Complex Processing Patterns of mRNAs of the Large ATP Synthase Operon in Arabidopsis Chloroplasts

Mustafa Malik Ghulam,Florence Courtois,Silva Lerbs-Mache,Livia Merendino,Keqiang Wu

doi:10.1371/journal.pone.0078265

Abstract

Chloroplasts are photosynthetic cell organelles which have evolved from endosymbiosis of the cyanobacterial ancestor. In chloroplasts, genes are still organized into transcriptional units as in bacteria but the corresponding poly-cistronic mRNAs undergo complex processing events, including inter-genic cleavage and 5′ and 3′ end-definition. The current model for processing proposes that the 3′ end of the upstream cistron transcripts and the 5′ end of the downstream cistron transcripts are defined by the same RNA-binding protein and overlap at the level of the protein-binding site.We have investigated the processing mechanisms that operate within the large ATP synthase (atp) operon, in Arabidopsis thaliana chloroplasts. This operon is transcribed by the plastid-encoded RNA polymerase starting from two promoters, which are upstream and within the operon, respectively, and harbors four potential sites for RNA-binding proteins. In order to study the functional significance of the promoters and the protein-binding sites for the maturation processes, we have performed a detailed mapping of the atp transcript ends. Our data indicate that in contrast to maize, atpI and atpH transcripts with overlapping ends are very rare in Arabidopsis. In addition, atpA mRNAs, which overlap with atpF mRNAs, are even truncated at the 3′ end, thus representing degradation products. We observe, instead, that the 5′ ends of nascent poly-cistronic atp transcripts are defined at the first protein-binding site which follows either one of the two transcription initiation sites, while the 3′ ends are defined at the subsequent protein-binding sites or at hairpin structures that are encountered by the progressing RNA polymerase. We conclude that the overlapping mechanisms of mRNA protection have only a limited role in obtaining stable processed atp mRNAs in Arabidopsis. Our findings suggest that during evolution of different plant species as maize and Arabidopsis, chloroplasts have evolved multiple strategies to produce mature transcripts suitable for translation.

Highlights

Chloroplasts are cell organelles in photosynthetic organisms where essential functions as photosynthesis, synthesis of lipids, pigments, vitamins and amino acids occur
In order to characterize primary transcripts, RNA self-ligation was preceded by treatment with Tobacco acid pyro-phosphatase (TAP) (Figure 1C)
Our study demonstrates that the 59 ends of processed atpI and atpH mRNAs and the 39 ends of processed atpH and atpH/F mRNAs map close to the short RNA (sRNA) sites (Figure 3A), which were identified as footprints of RNA-binding proteins [3,4]

Summary

Introduction

Chloroplasts are cell organelles in photosynthetic organisms where essential functions as photosynthesis, synthesis of lipids, pigments, vitamins and amino acids occur. They have evolved from endosymbiosis of photosynthetic cyanobacteria and from those they have inherited many prokaryotic-like elements. During the evolution process from bacteria, chloroplasts have acquired eukaryotic features and plastid-specific characteristics [1]. The gene-expression system represents a good example for the acquirement of complexity in plastids. Genes are still organized into transcriptional units or operons as in bacteria. Differently from the bacterial counterparts, the corresponding poly-cistronic mRNAs undergo complex processing events and only upon intron splicing, editing, inter-cistronic cleavage and termini definition, the mature mRNAs are ready for translation

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Conclusion