The chloroplast RNA helicase ISE 2 is required for multiple chloroplast RNA processing steps in Arabidopsis thaliana

Summary INCREASED SIZE EXCLUSION LIMIT 2 ( ISE 2) is a chloroplast‐localized RNA helicase that is indispensable for proper plant development. Chloroplasts in leaves with reduced ISE 2 expression have previously been shown to exhibit reduced thylakoid contents and increased stromal volume, indicative of defective development. It has recently been reported that ISE 2 is required for the splicing of group II introns from chloroplast transcripts. The current study extends these findings, and presents evidence for ISE 2's role in multiple aspects of chloroplast RNA processing beyond group II intron splicing. Loss of ISE 2 from Arabidopsis thaliana leaves resulted in defects in C‐to‐U RNA editing, altered accumulation of chloroplast transcripts and chloroplast‐encoded proteins, and defective processing of chloroplast ribosomal RNA s. Potential ISE 2 substrates were identified by RNA immunoprecipitation followed by next‐generation sequencing ( RIP ‐seq), and the diversity of RNA species identified supports ISE 2's involvement in multiple aspects of chloroplast RNA metabolism. Comprehensive phylogenetic analyses revealed that ISE 2 is a non‐canonical Ski2‐like RNA helicase that represents a separate sub‐clade unique to green photosynthetic organisms, consistent with its function as an essential protein. Thus ISE 2's evolutionary conservation may be explained by its numerous roles in regulating chloroplast gene expression.