Biology subjects

Colas des Francs-Small, C.

Publications and source records attributed to Colas des Francs-Small, C..

4 recordsLinked to original sources

bioRxiv2025

GRASP: a modular toolkit for synthetic pentatricopeptide repeat RNA-binding proteins

Pentatricopeptide repeat (PPR) proteins are eukaryotic RNA binding proteins with multiple roles in mitochondrial and chloroplast transcript processing. PPR proteins are naturally modular and hold great potential for development into tools for RNA processing or controlling RNA folding or expression. However, construction of synthetic PPR proteins is challenging due to their highly repetitive sequences. Here, we present the GRASP kit for assembly of synthetic PPR proteins. Utilising the S-variant of PPR motifs, we designed a library of 42 plasmids which can be combined to assemble synthetic PPR proteins with 9, 14 or 19 motifs to target any RNA sequence of the same length. The GRASP kit enables rapid design and construction of PPR proteins of any desired specificity and is compatible with the MoClo assembly standard. To demonstrate the capabilities of GRASP, we assembled a synthetic PPR RNA editing protein and variants with altered sequence specificity. We tested the functionality of 31 synthetic PPR protein variants against a set of 46 RNA targets and used RNA sequencing to determine levels of RNA editing. The variations in editing provide a wealth of insights into PPR-RNA interactions. The GRASP kit provides a foundation for further development of synthetic PPR protein technologies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/661641v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@d8c1d2org.highwire.dtl.DTLVardef@9ccba8org.highwire.dtl.DTLVardef@1feadfeorg.highwire.dtl.DTLVardef@150ffb7_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

A unique C-terminal domain contributes to the molecular function of restorer-of-fertility proteins in plant mitochondria

Restorer-of-fertility (Rf) genes have practical applications in hybrid seed production as a means to control self-pollination. They encode pentatricopeptide repeat (PPR) proteins that are targeted to mitochondria where they specifically bind to transcripts that induce cytoplasmic male sterility and repress their expression. In searching for a molecular signature unique to this class of proteins, we found that a majority of known Rf proteins have a unique domain, which we called RfCTD (Restorer-of-fertility C-terminal domain), and its presence correlates with the ability to induce cleavage of the mitochondrial RNA target. We constructed a sequence profile that can quickly and accurately identify RfCTD sequences in plant genomes or transcriptomes. We screened 219 angiosperm genomes from 123 genera and found that each diploid genome encodes, on average, 25 Rf-like (RFL) proteins, of which approximately 55% contain the C-terminal signature domain. This screen also revealed considerable variation in RFL gene numbers across flowering plants. We observed that plant genera with bisexual flowers have significantly higher numbers of RFL genes compared to those with unisexual flowers, consistent with a role of these proteins in restoration of male fertility. Finally, we show that removing the RfCTD from the RFL protein RNA PROCESSING FACTOR 2-nad6 prevented cleavage of its RNA target, the nad6 transcript, in Arabidopsis thaliana mitochondria. This research provides a simple way of identifying putative Rf candidates in genome sequences, new insights into the molecular mode of action of Rf proteins in plant mitochondria and expands our understanding of the evolution of fertility restoration in flowering plants.

plant biology↗

bioRxiv2022

MSP1 encodes an essential RNA-binding PPR factor required for nad1 maturation and complex I biogenesis in Arabidopsis mitochondria

SummaryMitochondria are semi-autonomous organelles that serve as hubs for aerobic energy metabolism. The biogenesis of the respiratory (OXPHOS) system relies on nuclear-encoded factors, which regulate the transcription, processing and translation of mitochondrial (mt)RNAs. These include proteins of primordial origin, as well as eukaryotic-type RNA-binding families recruited from the host genomes to function in mitogenome expression. Pentatricopeptide repeat (PPR) proteins constitute a major gene-family in angiosperms that is pivotal in many aspects of mtRNA metabolism, such as editing, splicing or stability. Here, we report the analysis of MITOCHONDRIA STABILITY/PROCESSING PPR FACTOR1 (MSP1, At4g20090), a canonical mitochondria-localized PPR protein that is necessary for mitochondrial biogenesis and embryo-development. Functional complementation confirmed that the phenotypes result from a disruption of the MSP1 gene. As a loss-of-function allele of Arabidopsis MSP1 leads to seed abortion, we employed an embryo-rescue method for the molecular characterization of msp1 mutants. Our data show that msp1 embryo-development fails to proceed beyond the heart-torpedo transition stage as a consequence of a severe nad1 pre-RNA processing-defect, resulting in the loss of respiratory complex I (CI) activity. The maturation of nad1 involves the processing of three RNA-fragments, nad1.1, nad1.2 and nad1.3. Based on biochemical analyses and the mtRNA profiles in wild-type and msp1 plants, we concluded that through its association with a specific site in nad1.1, MSP1 facilitates the generation of its 3-terminus and stabilizes it -a prerequisite for nad1 exons a-b splicing. Our data substantiate the importance of mtRNA metabolism for the biogenesis of the respiratory machinery during early-plant development.

plant biology↗

bioRxiv2022

Knockdown of mitochondrial atp1 mRNA by a custom-designed pentatricopeptide repeat protein alters F1Fo ATP synthase

We show that a custom-designed RNA-binding protein binds and specifically induces cleavage of atp1 RNA in mitochondria, significantly decreasing the abundance of the Atp1 protein and the assembled F1Fo ATP synthase in Arabidopsis thaliana. The transformed plants are characterized by delayed vegetative growth and reduced fertility. Five-fold depletion of Atp1 level was accompanied by a decrease in abundance of other ATP synthase subunits, lowered ATP synthesis rate of isolated mitochondria, but no change to mitochondrial electron transport chain complexes, adenylates or energy charge in planta. Transcripts for amino acid transport and a variety of stress response processes were differentially expressed in lines containing the PPR protein, indicating changes to achieve cellular homeostasis when ATP synthase was highly depleted. Leaves of ATP-synthase-depleted lines showed higher respiratory rates and elevated levels of most amino acids at night, most notably serine family amino acids. The results show the value of using custom-designed PPR proteins to influence expression of specific mitochondrial transcripts to carry out reverse genetics studies on mitochondrial gene functions and the consequences of ATP synthase depletion on cellular functions in Arabidopsis. One sentence SummaryKnockdown of mitochondrial atp1 mRNA by a custom-designed pentatricopeptide repeat protein alters F1Fo ATP synthase, plant growth and amino acid metabolism and ATP synthesis in Arabidopsis thaliana

plant biology↗