Bentolila, S. | Search bioRxiv

bioRxiv2024

Accessory proteins increase the efficiency of RNA editing by Arabidopsis chloroplast editosomes

RNA editing modifies cytidines to uridines in plant organelle transcripts so that their sequences differ from the ones predicted from the genomic DNA. This process, conserved across most land plants, involves a family of RNA-binding proteins that has significantly expanded, the pentatricopeptide repeat (PPR)-containing proteins. In angiosperms, PPR proteins are found in editosomes associated with accessory proteins. The exact function of these accessory proteins has been unclear. Bacterial co-expression of an angiosperm synthetic factor and different accessory proteins, RIP2, RIP9, ORRM1 demonstrates their essential role in editing of an RNA target. The presence of ORRM1 and RIP2 or ORRM1 and RIP9 in bacteria with the PPR factor results in a target editing extent of 80%, which is similar to what is observed in planta. Accessory proteins increase the affinity of the PPR factor for the target RNA, likely the explanation of their role in improving editing efficiency. RNA-seq analysis of bacterial transcriptome in samples expressing various combinations of accessory proteins with the synthetic factor identified a total of 34 off-target editing events. Investigation of their upstream sequences that are recognized and bound by the synthetic factor allows the optimization of future designs to improve the specificity of this programmable RNA-editing factor.

molecular biology↗

bioRxiv2023

Detection and editing of the updated plastid- and mitochondrial-encoded proteomes for Arabidopsis with PeptideAtlas

Arabidopsis thaliana Col-0 has plastid and mitochondrial genomes encoding for over one hundred proteins and several ORFs. Public databases (e.g. Araport11) have redundancy and discrepancies in gene identifiers for these organelle-encoded proteins. RNA editing results in changes to specific amino acid residues or creation of start and stop codons for many of these proteins, but the impact of such RNA editing at the protein level is largely unexplored due to the complexities of detection. This study first assembled the non-redundant set of identifiers, their correct protein sequences, and 452 predicted non-synonymous editing sites of which 56 are edited at lower frequency. Accumulation of edited and/or unedited proteoforms was then determined by searching [~]259 million raw MSMS spectra from ProteomeXchange as part of Arabidopsis PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/). All mitochondrial proteins and all except three plastid-encoded proteins (NDHG/NDH6, PSBM, RPS16), but none of the ORFs, were identified; we suggest that all ORFs and RPS16 are pseudogenes. Detection frequencies for each edit site and type of edit (e.g. S to L/F) were determined at the protein level, cross-referenced against the metadata (e.g. tissue), and evaluated for technical challenges of detection.167 predicted edit sites were detected at the proteome level. Minor frequency sites were indeed also edited at low frequency at the protein level. However, except for sites RPL5-22 and CCB382-124, proteins only accumulate in edited form (>98 -100% edited) even if RNA editing levels are well below 100%. This study establishes that RNA editing for major editing sites is required for stable protein accumulation.

plant biology↗