Mitochondrial Heat Shock Protein 60s Interact with What's This Factor 9 to Regulate RNA Splicing of ccmFC and rpl2.

Mitochondrial intron splicing is a plant-specific feature that was acquired during the co-evolution of eukaryotic host cells and a-proteobacteria. The elimination of these introns is facilitated by mitochondrial-targeted proteins encoded by its host, nucleus. What's this factor 9 (WTF9), a nuclear-encoded plant organelle RNA recognition (PORR) protein, is involved in the splicing of the mitochondrial group II introns rpl2 and ccmFC. Disruption of WTF9 causes developmental defects associated with the loss of Cyt c and Cyt c1 in Arabidopsis. In the present study, using a co-immunoprecipitation assay, we found that HSP60s interacted with WTF9, which was further confirmed by a pull-down assay. HSP60s are molecular chaperones that assist with protein folding in both eukaryotic and prokaryotic cells. However, accumulating evidence suggests that HSP60s also participate in other biological functions such as RNA metabolism and RNA protection. In this study, we found that consistently with their interaction with WTF9, HSP60s interacted with 48 nucleotides of the ccmFC intron. In mutant studies, the double mutant hsp60-3a1hsp60-3b1 exhibited a small stature phenotype and reduced splicing efficiency for rpl2 and ccmFC. These observations were similar to those in wtf9 mutants and suggest that HSP60s are involved in the RNA splicing of rpl2 and ccmFC introns in mitochondria. Our findings suggest that HSP60s participate in mitochondrial RNA splicing through their RNA-binding ability.

Arabidopsis CHROMOSOME TRANSMISSION FIDELITY 7 (AtCTF7/ECO1) is required for DNA repair, mitosis and meiosis.

The proper transmission of DNA in dividing cells is crucial for the survival of eukaryotic organisms. During cell division, faithful segregation of replicated chromosomes requires their tight attachment, known as sister chromatid cohesion, until anaphase. Sister chromatid cohesion is established during S-phase in a process requiring an acetyltransferase that in yeast is known as Establishment of cohesion 1 (Eco1). Inactivation of Eco1 typically disrupts chromosome segregation and homologous recombination-dependent DNA repair in dividing cells, ultimately resulting in lethality. We report here the isolation and detailed characterization of two homozygous T-DNA insertion mutants for the Arabidopsis thaliana Eco1 homolog, CHROMOSOME TRANSMISSION FIDELITY 7/ESTABLISHMENT OF COHESION 1 (CTF7/ECO1), called ctf7-1 and ctf7-2. Mutants exhibited dwarfism, poor anther development and sterility. Analysis of somatic tissues by flow cytometry, scanning electron microscopy and quantitative real-time PCR identified defects in DNA repair and cell division, including an increase in the area of leaf epidermal cells, an increase in DNA content and the upregulation of genes involved in DNA repair including BRCA1 and PARP2. No significant change was observed in the expression of genes that influence entry into the endocycle. Analysis of meiocytes identified changes in chromosome morphology and defective segregation; the abundance of chromosomal-bound cohesion subunits was also reduced. Transcript levels for several meiotic genes, including the recombinase genes DMC1 and RAD51C and the S-phase licensing factor CDC45 were elevated in mutant anthers. Taken together our results demonstrate that Arabidopsis CTF7/ECO1 plays important roles in the preservation of genome integrity and meiosis.