate lateral roots, whereas PNPase-depleted Chlamydomonas cells cannot survive in the same condition (Yehudai-Resheff et al, 2007;Marchive et al, 2009). PNPase was the first chloroplastic RNase whose influence on RNA maturation was investigated using RNA-seq (Hotto et al, 2011;Castandet et al, 2013;Castandet et al, 2019). This analysis, in association with traditional molecular biology techniques, showed that PNPase-deficient Arabidopsis plants accumulate numerous RNAs with 39 extensions along with additional defects in RNA splicing and editing.…”

“…This analysis, in association with traditional molecular biology techniques, showed that PNPase-deficient Arabidopsis plants accumulate numerous RNAs with 39 extensions along with additional defects in RNA splicing and editing. Studies in Arabidopsis and maize determined that all types of RNAs are affected, including mRNAs, rRNAs, tRNA precursors, antisense RNAs, and spliced intron products (Walter et al, 2002;Marchive et al, 2009;Williams-Carrier et al, 2010;Germain et al, 2011;Hotto et al, 2011;Castandet et al, 2013;Ruwe et al, 2013;Castandet et al, 2019).…”

“…Additionally, mutants for the endoribonuclease RNase E, its specificity partner RHON1, RNase J, and others, accumulate unique and apparently unprocessed transcripts, but the precise sites of cleavage are still unknown (Walter et al, 2010;Luro et al, 2013). These limitations have recently started to be overcome with the use of RNA-seq based approaches that have been instrumental in understanding the global role of two chloroplast ribonucleases, the exoribonuclease PNPase and the endoribonuclease mini-III (Hotto et al, 2011;Castandet et al, 2013;Ruwe et al, 2013;Hotto et al, 2015;Castandet et al, 2019). Combining a transcriptome-wide view to a single-nt resolution map of the chloroplast RNA processing sites is likely to speed up any future functional analysis of poorly studied chloroplastic ribonucleases whose RNA targets are still unknown.…”