Hanson Lab
 
RNA Editing in Plant Organelles
 

Particular cytidines in transcripts in chloroplasts and mitochondria of plant cells undergo modification to uridine by RNA editing (1).  These C-to-U changes can create start and stop codons or change the encoded amino acid from the one predicted from the genomic sequence. RNA editing is known to occur in a variety of organisms, including mammals, insects, plants and microorganisms. In humans and other animals, RNA editing creates diversity in certain classes of proteins encoded by the nuclear genome. Editing in plants appears to correct defective RNA post-transcriptionally.

A long-term goal of the lab has been to identify all of the components of the chloroplast and mitochondrial editosomes, the molecular machines that carry out RNA editing.  Initially we characterized the elements present on the chloroplast transcripts that signaled which C was to be targeted for editing.  We took advantage of technology for chloroplast genome transformation to introduce chimeric minigenes expressing transcripts with altered or divergent cis-elements. (2-4)  

We have also used chloroplast extracts capable of editing exogenous RNA (5-7) to probe the features of transcripts that are important in RNA editing.   Regions surrounding chloroplast editing sites could be mutated in vitro and RNA transcribed from mutated templates.  These RNAs could be introduced into chloroplast extracts and the extent of conversion of C to U was assayed.

 

 

Research in a variety of labs has shown that pentatricopeptide repeat (PPR)-containing proteins mediate specific recognition of the C target of editing in both chloroplasts and mitochondria.  We have identified PPR protein RNA editing recognition factors by comparative genomics or by exploiting natural variation in editing extent. (8-10)

 

Some members of the PPR protein family contain a DYW domain at the C-terminus.  This domain has been hypothesized to provide the enzymatic activity needed to convert C to U because it carries motifs characteristic of cytidine deaminases.  We performed site-specific mutagenesis of these motifs in two PPR proteins and demonstrated that editing activity was lost. (11)  This finding supports the idea that DYW domains carry cytidine deaminase activity.

 

 

 

An important breakthrough in our efforts to identify additional nuclear-encoded components of the editosome resulted from immunoprecipitation of a FLAG-tagged PPR protein, RARE1, followed by mass spectrometric analysis by the collaborating lab of Klaas Van Wijk (Cornell Section of Plant Biology).  Further analysis of a candidate protein resulted in the identification of RIP1 as a dual-targeted protein that affects RNA editing in both chloroplasts and mitochondria (12).

RIP1 is a member of a small gene family in Arabidopsis.  Analysis of mutants in additional members of the gene family revealed that RIP2 and RIP9 are required for efficient editing of many chloroplast C targets, while RIP3 and RIP8 control the editing of many mitochondrial transcripts.  These studies were facilitation by a new method for multiplex analysis of the editing extent of targets of numerous transcripts from multiple genotypes. (13)

The STS-PCRseq method pioneered by Stephane Bentolila provides a global method to analyze RNA editing in plant mitochondrial and chloroplast transcripts, and can be adapted for use in any biological system in order to sequence a selected subset of transcripts.  A protocol for application of the method to Arabidopsis organelle transcripts can be found here.

We have also developed a method named Multiplex RT-PCR Mass Spectrometry (MRMS), allows analysis of editing extent of up to 40 nucleotides in a 96- or 384-well format.  While more limited than STS-PCRseq, this method is simpler and particular valuable for assessing editing extent in vascular plant chloroplasts. (14)

Homology search revealed that a protein that we have named ORRM1 (Organelle RRM-domain containing protein 1) contained domains characteristic of the RIP family, but also exhibited an RNA Recognition Motif (RRM), unlike any of the RIP proteins. (15)  An Arabidopsis orrm1 mutant exhibited defects in editing of multiple chloroplast editing sites.  ORRM1 belongs to a clade within the large RRM family of Arabidopsis proteins.

 

 

 

 

 

 

 

 

 

 

 

 

By analyzing mutants and silenced tissue, we have subsequently demonstrated that two members of the ORRM clade, ORRM2 and ORMM3 are mitochondrial RNA editing factors.  While the ORRM3 protein contains a glycine-rich domain as well as an RRM domain, only the RRM domain is required for RNA editing activity. (16)

We have identified another family required for RNA editing by immunoprecipitating ORRM1 and performing proteomic analysis in collaboration with the Van Wijk lab.  We identified the protein Organelle Zinc Finger Protein 1 (OZ1) as required for editing of 14 targets in Arabidopsis. (17)  Arabidopsis contains three additional members of the zinc finger family, all predicted to be targeted to organelles.

The recent identification of additional nuclear-encoded proteins that are involved in editing in either chloroplasts or mitochondria, or in both organelles, has complicated our view of the molecular apparatus for RNA editing.  Further work should  complete the picture of the multiple configurations of RNA editosomes which are required to convert Cs to Us in plant organelles.

