RNase E is an endoribonuclease that has been studied primarily in Escherichia coli, where it is prominently involved in the processing and degradation of RNA. Homologs of bacterial RNase E are encoded in the nuclear genome of higher plants. RNA degradation in the chloroplast, an organelle that originated from a prokaryote similar to cyanobacteria, occurs via the polyadenylation-assisted degradation pathway. In E. coli, this process is probably initiated with the removal of 59-end phosphates followed by endonucleolytic cleavage by RNase E. The plant homolog has been proposed to function in a similar way in the chloroplast. Here we show that RNase E of Arabidopsis is located in the soluble fraction of the chloroplast as a high molecular weight complex. In order to characterize its endonucleolytic activity, Arabidopsis RNase E was expressed in bacteria and analyzed. Similar to its E. coli counterpart, the endonucleolytic activity of the Arabidopsis enzyme depends on the number of phosphates at the 59 end, is inhibited by structured RNA, and preferentially cleaves A/U-rich sequences. The enzyme forms an oligomeric complex of ;680 kDa. The chloroplast localization and the similarity in the two enzymes' characteristics suggest that plant RNase E participates in the initial endonucleolytic cleavage of the polyadenylation-stimulated RNA degradation process in the chloroplast, perhaps in collaboration with the two other chloroplast endonucleases, RNase J and CSP41.
Autophagy is the mechanism by which cytoplasmic components and organelles are degraded by the lysosomal machinery in response to diverse stimuli including nutrient deprivation, intracellular pathogens, and multiple forms of cellular stress. Here, we show that the membrane-associated E3 ligase RNF5 regulates basal levels of autophagy by controlling the stability of a select pool of the cysteine protease ATG4B. RNF5 controls the membranal fraction of ATG4B and limits LC3 (ATG8) processing, which is required for phagophore and autophagosome formation. The association of ATG4B with—and regulation of its ubiquitination and stability by—RNF5 is seen primarily under normal growth conditions. Processing of LC3 forms, appearance of LC3-positive puncta, and p62 expression are higher in RNF5−/− MEF. RNF5 mutant, which retains its E3 ligase activity but does not associate with ATG4B, no longer affects LC3 puncta. Further, increased puncta seen in RNF5−/− using WT but not LC3 mutant, which bypasses ATG4B processing, substantiates the role of RNF5 in early phases of LC3 processing and autophagy. Similarly, RNF-5 inactivation in Caenorhabditis elegans increases the level of LGG-1/LC3::GFP puncta. RNF5−/− mice are more resistant to group A Streptococcus infection, associated with increased autophagosomes and more efficient bacterial clearance by RNF5−/− macrophages. Collectively, the RNF5-mediated control of membranalATG4B reveals a novel layer in the regulation of LC3 processing and autophagy.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.