RNS2, a conserved member of the RNase T2 family, is necessary for ribosomal RNA decay in plants

Abstract

RNase T2 enzymes are conserved in most eukaryotic genomes, and expression patterns and phylogenetic analyses suggest that they may carry out an important housekeeping role. However, the nature of this role has been elusive. Here we show that RNS2, an intracellular RNase T2 from Arabidopsis thaliana, is essential for normal ribosomal RNA recycling. This enzyme is the main endoribonuclease activity in plant cells and localizes to the endoplasmic reticulum (ER), ER-derived structures, and vacuoles. Mutants lacking RNS2 activity accumulate RNA intracellularly, and rRNA in these mutants has a longer half-life. Normal rRNA turnover seems essential to maintain cell homeostasis because rns2 mutants display constitutive autophagy. We propose that RNS2 is part of a process that degrades rRNA to recycle its components. This process appears to be conserved in all eukaryotes. ribophagy R ibonucleases (RNases) belonging to the RNase T2 family are acidic endonucleases without base specificity that are either extracellular or targeted to the secretory pathway (1). This family is conserved in the genome of almost all eukaryotic organisms so far analyzed, suggesting that it performs an important function that has been maintained throughout evolution (2, 3). Phylogenetic analyses have identified three subclasses present in plant genomes (3, 4). Class I proteins are highly diversified and show evidence of gene sorting, and their expression is commonly tissue-specific and/or regulated by biotic and abiotic stress (3). Class III includes mostly members of the S-RNases, enzymes involved in the process of gametophytic self-incompatibility in three plant families (5). Finally, class II includes proteins that are highly conserved in all plant genomes and are normally expressed at high levels in most plant tissues (3). On the basis of their conservation and gene expression characteristics, we proposed that class II RNases may have a housekeeping role in plant cells (3). Similar gene expression and phylogenetic studies in animals led us to the hypothesis that metazoan RNase T2 enzymes also have a housekeeping role and may be equivalent to class II enzymes from plants (2).RNS2, one of five RNase T2 genes in the Arabidopsis genome, encodes the only class II protein in this model organism (3,4). This RNase is present in all tissues at high levels (6, 7) and is localized in an intracellular compartment. RNS2 expression is increased even further during senescence and during inorganic phosphate (Pi) starvation (6, 8). Thus, it was hypothesized that RNS2 is part of a phosphate scavenging system that rescues plants that are under nutritional stress (9). However, because RNS2 and other class II RNase T2 proteins accumulate to high levels even under optimal growth conditions, this rescue function is unlikely to be the main role of these enzymes. Moreover, in vertebrates, in which RNase T2 enzymes are absolutely conserved (2), the mechanisms that control the response to phosphate starvation seem to be specific to the intestine and kidney (10), wherea...

Evidence for autophagy-dependent pathways of rRNA turnover inArabidopsis

“…RNS2 activity was verified in the transgenic lines by an in-gel ribonuclease activity assay using high-molecular weight torula yeast RNA as substrate. 19,32 Even though RNS2 event #1 had much lower RNS2 activity than WT, both lines showed complementation of the constitutive autophagy phenotype, indicating that relatively low RNS2 activity is sufficient for complementation (Fig. 2C).…”

“…The Arabidopsis thaliana RNS2 null mutant (rns2-2) was previously shown to accumulate MDC-stained structures even under normal nutrient-rich conditions, 19 which were hypothesized to be autophagosomes. Since this assumption is central to the hypothesis that ribosomes are recycled through a ribophagy-like mechanism in Arabidopsis, and since MDC can stain other acidic organelles in addition to autophagosomes, 24 our first objective was to determine whether the MDC-stained structures observed in rns2-2 are autophagosomes.…”

“…Disruption of this mechanism would lead to accumulation of RNA and the induction of nonselective autophagy as a compensatory mechanism. 19 This mechanism seems to be conserved in eukaryotes. Mutations affecting Danio rerio (zebrafish) RNASET2 also resulted in animals that accumulate rRNA in lysosomes, particularly in neurons, and this accumulation caused white-matter lesions in the fish brain, again indicating that rRNA degradation is necessary for cellular homeostasis in nonstarvation conditions.…”

“…In Arabidopsis thaliana mutants lacking RNS2 (At2g39780), a nonspecific endoribonuclease of the RNase T2 family that resides in the endoplasmic reticulum and the vacuole, rRNA has a longer half-life than in wild-type (WT) plants, indicating that this enzyme is necessary for normal rRNA decay. 19 Moreover, the rns2-2 mutants accumulated RNA intracellularly and mutant cells accumulated acidic vesicles revealed by monodansylcadaverine (MDC) staining, hypothesized to be autophagosomes, even under nonstarvation conditions. These results led to the hypothesis that RNS2 participates in a ribophagy-like mechanism that targets ribosomes for degradation as a housekeeping function that maintains cellular homeostasis.…”

“…16 While ribophagy has not yet been specifically studied in other eukaryotes, mounting evidence suggests that the process could be conserved in plants and animals and that starvation is not required for ribophagy to occur. [17][18][19] In addition to an important role in response to nutrient deficiency, rRNA turnover also seems to be necessary for maintenance of cellular homeostasis during periods of normal growth. In Arabidopsis thaliana mutants lacking RNS2 (At2g39780), a nonspecific endoribonuclease of the RNase T2 family that resides in the endoplasmic reticulum and the vacuole, rRNA has a longer half-life than in wild-type (WT) plants, indicating that this enzyme is necessary for normal rRNA decay.…”