ABSTRACT RNase J is a ribonuclease found in bacteria, archaea, and plant chloroplasts, and plays diverse roles in RNA maturation and stability. Chloroplast RNase J is encoded by the nuclear RNJ locus and is essential for embryo maturation. Arabidopsis or tobacco plants depleted for RNase J accumulate massive amounts of double-stranded RNA, which interferes with translation and causes chlorosis. Land plant RNase J uniquely contains a C-terminal GT1 domain, a DNA- binding motif found in transcription factors. Here, we have used complementation of an Arabidopsis rnj mutant with versions of RNase J with a mutated or deleted GT1 domain to investigate its role in RNase J function. We show that in vitro , the recombinant GT1 domain binds both double-stranded RNA and DNA, but not single-stranded nucleic acids, with no sequence specificity. Furthermore, while RNase J lacking GT1 binding complements the rnj mutant, these plants accumulate high levels of dsRNA as detected by immunolocalization and RNA-Seq. GT1 mutations also change RNase J solubility in vivo , suggesting that the GT1 domain is involved in localization within the plastid. Taken together, our results suggest that the GT1 domain plays a key role in dsRNA removal through localizing the enzyme and/or selectively binding the dsRNA substrate. GRAPHICAL ABSTRACT