Stable native RIP9 complexes associate with C-to-U RNA editing activity, PPRs, RIPs, OZ1, ORRM1 and ISE2

Rafael Sandoval, Robert D. Boyd, Alena N. Kiszter, Yeva Mirzakhanyan, Paola Santibańez, Paul D. Gershon, Michael L. Hayes

Research output: Contribution to journalArticleResearchpeer-review

Abstract

The mitochondrial and chloroplast mRNAs 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 OZ1, RIP2, RIP9 and ORRM1 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 RNase A abolished the relationship of editing activity with high-MW fractions, suggesting a structural RNA component in native complexes. By immunoblotting, RIP9, OTP86, OZ1 and ORRM1 were shown to be present in active gel filtration fractions, though OZ1 and ORRM1 were mainly found in low-MW inactive fractions. Active editing factor complexes were affinity-purified using anti-RIP9 antibodies, and orthologs to putative Arabidopsis thaliana RNA editing factor PPR proteins, RIP2, RIP9, RIP1, OZ1, ORRM1 and ISE2 were identified via mass spectrometry. Western blots from co-IP studies revealed the mutual association of OTP86 and OZ1 with native RIP9 complexes. Thus, RIP9 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.

Original languageEnglish
Pages (from-to)1116-1126
Number of pages11
JournalPlant Journal
Volume99
Issue number6
DOIs
Publication statusPublished - 1 Sep 2019

Fingerprint

RNA Editing
RNA editing
cytidine
Cytidine
uridine
Uridine
Chloroplasts
chloroplasts
Immunoprecipitation
Gel Chromatography
RNA
Embryophyta
Reverse Genetics
Pancreatic Ribonuclease
embryophytes
immunoblotting
Immunoblotting
Arabidopsis
vascular plants
Blood Vessels

Keywords

  • chloroplasts
  • complexes
  • gel filtration
  • immunoprecipitation
  • in vitro assay
  • maize
  • pentatricopeptide repeat proteins
  • RIP9
  • RNA editing
  • tandem MS

ASJC Scopus subject areas

  • Genetics
  • Plant Science
  • Cell Biology

Cite this

Sandoval, R., Boyd, R. D., Kiszter, A. N., Mirzakhanyan, Y., Santibańez, P., Gershon, P. D., & Hayes, M. L. (2019). Stable native RIP9 complexes associate with C-to-U RNA editing activity, PPRs, RIPs, OZ1, ORRM1 and ISE2. Plant Journal, 99(6), 1116-1126. https://doi.org/10.1111/tpj.14384

Stable native RIP9 complexes associate with C-to-U RNA editing activity, PPRs, RIPs, OZ1, ORRM1 and ISE2. / Sandoval, Rafael; Boyd, Robert D.; Kiszter, Alena N.; Mirzakhanyan, Yeva; Santibańez, Paola; Gershon, Paul D.; Hayes, Michael L.

In: Plant Journal, Vol. 99, No. 6, 01.09.2019, p. 1116-1126.

Research output: Contribution to journalArticleResearchpeer-review

Sandoval, R, Boyd, RD, Kiszter, AN, Mirzakhanyan, Y, Santibańez, P, Gershon, PD & Hayes, ML 2019, 'Stable native RIP9 complexes associate with C-to-U RNA editing activity, PPRs, RIPs, OZ1, ORRM1 and ISE2' Plant Journal, vol. 99, no. 6, pp. 1116-1126. https://doi.org/10.1111/tpj.14384
Sandoval R, Boyd RD, Kiszter AN, Mirzakhanyan Y, Santibańez P, Gershon PD et al. Stable native RIP9 complexes associate with C-to-U RNA editing activity, PPRs, RIPs, OZ1, ORRM1 and ISE2. Plant Journal. 2019 Sep 1;99(6):1116-1126. https://doi.org/10.1111/tpj.14384
Sandoval, Rafael ; Boyd, Robert D. ; Kiszter, Alena N. ; Mirzakhanyan, Yeva ; Santibańez, Paola ; Gershon, Paul D. ; Hayes, Michael L. / Stable native RIP9 complexes associate with C-to-U RNA editing activity, PPRs, RIPs, OZ1, ORRM1 and ISE2. In: Plant Journal. 2019 ; Vol. 99, No. 6. pp. 1116-1126.
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