 

 

 
Citations
 

1.    Stern, D. B., Goldschmidt-Clermont, M., and Hanson, M. R. (2010) Chloroplast RNA metabolism. Annual Rev Plant Biol 61, 125-155.

2.    Reed, M. L., and Hanson, M. R. (1997) A heterologous maize rpoB editing site is recognized by transgenic tobacco chloroplasts. Mol Cell Biol 17, 6948-6952.

3.    Reed, M. L., Lyi, S. M., and Hanson, M. R. (2001) Edited transcripts compete with unedited mRNAs for trans-acting editing factors in higher plant chloroplasts. Gene 272, 165-171.

4.    Reed, M. L., Peeters, N. M., and Hanson, M. R. (2001) A single alteration 20 nt 5' to an editing target inhibits chloroplast RNA editing in vivo. Nucleic Acids Res 29, 1507-1513.

5.    Hegeman, C. E., Hayes, M. L., and Hanson, M. R. (2005) Substrate and cofactor requirements for RNA editing of chloroplast transcripts in Arabidopsis in vitro. Plant Journal 42, 124-132.

6.    Heller, W. P., Hayes, M. L., and Hanson, M. R. (2008) Cross-competition in editing of chloroplast RNA transcripts in vitro implicates sharing of trans-factors between different C targets, J Biol Chem 283, 7314-7319.

7.    Hayes, M. L., and Hanson, M. R. (2007) Identification of a sequence motif critical for editing of a tobacco chloroplast transcript. RNA 13, 281-288.

8.    Bentolila, S., Babina, A. M., Germain, A., and Hanson, M. R. (2013) Quantitative trait locus mapping identifies REME2, a PPR-DYW protein required for editing of specific C targets in Arabidopsis mitochondria. RNA Biol 10.

9.    Bentolila, S., Knight, W., and Hanson, M. (2010) Natural variation in Arabidopsis leads to the identification of REME1, a pentatricopeptide repeat-DYW protein controlling the editing of mitochondrial transcripts, Plant physiology 154, 1966-1982.

10.    Robbins, J. C., Heller, W. P., and Hanson, M. R. (2009) A comparative genomics approach identifies a PPR-DYW protein that is essential for C-to-U editing of the Arabidopsis chloroplast accD transcript. RNA 15, 1142-1153.

11.   Wagoner,  J.A., Sun, T., Lin, L., Hanson, M.R.  (2015) Cytidine deaminase motifs within the DYW domain of two pentatricopeptide repeat-containing proteins are required for site- specific chloroplast RNA editing.  J Biol Chem 290:2957-68.

12.    Bentolila, S., Heller, W. P., Sun, T., Babina, A. M., Friso, G., van Wijk, K. J., and Hanson, M. R. (2012) RIP1, a member of an Arabidopsis protein family, interacts with the protein RARE1 and broadly affects RNA editing. Proc Natl Acad Sci USA 109, E1453-1461.

13.    Bentolila, S., Oh, J., Hanson, M. R., and Bukowski, R. (2013) Comprehensive high-resolution analysis of the role of an Arabidopsis gene family in RNA editing, PLoS Genetics 9, e1003584.

14.  Germain A, Hanson MR, Bentolila S (2015) High‐throughput quantification of chloroplast RNA editing extent using Multiplex RT‐PCR Mass Spectrometry (MRMS).  Plant Journal  83:546-54.

15.    Sun, T., Germain, A., Giloteaux, L., Hammani, K., Barkan, A., Hanson, M. R., and Bentolila, S. (2013) An RNA recognition motif-containing protein is required for plastid RNA editing in Arabidopsis and maize. Proc Natl Acad Sci USA 110, E1169-1178.

16. Shi, X., Hanson, M.R., and Bentolila S. (2015)  Two RNA recognition motif-containing proteins are plant mitochondrial editing factors  Nucleic Acids Res 43:3814-3825

17. Sun T, Shi X, Friso G, Van Wijk K, Bentolila S, Hanson MR.  (2015) A zinc finger motif-containing protein is essential for chloroplast RNA editing. PLoS Genet. 11:e1005028.

 

 
Project Sponsors
 

 
Maureen R. Hanson
Liberty Hyde Bailey Professor
Phone: 607-254-4833
Fax: 607-255-6249

Hanson Laboratory
Department of Molecular Biology and Genetics
321 Biotechnology Building
Cornell University
Ithaca, NY 14853
Phone: 607-254-4832

View All Publications:
Maureen Hanson at NCBI Pubmed   Maureen Hanson at Google Scholar