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Title: Stable native RIP 9 complexes associate with C‐to‐U RNA editing activity, PPR s, RIP s, OZ 1, ORRM 1 and ISE 2

The mitochondrial and chloroplastmRNAs of the majority of land plants are modified through cytidine to uridine (C‐to‐U)RNAediting. Previously, forward and reverse genetic screens demonstrated a requirement for pentatricopeptide repeat (PPR) proteins forRNAediting. Moreover, chloroplast editing factorsOZ1,RIP2,RIP9 andORRM1 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 siterps14C80.RNAcontent peaked in the ≥ 670 kDa fraction. Treatment of active chloroplast extracts withRNase A abolished the relationship of editing activity with high‐MWfractions, suggesting a structuralRNAcomponent in native complexes. By immunoblotting,RIP9,OTP86,OZ1 andORRM1 were shown to be present in active gel filtration fractions, thoughOZ1 andORRM1 were mainly found in low‐MWinactive fractions. Active editing factor complexes were affinity‐purified using anti‐RIP9 antibodies, and orthologs to putativeArabidopsis thalianaRNAediting factorPPRproteins,RIP2,RIP9,RIP1,OZ1,ORRM1 andISE2 were identified via mass spectrometry. Western blots from co‐IP studies revealed the mutual association ofOTP86 andOZ1 with nativeRIP9 complexes. Thus,RIP9 complexes were discovered to be highly associated with C‐to‐URNAediting activity and other editing factors indicative of their critical role in vascular plant editosomes.

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Author(s) / Creator(s):
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Publisher / Repository:
Date Published:
Journal Name:
The Plant Journal
Page Range / eLocation ID:
p. 1116-1126
Medium: X
Sponsoring Org:
National Science Foundation
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