Anthony Gobert

An Arabidopsis Dual-Localized Pentatricopeptide Repeat Protein Interacts with Nuclear Proteins Involved in Gene Expression Regulation

This work examines a novel PPR protein that localizes to both mitochondria and nuclei in Arabidopsis. In mitochondria, it associates with polysomes, and in the nucleus, it interacts with a nucleosome assembly protein and a TCP transcription factor. This PPR protein might play a role in gene expression adjustments between mitochondria and the nucleus. Following the endosymbiotic acquisition of mitochondria by eukaryotic cells, most of the genes in this organelle were transferred to the nucleus. To maintain mitochondrial biogenesis and function, nuclear and mitochondrial genomes require regulated and coordinated expression. In plant organelles, nuclear-encoded proteins targeted to the organelles control posttranscriptional and posttranslational mechanisms. Pentatricopeptide repeat (PPR) proteins are good candidates to play such regulatory roles. Here, we identify PNM1 (for PPR protein localized to the nucleus and mitochondria 1), a novel PPR protein that is dual localized to mitochondria and nuclei in Arabidopsis thaliana, as observed by green fluorescent protein fusions and immunodetection on subcellular fractions and on histological sections. Genetic complementation showed that loss of PNM1 function in mitochondria, but not in nuclei, is lethal for the embryo. In mitochondria, it is associated with polysomes and may play a role in translation. A genetic screen in yeast identified protein partners of PNM1. These partners, the nucleosome assembly protein NAP1, and the transcription factor TCP8 interact with PNM1 in the nucleus in planta. Furthermore, TCP8 can bind the promoter of PNM1. This suggests that PNM1 might be involved in the regulation of its own gene expression in the nucleus and could thus play a role in gene expression adjustments between mitochondria and the nucleus.

Structural insights into protein-only RNase P complexed with tRNA

RNase P is the essential activity removing 5′-leader sequences from transfer RNA precursors. RNase P was always associated with ribonucleoprotein complexes before the discovery of protein-only RNase P enzymes called PRORPs (PROteinaceous RNase P) in eukaryotes. Here we provide biophysical and functional data to understand the mode of action of PRORP enzymes. Activity assays and footprinting experiments show that the anticodon domain of transfer RNA is dispensable, whereas individual residues in D and TψC loops are essential for PRORP function. PRORP proteins are characterized in solution and a molecular envelope is derived from small-angle X-ray scattering. Conserved residues are shown to be involved in the binding of one zinc atom to PRORP. These results facilitate the elaboration of a model of the PRORP/transfer RNA interaction. The comparison with the ribonucleoprotein RNase P/transfer RNA complex suggests that transfer RNA recognition by PRORP proteins is similar to that by ribonucleoprotein RNase P.

Helical repeats modular proteins are major players for organelle gene expression.

Mitochondria and chloroplasts are often described as semi-autonomous organelles because they have retained a genome. They thus require fully functional gene expression machineries. Many of the required processes going all the way from transcription to translation have specificities in organelles and arose during eukaryote history. Most factors involved in these RNA maturation steps have remained elusive for a long time. The recent identification of a number of novel protein families including pentatricopeptide repeat proteins, half-a-tetratricopeptide proteins, octotricopeptide repeat proteins and mitochondrial transcription termination factors has helped to settle long-standing questions regarding organelle gene expression. In particular, their functions have been related to replication, transcription, RNA processing, RNA editing, splicing, the control of RNA turnover and translation throughout eukaryotes. These families of proteins, although evolutionary independent, seem to share a common overall architecture. For all of them, proteins contain tandem arrays of repeated motifs. Each module is composed of two to three α-helices and their succession forms a super-helix. Here, we review the features characterising these protein families, in particular, their distribution, the identified functions and mode of action and propose that they might share similar substrate recognition mechanisms.

PlantRNA, a database for tRNAs of photosynthetic eukaryotes

PlantRNA database (http://plantrna.ibmp.cnrs.fr/) compiles transfer RNA (tRNA) gene sequences retrieved from fully annotated plant nuclear, plastidial and mitochondrial genomes. The set of annotated tRNA gene sequences has been manually curated for maximum quality and confidence. The novelty of this database resides in the inclusion of biological information relevant to the function of all the tRNAs entered in the library. This includes 5′- and 3′-flanking sequences, A and B box sequences, region of transcription initiation and poly(T) transcription termination stretches, tRNA intron sequences, aminoacyl-tRNA synthetases and enzymes responsible for tRNA maturation and modification. Finally, data on mitochondrial import of nuclear-encoded tRNAs as well as the bibliome for the respective tRNAs and tRNA-binding proteins are also included. The current annotation concerns complete genomes from 11 organisms: five flowering plants (Arabidopsis thaliana, Oryza sativa, Populus trichocarpa, Medicago truncatula and Brachypodium distachyon), a moss (Physcomitrella patens), two green algae (Chlamydomonas reinhardtii and Ostreococcus tauri), one glaucophyte (Cyanophora paradoxa), one brown alga (Ectocarpus siliculosus) and a pennate diatom (Phaeodactylum tricornutum). The database will be regularly updated and implemented with new plant genome annotations so as to provide extensive information on tRNA biology to the research community.

