Gene expression in chloroplasts involves core transcription, translation and RNA turnover machineries that were acquired from the chloroplast's cyanobacterial ancestor (1
). These ancient mechanisms function in concert with more recently evolved RNA processing steps that include RNA editing, the processing of mRNA termini and the protein-facilitated splicing of group II introns. In land plant chloroplasts, the majority of protein-coding genes are found in polycistronic transcription units that give rise to complex transcript populations via processing between coding regions (intercistronic processing) and upstream of the 5′ open reading frame (5′-processing). Where orthologous transcription units have been examined, the populations of processed transcripts are highly conserved between monocot, dicot and even non-vascular plants (2–4
). However, the mechanisms and functional consequences of these widespread and conserved RNA processing events remain subjects of debate.
Genetic analyses have highlighted members of the pentatricopeptide repeat (PPR) family as effectors of intercistronic and 5′ RNA processing in chloroplasts. PPR proteins are defined by tandem arrays of a degenerate 35 amino acid repeating unit, which are predicted to form an elongated solenoid consisting of stacked helical repeats (5
). The PPR proteins CRP1, PPR10 and HCF152 are each required for the accumulation of chloroplast RNAs with processed 5′- or 3′-ends mapping in specific intergenic regions (6–9
). The underlying mechanism has been described for PPR10, which binds RNA segments in each of two intergenic regions and impedes exoribonucleases intruding from either direction (7
). Genetic data implicate other PPR proteins as well as ‘PPR-like’ proteins with distinct helical repeat architectures in stabilizing chloroplast RNAs with specific 5′ termini (11–16
). Together, these observations suggest that intercistronic RNA processing, 5′ RNA processing and 5′ RNA stabilization in chloroplasts involve similar mechanisms: in each case a helical repeat protein binds a specific RNA segment and protects the adjacent RNA by serving as a barrier to exoribonucleases.
Although there is considerable evidence that this mechanism accounts for the processing of several chloroplast mRNAs, its global impact on the chloroplast transcriptome is unknown. In fact, stable RNA structures provide an alternative mechanism for impeding the vectorial degradation of chloroplast mRNAs from both the 5′ and 3′ directions (17
), and the involvement of site-specific endonucleases in intercistronic processing has typically been invoked. In this study, we provide evidence that protection by PPR or PPR-like proteins is the predominant mechanism for defining the positions of processed 5′ and intercistronic mRNA termini in land plant chloroplasts. In addition, we use the attributes of known PPR binding sites to infer likely binding sites for PPR (or PPR-like) proteins on chloroplast mRNAs for which stabilizing proteins have not been identified.