The Arabidopsis chloroplast RNase J displays both exo- and robust endonucleolytic activities
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Arabidopsis chloroplast RNase J displaces both exo- and endo-ribonucleolytic activities and contains a unique GT-1 DNA binding domain.
Control of chloroplast gene expression is predominantly at the post-transcriptional level via the coordinated action of nuclear encoded ribonucleases and RNA-binding proteins. The 5′ end maturation of mRNAs ascribed to the combined action of 5′→3′ exoribonuclease and gene-specific RNA-binding proteins of the pentatricopeptide repeat family and others that impede the progression of this nuclease. The exo- and endoribonuclease RNase J, the only prokaryotic 5′→3′ ribonuclease that is commonly present in bacteria, Archaea, as well as in the chloroplasts of higher plants and green algae, has been implicated in this process. Interestingly, in addition to the metalo-β-lactamase and β-CASP domains, RNase J of plants contains a conserved GT-1 domain that was previously characterized in transcription factors that function in light and stress responding genes. Here, we show that the Arabidopsis RNase J (AtRNase J), when analyzed in vitro with synthetic RNAs, displays both 5′→3′ exonucleolytic activity, as well as robust endonucleolytic activity as compared to its bacterial homolog RNase J1 of Bacillus subtilis. AtRNase J degraded single-stranded RNA and DNA molecules but displays limited activity on double stranded RNA. The addition of three guanosines at the 5′ end of the substrate significantly inhibited the degradation activity, indicating that the sequence and structure of the RNA substrate modulate the ribonucleolytic activity. Mutation of three amino acid in the catalytic reaction center significantly inhibited both the endonucleolytic and exonucleolytic degradation activities, while deletion of the carboxyl GT-1 domain that is unique to the plant RNAse J proteins, had a little or no significant effect. The robust endonucleolytic activity of AtRNase J suggests its involvement in the processing and degradation of RNA in the chloroplast.
KeywordsRNA-processing in the chloroplast Metallo-beta-lactamase ribonuclease GT-1 domain 5′ to 3′ exoribonuclease
We gratefully thank Ciarán Condon from the Institute de Biologie Physico-Chimique for the plasmid and protocol to express the Bacillus Subtilis RNase J1. Alice Barkan, David Stern and Ciarán Condon for discussions and ideas. This work was supported by a grant from the Binational Science Foundation (BSF) (2009253).
MH—designed and performed the experiments. VL—designed and performed some of the experiments. FG—performed the bioinformatics analysis of the GT-1 domain. GS—designed the research, secured funding and wrote the manuscript.
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