Plant organellar RNA maturation

Abstract

Plant organellar RNA metabolism is run by a multitude of nucleus-encoded RNA-binding proteins (RBPs) that control RNA stability, processing, and degradation. In chloroplasts and mitochondria, these post-transcriptional processes are vital for the production of a small number of essential components of the photosynthetic and respiratory machinery-and consequently for organellar biogenesis and plant survival. Many organellar RBPs have been functionally assigned to individual steps in RNA maturation, often specific to selected transcripts. While the catalog of factors identified is ever-growing, our knowledge of how they achieve their functions mechanistically is far from complete. This review summarizes the current knowledge of plant organellar RNA metabolism taking an RBP-centric approach and focusing on mechanistic aspects of RBP functions and the kinetics of the processes they are involved in.

Figure 1.

Abundance of RBPs in different organisms. RBP numbers are set in relation to gene numbers for humans and several model species as well as chloroplasts of Arabidopsis as a representative of embryophytes. For gene and RBP numbers as well as references, see Supplemental Table S1.

Figure 2.

Helical repeat proteins extend half-lives of organellar mRNAs. 1, Organellar RNAs are under attack by a variety of RNases (e.g. RNAse J, Rnase E, RNase II, and PNPase) during and after transcription. 2, If not protected, RNAs decay rapidly. 3, Helical repeat proteins protect RNAs against exo- and endonucleases. 4a, In mitochondria, PPR proteins bind to 5′ UTR regions and guide endonucleolytic cleavage, for instance via RNase P. 4b, Binding of helical repeat proteins to RNAs can also impact the local RNA structure since they keep their binding site in a single-stranded conformation. This can, for example, aid in intron folding and thus splicing. 5, The protective action of helical repeat proteins increases target RNA half-lives and thus opens an extended window of opportunity for translation and other RNA processing events, for example, splicing. 5a, Stochastic endonucleolytic cuts initiate exonucleolytic degradation up to helical repeat proteins, which leads to the complex transcript patterns known from plant organelles. Adjacent RNAs overlap at helical repeat protein BS. 5b and 6, The stabilization of RNAs allows slow processes like group II intron folding to happen. Splicing factors like CRM or MatK have time to aid in folding as RNA chaperones to eventually complete the catalytically active intron structure. 7, Helical repeat proteins remodel RNA structures in 5′-UTRs to allow ribosome entry at start codons. 8, flowchart of RNA editing and RNA splicing in plant organelles. Four RNA species can be distinguished for any RNA that is both spliced and edited. The gray arrows indicate that all RNA isoforms are subject to RNA degradation. This flowchart formed the basis for a mathematical model (using Catalyst.jl, Loman et al. 2022) to explore the effects of different RNA turnover rates on the splicing and editing status. 9 and 10, Ratios of spliced to unspliced and edited to unedited RNAs are shown at different degradation rates as electronic RNA gel blot results and modeled Sanger sequencing results. Each “+” indicates an order of magnitude increase in RNA degradation rate.

Figure 3.

Structure of a DYW–PPR protein. PPR proteins are generally characterized by their extended tract of helical elements that is flanked at its N-terminus by an unstructured targeting peptide. While the PPR tract is responsible for base-specific RNA recognition of all PPR proteins, DYW–PPR proteins have an additional, C-terminal DYW domain with base deamination activity. The structure shown is of MEF1 (Zehrmann et al. 2009) as predicted by AlphaFold (Jumper et al. 2021).

Figure 4.

Examples for the evolution of RNA:protein interactions according to the CNE theory. A) Suppression of a loss of an important RNA secondary structure by a helical repeat protein. The 5′-terminus of an mRNA is protected against exonucleolytic degradation by RNase J via a stem-loop. 1, A helical repeat protein that can bind in the vicinity of the stem-loop (binding site marked in orange) emerges in evolution. The helical repeat protein can serve as an alternative to the stem-loop to protect the mRNA against 5′-to-3′ degradation. 2, A mutation destroys the stem-loop (marked in red). 3, This mutation is, however, neutral in the background of the helical repeat protein stabilizing the RNA independently. B) Suppression of a T-to-C mutation by RNA editing. A coding sequence requires a uridine (large U) at a particular site to encode the correct amino acid and thus keep the corresponding protein functional. 1, A DYW–PPR protein (hexagon = DWY-domain) emerges in evolution that can bind upstream of the critical U (binding site in orange). 2, Whether or not the DYW–PPR protein binds to the target sequence is at first irrelevant—all messages encode the correct protein. A T-to-C mutation at the DNA-level leads to a codon change that is detrimental to the corresponding protein. 3, The mutation is however neutral in the background of the DYW–PPR protein that performs C-to-U base deamination at the RNA level and thus restores the correct codon. C) Suppression of a mutation negatively affecting the structure of a group II intron. A group II intron requires folding into the correct structure (a simplified 2D-representation is shown) to allow forming of the ribozyme catalytic center and thus intron removal. 1, A helical repeat protein emerges in evolution that can bind to a single-stranded region of the intron (binding site shown in orange). 2, Whether interaction with this helical repeat protein occurs or not is in the beginning irrelevant for splicing. 3, A mutation leads to a sequence element that is prone to base-pair with the sequence marked in orange. 4, Base-pairing between the mutated (red) sequence and the orange sequence leads to an unproductive alternative intron structure that blocks splicing. However, binding of the helical repeat protein forces the orange sequence element into single-strandedness, preventing detrimental misfolding, and fostering continuous splicing.

Figure 5.

Helical repeat proteins for an optimized, synchronized, and regulated expression of transgenes in organelles. The expression of native or designer helical repeat proteins encoded in the nucleus under control of the c6 promoter can be induced specifically by copper depletion. After its translation in the cytosol, the protein is imported into the organelle, where it interacts with BS to regulate the expression of a synthetic operon encoding the proteins X, Y, and Z.