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

   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. ( June 2019 , The Plant Journal)

   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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2. The dicot homolog of maize PPR103 carries a C-terminal DYW domain and may have a role in C-to-U editing of some chloroplast RNA transcripts

   https://doi.org/10.1007/s11103-024-01424-1  

   McCray, Tyra N. ; Azim, Mohammad F. ; Burch-Smith, Tessa M. ( March 2024 , Plant Molecular Biology)

   In plants, cytidine-to-uridine (C-to-U) editing is a crucial step in processing mitochondria- and chloroplast-encoded transcripts. This editing requires nuclear-encoded proteins including members of the pentatricopeptide (PPR) family, especially PLS-type proteins carrying the DYW domain.IPI1/emb175/PPR103is a nuclear gene encoding a PLS-type PPR protein essential for survival inArabidopsis thalianaand maize. Arabidopsis IPI1 was identified as likely interacting with ISE2, a chloroplast-localized RNA helicase associated with C-to-U RNA editing in Arabidopsis and maize. Notably, while the Arabidopsis andNicotianaIPI1 orthologs possess complete DYW motifs at their C-termini, the maize homolog, ZmPPR103, lacks this triplet of residues which are essential for editing. In this study we examined the function of IPI1 in chloroplast RNA processing inN. benthamianato gain insight into the importance of the DYW domain to the function of the EMB175/PPR103/ IPI1 proteins. Structural predictions suggest that evolutionary loss of residues identified as critical for catalyzing C-to-U editing in other members of this class of proteins, were likely to lead to reduced or absent editing activity in theNicotianaand Arabidopsis IPI1 orthologs. Virus-induced gene silencing ofNbIPI1led to defects in chloroplast ribosomal RNA processing and changes to stability ofrpl16transcripts, revealing conserved function with its maize ortholog.NbIPI1-silenced plants also had defective C-to-U RNA editing in several chloroplast transcripts, a contrast from the finding that maize PPR103 had no role in editing. The results indicate that in addition to its role in transcript stability, NbIPI1 may contribute to C-to-U editing inN. benthamianachloroplasts.

    

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3. An Agrobacterium ‐mediated stable transformation technique for the hornwort model Anthoceros agrestis

   https://doi.org/10.1111/nph.17524  

   Frangedakis, Eftychios ; Waller, Manuel ; Nishiyama, Tomoaki ; Tsukaya, Hirokazu ; Xu, Xia ; Yue, Yuling ; Tjahjadi, Michelle ; Gunadi, Andika ; Van Eck, Joyce ; Li, Fay‐Wei ; et al ( July 2021 , New Phytologist)

   Despite their key phylogenetic position and their unique biology, hornworts have been widely overlooked. Until recently there was no hornwort model species amenable to systematic experimental investigation.Anthoceros agrestishas been proposed as the model species to study hornwort biology.

   We have developed anAgrobacterium‐mediated method for the stable transformation ofA. agrestis, a hornwort model species for which a genetic manipulation technique was not yet available.

   High transformation efficiency was achieved by using thallus tissue grown under low light conditions. We generated a total of 274 transgenicA. agrestislines expressing the β‐glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under control of the CaMV 35S promoter and several endogenous promoters. Nuclear and plasma membrane localization with multiple color fluorescent proteins was also confirmed.

   The transformation technique described here should pave the way for detailed molecular and genetic studies of hornwort biology, providing much needed insight into the molecular mechanisms underlying symbiosis, carbon‐concentrating mechanism, RNA editing and land plant evolution in general.

    

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4. Two organelle RNA recognition motif proteins affect distinct sets of RNA editing sites in the Arabidopsis thaliana plastid

   https://doi.org/10.1002/pld3.213  

   Searing, Audrey M. ; Satyanarayan, Manasa B. ; O′Donnell, James P. ; Lu, Yan ( April 2020 , Plant Direct)

   Plastid and mitochondrial RNAs in vascular plants are subjected to cytidine‐to‐uridine editing. The model plant speciesArabidopsis thaliana(Arabidopsis) has two nuclear‐encoded plastid‐targeted organelle RNA recognition motif (ORRM) proteins: ORRM1 and ORRM6. In theorrm1mutant, 21 plastid RNA editing sites were affected but none are essential to photosynthesis. In theorrm6mutants, two plastid RNA editing sites were affected:psbF‐C77 andaccD‐C794. BecausepsbFencodes the β subunit of cytochromeb₅₅₉in photosystem II, which is essential to photosynthesis, theorrm6mutants were much smaller than the wild type. In addition, theorrm6mutants had pale green leaves and reduced photosynthetic efficiency. To investigate the functional relationship between ORRM1 and ORRM6, we generatedorrm1 orrm6double homozygous mutants. Morphological and physiological analyses showed that theorrm1 orrm6double mutants had a smaller plant size, reduced chlorophyll contents, and decreased photosynthetic efficiency, similar to theorrm6single mutants. Although theorrm1 orrm6double mutants adopted the phenotype of theorrm6single mutants, the total number of plastid RNA editing sites affected in theorrm1 orrm6double mutants was the sum of the sites affected in theorrm1andorrm6single mutants. These data suggest that ORRM1 and ORRM6 are in charge of distinct sets of plastid RNA editing sites and that simultaneous mutations inORRM1andORRM6genes do not cause additional reduction in editing extent at other plastid RNA editing sites.

    

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5. FLARE: a fast and flexible workflow for identifying RNA editing foci

   https://doi.org/10.1186/s12859-023-05452-4  

   Kofman, Eric ; Yee, Brian ; Medina-Munoz, Hugo C. ; Yeo, Gene W. ( October 2023 , BMC Bioinformatics)

   Fusion of RNA-binding proteins (RBPs) to RNA base-editing enzymes (such as APOBEC1 or ADAR) has emerged as a powerful tool for the discovery of RBP binding sites. However, current methods that analyze sequencing data from RNA-base editing experiments are vulnerable to false positives due to off-target editing, genetic variation and sequencing errors.

   We present FLagging Areas of RNA-editing Enrichment (FLARE), a Snakemake-based pipeline that builds on the outputs of the SAILOR edit site discovery tool to identify regions statistically enriched for RNA editing. FLARE can be configured to analyze any type of RNA editing, including C to U and A to I. We applied FLARE to C-to-U editing data from a RBFOX2-APOBEC1 STAMP experiment, to show that our approach attains high specificity for detecting RBFOX2 binding sites. We also applied FLARE to detect regions of exogenously introduced as well as endogenous A-to-I editing.

   FLARE is a fast and flexible workflow that identifies significantly edited regions from RNA-seq data. The FLARE codebase is available athttps://github.com/YeoLab/FLARE.

    

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