Abstract
Most chloroplast genes in land plants are represented by multiple transcript isoforms that arise via differential splicing, endo- and exo-nucleolytic processing, and/or RNA editing. Exploration of the functional significance and mechanisms of these processing events is an active area of current research. This chapter focuses on methods that can be used to define the termini of chloroplast RNAs, quantify the relative levels of alternative processed RNA isoforms, and identify the binding sites of proteins that mediate chloroplast RNA processing. Various approaches for defining the sequence specificity of chloroplast RNA binding proteins are discussed, as are the parameters to consider in designing in vitro assays for RNA binding activities. A protocol is provided for a poisoned-primer extension assay for quantifying different splice isoforms.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Stern, D.B., Goldschmidt-Clermont, M., and Hanson, M.R. (2010) Chloroplast RNA metabolism. Annu. Rev. Plant. Biol. 61, 125–155.
Barkan, A., Klipcan, L., Ostersetzer, O., Kawamura, T., Asakura, Y., and Watkins, K. (2007) The CRM domain: an RNA binding module derived from an ancient ribosome-associated protein. RNA 13, 55–64.
Kroeger, T., Watkins, K., Friso, G., Wijk, K.v., and Barkan, A. (2009) A plant-specific RNA binding domain revealed through analysis of chloroplast group II intron splicing. Proc. Natl. Acad. Sci. USA 106, 4537–4542.
Schmitz-Linneweber, C., and Small, I. (2008) Pentatricopeptide repeat proteins: a socket set for organelle gene expression. Trends Plant Sci. 13, 663–670.
Barkan, A. (1998) Approaches to investigating nuclear genes that function in chloroplast biogenesis in land plants. Methods Enzymol. 297, 38–57.
Pfalz, J., Bayraktar, O., Prikryl, J., and Barkan, A. (2009) Site-specific binding of a PPR Âprotein defines and stabilizes 5’ and 3’ mRNA termini in chloroplasts. EMBO J. 28, 2042–2052.
Kuhn, J., and Binder, S. (2002) RT-PCR analysis of 5’ to 3’-end-ligated mRNAs identifies the extremities of cox2 transcripts in pea mitochondria. Nucleic Acids Res. 30, 439–446.
Slomovic, S., Portnoy, V., and Schuster, G. (2008) Detection and characterization of Âpolyadenylated RNA in Eukarya, Bacteria, Archaea, and organelles. Methods Enzymol. 447, 501–520.
Falcon de Longevialle, A., Hendrickson, L., Taylor, N., Delannoy, E., Lurin, C., Badger, M., Millar, A.H., and Small, I. (2008) The pentatricopeptide repeat gene OTP51 with two LAGLIDADG motifs is required for the cis-splicing of plastid ycf3 intron 2 in Arabidopsis thaliana. Plant J. 56, 157–168.
Jenkins, B., Kulhanek, D., and Barkan, A. (1997) Nuclear mutations that block group II RNA splicing in maize chloroplasts reveal several intron classes with distinct requirements for splicing factors. Plant Cell 9, 283–296.
Watkins, K., Kroeger, T., Cooke, A., Williams-Carrier, R., Friso, G., Belcher, S., Wijk, K.v., and Barkan, A. (2007) A ribonuclease III domain protein functions in group II intron splicing in maize chloroplasts. Plant Cell 19, 2606–2623.
Beick, S., Schmitz-Linneweber, C., Williams-Carrier, R., Jensen, B., and Barkan, A. (2008) The pentatricopeptide repeat protein PPR5 stabilizes a specific tRNA precursor in maize chloroplasts. Mol. Cell Biol. 28, 5337–5347.
Schmitz-Linneweber, C., Williams-Carrier, R.E., Williams-Voelker, P.M., Kroeger, T.S., Vichas, A., and Barkan, A. (2006) A pentatricopeptide repeat protein facilitates the trans-splicing of the maize chloroplast rps12 pre-mRNA. Plant Cell 18, 2650–2663.
Ostheimer, G., Williams-Carrier, R., Belcher, S., Osborne, E., Gierke, J., and Barkan, A. (2003) Group II intron splicing factors derived by diversification of an ancient RNA binding module. EMBO J. 22, 3919–3929.
Schmitz-Linneweber, C., Williams-Carrier, R., and Barkan, A. (2005) RNA immunoprecipitation and microarray analysis show a chloroplast pentatricopeptide repeat protein to be associated with the 5’-region of mRNAs whose translation it activates. Plant Cell 17, 2791–2804.
Barkan, A. (2008) Genome-wide analysis of RNA-protein interactions in plants. In, Plant Systems Biology (Belostotsky, D., ed.) Humana Press, Totowa, NJ, USA, pp. 13–37.
Williams-Carrier, R., Kroeger, T., and Barkan, A. (2008) Sequence-specific binding of a chloroplast pentatricopeptide repeat protein to its native group II intron ligand. RNA 14, 1930–1941.
Wong, I., and Lohman, T. (1993) A double-filter method for nitrocellulose-filter binding: application to protein-nucleic acid interactions. Proc. Natl. Acad. Sci. USA 90, 5428–5432.
Setzer, D.R. (1999) Measuring equilibrium and kinetic constants using gel retardation assays. Methods Mol. Biol. 118, 115–128.
Ostersetzer, O., Watkins, K., Cooke, A., and Barkan, A. (2005) CRS1, a chloroplast group II intron splicing factor, promotes intron folding through specific interactions with two intron domains. Plant Cell 17, 241–255.
Draper, D.E. (2008) RNA folding: thermodynamic and molecular descriptions of the roles of ions. Biophys. J. 95, 5489–5495.
Asakura, Y., and Barkan, A. (2006) Arabidopsis orthologs of maize chloroplast splicing factors promote splicing of orthologous and species-specific group II introns. Plant Physiol. 142, 1656–1663.
Acknowledgments
I thank Yukari Asakura and Kenny Watkins for their input into the development of the protocol for poisoned-primer extension, and Yukari Asakura for permission to present the data in Fig. 1. The guidelines for RNA-binding assays evolved from discussions with and data obtained by Kenny Watkins, Rosalind Williams-Carrier, Oren Ostersetzer, and Margarita Rojas.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Barkan, A. (2011). Studying the Structure and Processing of Chloroplast Transcripts. In: Jarvis, R. (eds) Chloroplast Research in Arabidopsis. Methods in Molecular Biology, vol 774. Humana Press. https://doi.org/10.1007/978-1-61779-234-2_12
Download citation
DOI: https://doi.org/10.1007/978-1-61779-234-2_12
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-233-5
Online ISBN: 978-1-61779-234-2
eBook Packages: Springer Protocols