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Studying the Structure and Processing of Chloroplast Transcripts

  • Alice Barkan
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 774)

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.

Key words

Chloroplast Plastid RNA processing RNA binding protein RNA binding assay 

Notes

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.

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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Institute of Molecular BiologyUniversity of OregonEugeneUSA

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