PPR proteins shed a new light on RNase P biology

A fast growing number of studies identify pentatricopeptide repeat (PPR) proteins as major players in gene expression processes. Among them, a subset of PPR proteins called PRORP possesses RNase P activity in several eukaryotes, both in nuclei and organelles. RNase P is the endonucleolytic activity that removes 5′ leader sequences from tRNA precursors and is thus essential for translation. Before the characterization of PRORP, RNase P enzymes were thought to occur universally as ribonucleoproteins, although some evidence implied that some eukaryotes or cellular compartments did not use RNA for RNase P activity. The characterization of PRORP reveals a two-domain enzyme, with an N-terminal domain containing multiple PPR motifs and assumed to achieve target specificity and a C-terminal domain holding catalytic activity. The nature of PRORP interactions with tRNAs suggests that ribonucleoprotein and protein-only RNase P enzymes share a similar substrate binding process.

Plant mitochondria use two pathways for the biogenesis of tRNAHis

All tRNAHis possess an essential extra G–1 guanosine residue at their 5′ end. In eukaryotes after standard processing by RNase P, G–1 is added by a tRNAHis guanylyl transferase. In prokaryotes, G–1 is genome-encoded and retained during maturation. In plant mitochondria, although trnH genes possess a G–1 we find here that both maturation pathways can be used. Indeed, tRNAHis with or without a G–1 are found in a plant mitochondrial tRNA fraction. Furthermore, a recombinant Arabidopsis mitochondrial RNase P can cleave tRNAHis precursors at both positions G+1 and G–1. The G–1 is essential for recognition by plant mitochondrial histidyl-tRNA synthetase. Whether, as shown in prokaryotes and eukaryotes, the presence of uncharged tRNAHis without G–1 has a function or not in plant mitochondrial gene regulation is an open question. We find that when a mutated version of a plant mitochondrial trnH gene containing no encoded extra G is introduced and expressed into isolated potato mitochondria, mature tRNAHis with a G–1 are recovered. This shows that a previously unreported tRNAHis guanylyltransferase activity is present in plant mitochondria.

A PPR protein involved in regulating nuclear genes encoding mitochondrial proteins

The novel pentatricopeptide repeat protein PNM1 has recently been shown to be dual localized to the nucleus and mitochondria. In the nucleus it binds proteins involved in regulating gene expression, especially a TCP transcription factor. This class of proteins was recently shown to control the expression of nuclear genes encoding mitochondrial proteins that contain cis-acting “site II” regulatory elements in their promoter regions. The analysis of mutant plants showed that some genes with site II elements have increased expression levels when PNM1 is not present in the nucleus. This suggests that PNM1 might act as a negative regulator for the expression of an unknown number of genes with site II elements. Altogether, PNM1 might act as a nuclear regulator and / or could be a retrograde messenger molecule from mitochondria to the nucleus for the fine-tuning of nuclear gene expression required for mitochondrial biogenesis.

RNA degradation by the plant RNA exosome involves both phosphorolytic and hydrolytic activities

The RNA exosome provides eukaryotic cells with an essential 3′–5′ exoribonucleolytic activity, which processes or eliminates many classes of RNAs. Its nine-subunit core (Exo9) is structurally related to prokaryotic phosphorolytic exoribonucleases. Yet, yeast and animal Exo9s have lost the primordial phosphorolytic capacity and rely instead on associated hydrolytic ribonucleases for catalytic activity. Here, we demonstrate that Arabidopsis Exo9 has retained a distributive phosphorolytic activity, which contributes to rRNA maturation processes, the hallmark of exosome function. High-density mapping of 3′ extremities of rRNA maturation intermediates reveals the intricate interplay between three exoribonucleolytic activities coordinated by the plant exosome. Interestingly, the analysis of RRP41 protein diversity across eukaryotes suggests that Exo9’s intrinsic activity operates throughout the green lineage, and possibly in some earlier-branching non-plant eukaryotes. Our results reveal a remarkable evolutionary variation of this essential RNA degradation machine in eukaryotes.The yeast and human RNA exosome is structurally related to prokaryotic phosphorylases but degrades RNA only via associated hydrolytic activities. Here the authors show that the RNA exosome of plants, and likely those of a few basal eukaryotes, combines phosphorolytic and hydrolytic activities to degrade RNA.

Biophysical analysis of Arabidopsis protein-only RNase P alone and in complex with tRNA provides a refined model of tRNA binding

RNase P is a universal enzyme that removes 5′ leader sequences from tRNA precursors. The enzyme is therefore essential for maturation of functional tRNAs and mRNA translation. RNase P represents a unique example of an enzyme that can occur either as ribonucleoprotein or as protein alone. The latter form of the enzyme, called protein-only RNase P (PRORP), is widespread in eukaryotes in which it can provide organellar or nuclear RNase P activities. Here, we have focused on Arabidopsis nuclear PRORP2 and its interaction with tRNA substrates. Affinity measurements helped assess the respective importance of individual pentatricopeptide repeat motifs in PRORP2 for RNA binding. We characterized the PRORP2 structure by X-ray crystallography and by small-angle X-ray scattering in solution as well as that of its complex with a tRNA precursor by small-angle X-ray scattering. Of note, our study reports the first structural data of a PRORP–tRNA complex. Combined with complementary biochemical and biophysical analyses, our structural data suggest that PRORP2 undergoes conformational changes to accommodate its substrate. In particular, the catalytic domain and the RNA-binding domain can move around a central hinge. Altogether, this work provides a refined model of the PRORP–tRNA complex that illustrates how protein-only RNase P enzymes specifically bind tRNA and highlights the contribution of protein dynamics to achieve this specific interaction.