Full Length Transcriptome Highlights the Coordination of Plastid Transcript Processing

Marine Guilcher,Etienne Delannoy,Thomas Blein,Arnaud Liehrmann,Benoit Castandet,Chloé Seyman,Guillem Rigaill

doi:10.3390/ijms222011297

Abstract

Plastid gene expression involves many post-transcriptional maturation steps resulting in a complex transcriptome composed of multiple isoforms. Although short-read RNA-Seq has considerably improved our understanding of the molecular mechanisms controlling these processes, it is unable to sequence full-length transcripts. This information is crucial, however, when it comes to understanding the interplay between the various steps of plastid gene expression. Here, we describe a protocol to study the plastid transcriptome using nanopore sequencing. In the leaf of Arabidopsis thaliana, with about 1.5 million strand-specific reads mapped to the chloroplast genome, we could recapitulate most of the complexity of the plastid transcriptome (polygenic transcripts, multiple isoforms associated with post-transcriptional processing) using virtual Northern blots. Even if the transcripts longer than about 2500 nucleotides were missing, the study of the co-occurrence of editing and splicing events identified 42 pairs of events that were not occurring independently. This study also highlighted a preferential chronology of maturation events with splicing happening after most sites were edited.

Highlights

Plastids are derived from the endosymbiosis between photosynthetic organisms and an ancestral Eukaryote
We describe the analysis of the A. thaliana plastid transcriptome by sequencing full-length non-polyadenylated and polyadenylated cDNAs using the Oxford Nanopore technology (ONT)
Sampling RNA from leaves of 5 week-old col-0 A. thaliana plants grown in long-day conditions at 20 ◦C, we mapped between 1.55 million and 2.69 million stranded reads to the A. thaliana genome including between 10% and 40% to the plastid genome and between 0.3% and 0.8% to the mitochondrial genome

Summary

Introduction

Plastids are derived from the endosymbiosis between photosynthetic organisms and an ancestral Eukaryote. Most of the initial symbiont genes have been transferred to the nucleus during the course of evolution, plastids of land plants and other photosynthetic Eukaryotes still maintain a small but essential genome It mainly encodes subunits of each of the photosynthetic complexes (Photosystem I and II, cytochrome b6/f, ATP synthase and Rubisco) and some of the plastid gene expression (PGE) machinery [1]. Most of the RNA binding proteins (RBP) or ribonucleases known to be involved in PGE are localized in a membraneless structure surrounding the plastome—the nucleoid [9] This close association between RNA maturation factors might be an explanation for the multiple pleiotropic effects observed in chloroplast mutants [7]

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Results

Conclusion