Mechanism Of Chloroplast RRNA Processing Mediated By Endoribonuclease YbeY In Arbidopsis Thaliana

Plastids (chloroplasts) of higher plants, evolved from cyanobacterial ancestors via endosymbiotic events, have their own genome and components of gene-expression system including transcription and translation machineries. Chloroplast ribosomes (chlororibosomes) are the translational machine, consisting of two subunits, and they are designated30S and50S subunit. Although structure and organization of chlororibosomes has been solved, much less is known about the ribosome biogenesis including ribosome maturation and assembly. Here we report functional characterization of a chloroplast-localized endoribonuclease YbeY in Arabidopsis, the homologue of highly conserved bacterial UPF0054family protein. The maining results are listed as following:(1) Bacterial YbeY homologues exists in plant. The Arabidopsis YbeY encoded by a single-copy nuclear gene contains two domains of UPF0054and HAD hydrolase-like, suggesting evolutional development of YbeY gene in higher plant.(2) AtYebY is localized in chloroplasts. AtYebY was regulated by light on its transcript levels in etiolated seedling from dark to light, but AtYbeY transcripts were not response to heat shock.(3) AtYbeY knockdown plants lead to the pale-green leaves and growth-slow phenotypes, while knockout mutants show seedling lethal, indicating the essentiality of chloroplast AtYbeY for viability and photoautotrophic growth. Physiological data reveal the significant decreases of chlorophyll contents and the impaired photosynthetic capacity in AtYbeY-deficient plants. Microscopy shows defective chloroplasts of atybeY-1mutant by AtYbeY deficiency.(4) Furthermore, we have found that photosynthetic complexes and their chloroplast-encoded protein components are impaired in the atybeY-1mutant and relevant transcripts were not affected significantly, suggesting chloroplast translation defect by AtYbeY deficiency. Analysis of RNA gel blot and circle RT-PCR clearly indicate the involvement of AtYbeY in processing of chloroplast16S,23S,4.5S, and5S rRNA maturation.(5) Besides, AtYbeY deficiency affects chloroplast RNA metabolism, for instance, the overaccumulation of mRNA (petB and clpPl) and tRNA (trnD, trnE and trnQ) transcripts and the maturation of trnl, trnA and trnR in rRNA operon.(6) AtYbeY deficiency affects rRNA metabolism, ribosome biogenesis, and consequent translation in chloroplasts.(7) Biochemical data confirm that AtYbeY in vitro cleaves rRNA, mRNA and tRNA, showing a chloroplast RNase.Finally, our findings reveal the plastid ribosome deficiency in knockdown mutants of AtYbeY, providing the molecular explanation for the observed ybe Y mutant phenotypes.