The Arabidopsis chloroplast RNase J displays both exo- and robust endonucleolytic activities.

KEY MESSAGE: 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-ß-lactamase and ß-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.

Chloroplast RNase J compensates for inefficient transcription termination by removal of antisense RNA.

Ribonuclease J is an essential enzyme, and the Bacillus subtilis ortholog possesses both endoribonuclease and 5' → 3' exoribonuclease activities. Chloroplasts also contain RNase J, which has been postulated to participate, as both an exo- and endonuclease, in the maturation of polycistronic mRNAs. Here we have examined recombinant Arabidopsis RNase J and found both 5' → 3' exoribonuclease and endonucleolytic activities. Virus-induced gene silencing was used to reduce RNase J expression in Arabidopsis and Nicotiana benthamiana, leading to chlorosis but surprisingly few disruptions in the cleavage of polycistronic rRNA and mRNA precursors. In contrast, antisense RNAs accumulated massively, suggesting that the failure of chloroplast RNA polymerase to terminate effectively leads to extensive symmetric transcription products that are normally eliminated by RNase J. Mung bean nuclease digestion and polysome analysis revealed that this antisense RNA forms duplexes with sense strand transcripts and prevents their translation. We conclude that a major role of chloroplast RNase J is RNA surveillance to prevent overaccumulation of antisense RNA, which would otherwise exert deleterious effects on chloroplast gene expression.

Rac-dependent doubling of HeLa cell area and impairment of cell migration and cell cycle by compounds from Iris germanica.

Plants are an infinite source of bioactive compounds. We screened the Israeli flora for compounds that interfere with the organization of the actin cytoskeleton. We found an activity in lipidic extract from Iris germanica that was able to increase HeLa cell area and adhesion and augment the formation of actin stress fibers. This effect was not observed when Ref52 fibroblasts were tested and was not the result of disruption of microtubules. Further, the increase in cell area was Rac1-dependent, and the iris extract led to slight Rac activation. Inhibitor of RhoA kinase did not interfere with the ability of the iris extract to increase HeLa cell area. The increase in HeLa cell area in the presence of iris extract was accompanied by impairment of cell migration and arrest of the cell cycle at G1 although the involvement of Rac1 in these processes is not clear. Biochemical verification of the extract based on activity-mediated fractionation and nuclear magnetic resonance analysis revealed that the active compounds belong to the group of iridals, a known group of triterpenoid. Purified iripallidal was able to increase cell area of both HeLa and SW480 cells.