Studying the Structure and Processing of Chloroplast Transcripts
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 wordsChloroplast Plastid RNA processing RNA binding protein RNA binding assay
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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