RNA processing, including splicing, RNA editing, and RNA stability, can control gene expression in chloroplasts and mitochondria. Not all the nuclear-encoded protein factors that mediate organelle RNA processing are known. We have discovered a small family of Arabidopsis proteins that includes ones that are essential for RNA processing in chloroplasts and mitochondria. This family is named the organelle zinc-finger (OZ) family, as the four members contain multiple RanBP2-type zinc finger domains. Using protein interaction assays, RNA analysis, and mutational studies, we have characterized OZ1, a chloroplast RNA editing factor essential for editing of nearly one-third of the C targets of chloroplast editing. We have also identified OZ2 as a mitochondrial splicing factor required for processing transcripts encoding rps3 and subunits of Complex I. Homozygous oz1 mutants exhibit delayed greening and poor growth, while oz2 mutants are embryo lethal. oz2 plants rescued by the embryonic expression of OZ2 under control of the ABI3 promoter exhibit slow growth and curly, dark leaves once OZ2 expression ceases. Complex I is impaired in the oz2 mutants. Mass spectrometry demonstrated that OZ2 binds two zinc ions, consistent with the presence of two RanBP2 zinc fingers. Using yeast two-hybrid and Bimolecular Fluorescence Complementation assays, the unique C-terminal region of OZ1 was identified as a domain that interacts with other chloroplast editing factors, while OZ2 interacts with known mitochondrial splicing factors. Point mutation of the zinc finger domains of OZ1 showed that they are essential for its function as an editing factor. Complementation of knockout mutants with the OZ1 zinc finger domains alone demonstrated that they are sufficient to restore editing. These discoveries represent a new function for the RanBP2 zinc finger domain, which is widespread in both the plant and animal kingdoms.
Coauthors: Stephane Bentolila – Cornell University;Michael Hayes – California State University, Los Angeles;Maureen Hanson – Cornell University