Overexpression of a natural chloroplast-encoded antisense RNA in tobacco destabilizes 5S rRNA and retards plant growth

AS5-overexpressing (AS5ox) plants obtained by chloroplast transformation exhibited slower growth and slightly pale green leaves. Analysis of AS5 transcripts revealed four distinct species in wild-type (WT) and AS5ox plants, and additional AS5ox-specific products. Of the corresponding sense strand transcripts, tRNAArg overaccumulated several-fold in transgenic plants whereas 5S rRNA was unaffected. However, run-on transcription showed that the 5S-trnR region was transcribed four-fold more in the AS5ox plants compared to WT, indicating that overexpression of AS5 was associated with decreased stability of 5S rRNA. In addition, polysome analysis of the transformants showed less 5S rRNA and rbcL mRNA associated with ribosomes.Our results suggest that AS5 can modulate 5S rRNA levels, giving it the potential to affect Chloroplast translation and plant growth. More globally, overexpression of asRNAs via chloroplast transformation may be a useful strategy for defining their functions.Chloroplasts originated around 1.5 billion years ago via an endosymbiotic event where a primitive eukaryote engulfed an ancestor of modern-day cyanobacteria. Subsequently, massive gene transfer to the nucleus occurred, resulting in a highly reduced plastid genome of 120-160 kb that possesses ~120 genes. These remaining genes are mostly organized into clusters, and their expression requires a combination of prokaryotic and eukaryotic-like post-transcriptional events including maturation of polycistronic transcripts, splicing, RNA editing, and 5' and 3' end trimming [reviewed in [1]]. These processes are catalyzed by nucleus-encoded proteins, many of which were originally encoded by the chloroplast ancestor [2].Post-transcriptional regulation in both prokaryotes and eukaryotes is also exerted by non-coding RNAs (ncRNAs), which include antisense RNAs (asRNAs) and transcribed intergenic sequences. The asRNAs can act in cis, on the cognizant sense strand transcript, or in trans, targeting one or more