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Stable native RIP 9 complexes associate with C‐to‐U RNA editing activity, PPR s, RIP s, OZ 1, ORRM 1 and ISE 2
Author(s) -
Sandoval Rafael,
Boyd Robert D.,
Kiszter Ale.,
Mirzakhanyan Yeva,
Santibańez Paola,
Gershon Paul D.,
Hayes Michael L.
Publication year - 2019
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.14384
Subject(s) - rna editing , rna , pentatricopeptide repeat , chemistry , immunoprecipitation , chloroplast , ribonuclease , biochemistry , microbiology and biotechnology , biology , gene
Summary The mitochondrial and chloroplast mRNA s of the majority of land plants are modified through cytidine to uridine (C‐to‐U) RNA editing. Previously, forward and reverse genetic screens demonstrated a requirement for pentatricopeptide repeat ( PPR ) proteins for RNA editing. Moreover, chloroplast editing factors OZ 1, RIP 2, RIP 9 and ORRM 1 were identified in co‐immunoprecipitation (co‐IP) experiments, albeit the minimal complex sufficient for editing activity was never deduced. The current study focuses on isolated, intact complexes that are capable of editing distinct sites. Peak editing activity for four sites was discovered in size‐exclusion chromatography (SEC) fractions ≥ 670 kDa, while fractions estimated to be approximately 413 kDa exhibited the greatest ability to convert a substrate containing the editing site rps14 C80. RNA content peaked in the ≥ 670 kDa fraction. Treatment of active chloroplast extracts with RN ase A abolished the relationship of editing activity with high‐ MW fractions, suggesting a structural RNA component in native complexes. By immunoblotting, RIP 9, OTP 86, OZ 1 and ORRM 1 were shown to be present in active gel filtration fractions, though OZ 1 and ORRM 1 were mainly found in low‐ MW inactive fractions. Active editing factor complexes were affinity‐purified using anti‐ RIP 9 antibodies, and orthologs to putative Arabidopsis thaliana RNA editing factor PPR proteins, RIP 2, RIP 9, RIP 1, OZ 1, ORRM 1 and ISE 2 were identified via mass spectrometry. Western blots from co‐IP studies revealed the mutual association of OTP 86 and OZ 1 with native RIP 9 complexes. Thus, RIP 9 complexes were discovered to be highly associated with C‐to‐U RNA editing activity and other editing factors indicative of their critical role in vascular plant editosomes.