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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...

The soybean aphid (Aphis glycines) is a major phloem-feeding pest of soybean (Glycine max). A. glycines feeding can cause the diversion of photosynthates and transmission of plant viruses, resulting in significant yield losses. In this study, we used oligonucleotide microarrays to characterize the long-term transcriptional response to soybean aphid colonization of two related soybean cultivars, one with the Rag1 aphid-resistance gene and one aphid-susceptible cultivar (without Rag1). Transcriptome profiles were determined after 1 and 7 days of aphid infestation. Our results revealed a susceptible response involving hundreds of transcripts, whereas only one transcript changed in the resistant response to aphids. This nonexistent resistance response might be explained by the fact that many defense-related transcripts are constitutively expressed in resistant plants, whereas these same genes are activated in susceptible plants only during aphid infestation. Analysis of phytohormone-related transcripts in the susceptible response showed different hormone profiles for the two time points, and suggest that aphids are able to suppress hormone signals in susceptible plants. A significant activation of abscissic acid, normally associated with abiotic stress responses, at day 7, might be a decoy strategy implemented by the aphid to suppress effective salicylic acid- and jasmonate-related defenses.

The S-like ribonucleases (RNases) RNS1 and RNS2 of Arabidopsis are members of the widespread T 2 ribonuclease family, whose members also include the S-RNases, involved in gametophytic selfincompatibility in plants. Both RNS1 and RNS2 mRNAs have been shown previously to be induced by inorganic phosphate (Pi) starvation. In our study we examined this regulation at the protein level and determined the effects of diminishing RNS1 and RNS2 expression using antisense techniques. The Pi-starvation control of RNS1 and RNS2 was confirmed using antibodies specific for each protein. These specific antibodies also demonstrated that RNS1 is secreted, whereas RNS2 is intracellular. By introducing antisense constructs, mRNA accumulation was inhibited by up to 90% for RNS1 and up to 65% for RNS2. These plants contained abnormally high levels of anthocyanins, the production of which is often associated with several forms of stress, including Pi starvation. This effect demonstrates that diminishing the amounts of either RNS1 or RNS2 leads to effects that cannot be compensated for by the actions of other RNases, even though Arabidopsis contains a large number of different RNase activities. These results, together with the differential localization of the proteins, imply that RNS1 and RNS2 have distinct functions in the plant.

The plant RNase T2 family is divided into two different subfamilies. S-RNases are involved in rejection of self-pollen during the establishment of self-incompatibility in three plant families. S-like RNases, on the other hand, are not involved in self-incompatibility, and although gene expression studies point to a role in plant defense and phosphate recycling, their biological roles are less well understood. Although S-RNases have been subjects of many phylogenetic studies, few have included an extensive analysis of S-like RNases, and genome-wide analyses to determine the number of S-like RNases in fully sequenced plant genomes are missing. We characterized the eight RNase T2 genes present in the Oryza sativa genome; and we also identified the full complement of RNase T2 genes present in other fully sequenced plant genomes. Phylogenetics and gene expression analyses identified two classes among the S-like RNase subfamily. Class I genes show tissue specificity and stress regulation. Inactivation of RNase activity has occurred repeatedly throughout evolution. On the other hand, Class II seems to have conserved more ancestral characteristics; and, unlike other S-like RNases, genes in this class are conserved in all plant species analyzed and most are constitutively expressed. Our results suggest that gene duplication resulted in high diversification of Class I genes. Many of these genes are differentially expressed in response to stress, and we propose that protein characteristics, such as the increase in basic residues can have a defense role independent of RNase activity. On the other hand, constitutive expression and phylogenetic conservation suggest that Class II S-like RNases may have a housekeeping role.

T2-family acidic endoribonucleases are represented in all genomes. A physiological role for RNase T2 has yet to be defined for metazoa. RNASET2 mutation in humans is linked with a leukoencephalopathy that arises in infancy characterized by cortical cysts and multifocal white matter lesions. We now show localization of RNASET2 within lysosomes. Further, we demonstrate that loss of rnaset2 in mutant zebrafish results in accumulation of undigested rRNA within lysosomes within neurons of the brain. Further, by using high field intensity magnetic resonance microimaging, we reveal white matter lesions in these animals comparable to those observed in RNASET2-deficient infants. This correlates with accumulation of Amyloid precursor protein and astrocytes at sites of neurodegeneration. Thus we conclude that familial cystic leukoencephalopathy is a lysosomal storage disorder in which rRNA is the best candidate for the noxious storage material.

Ribosomes account for a majority of the cell's RNA and much of its protein and represent a significant investment of cellular resources. The turnover and degradation of ribosomes has been proposed to play a role in homeostasis and during stress conditions. Mechanisms for the turnover of rRNA and ribosomal proteins have not been fully elucidated. We show here that the RNS2 ribonuclease and autophagy participate in RNA turnover in Arabidopsis thaliana under normal growth conditions. An increase in autophagosome formation was seen in an rns2-2 mutant, and this increase was dependent on the core autophagy genes ATG9 and ATG5. Autophagosomes and autophagic bodies in rns2-2 mutants contain RNA and ribosomes, suggesting that autophagy is activated as an attempt to compensate for loss of rRNA degradation. Total RNA accumulates in rns2-2, atg9-4, atg5-1, rns2-2 atg9-4, and rns2-2 atg5-1 mutants, suggesting a parallel role for autophagy and RNS2 in RNA turnover. rRNA accumulates in the vacuole in rns2-2 mutants. Vacuolar accumulation of rRNA was blocked by disrupting autophagy via an rns2-2 atg5-1 double mutant but not by an rns2-2 atg9-4 double mutant, indicating that ATG5 and ATG9 function differently in this process. Our results suggest that autophagy and RNS2 are both involved in homeostatic degradation of rRNA in the vacuole.