Localization of RNS2 ribonuclease to the vacuole is required for its role in cellular homeostasis

Abstract

Localization of the RNase RNS2 to the vacuole via a C-terminal targeting signal is essential for its function in rRNA degradation and homeostasis. RNase T2 ribonucleases are highly conserved enzymes present in the genomes of nearly all eukaryotes and many microorganisms. Their constitutive expression in different tissues and cell types of many organisms suggests a housekeeping role in RNA homeostasis. The Arabidopsis thaliana class II RNase T2, RNS2, is encoded by a single gene and functions in rRNA degradation. Loss of RNS2 results in RNA accumulation and constitutive activation of autophagy, possibly as a compensatory mechanism. While the majority of RNase T2 enzymes is secreted, RNS2 is located within the vacuole and in the endoplasmic reticulum (ER), possibly within ER bodies. As RNS2 has a neutral pH optimum, and the endomembrane organelles are connected by vesicle transport, the site within the endomembrane system at which RNS2 functions is unclear. Here we demonstrate that localization to the vacuole is essential for the physiological function of RNS2. A mutant allele of RNS2, rns2-1, results in production of an active RNS2 RNase but with a mutation that removes a putative C-terminal vacuolar targeting signal. The mutant protein is, therefore, secreted from the cell. This results in a constitutive autophagy phenotype similar to that observed in rns2 null mutants. These findings illustrate that the intracellular retention of RNS2 and localization within the vacuole are critical for its cellular function.

Differential degradation of RNA species by autophagy related pathways in plants

“…At the same time, RNA degradation products were decreased in these lines, as well as the extractable vacuolar RNase activity. However, about 30% of residual RNase activity was found in both RNS2 knockout lines, in line with similar observations by others [19]. This provides evidence for the presence of further RNases in Arabidopsis vacuoles.…”

“…However, the effect of a loss of RNS2 on vacuolar RNA composition and that of breakdown products and intermediates has not been analyzed so far. Rns2-2 (SALK_069588) was described as real knockout line of RNS2 but rns2-1 as expressing a C-terminal truncated version of RNS2 [19].…”

Molecular mechanisms underpinning phosphorus‐use efficiency in rice

“…A potential autophagy pathway for the movement of intact ribosomes to the vacuole for degradation has been described in maize root tip cells (Niki, Saito, & Gladish, ). Ultimately, the degradation of rRNA requires the RNS2 that localizes to vacuoles (Floyd, Mugume, Morriss, MacIntosh, & Bassham, ; Hillwig et al, ). RNS2 degrades RNA into nucleotide monophosphates (3'NMPs), which are subsequently dephosphorylated by vacuolar PAP such as AtPAP26 (Bassham & MacIntosh, ; Hillwig et al, ; Hurley, Tran, Snedden, & Plaxton, ; Robinson, Carson, Ying, Ellis, & Plaxton, ; Robinson, Park, et al, ).…”

The Ins and Outs of Autophagic Ribosome Turnover

“…Additionally, the rns2-2 mutant plants have constitutive autophagy [62,67], possibly as an attempt to compensate for loss of rRNA degradation. The localization of RNS2 to the vacuole is critical for normal ribosomal RNA turnover and homeostasis [63], as shown using a mutant (rns2-1) that lacks the C-terminal vacuolar targeting signal and is instead secreted from the cell. Although it retains RNase catalytic activity, rns2-1 has constitutive autophagy, similar to the null mutant rns2-2 [62,63].…”

“…The ribonuclease RNS2 is necessary for normal ribosomal RNA decay in Arabidopsis (Figure 1) [62]. RNS2 is a nonspecific endoribonuclease of the RNase T2 family that localizes to the vacuole and endoplasmic reticulum [62,63]. During senescence and phosphate starvation, RNS2 is upregulated, possibly to remobilize the phosphate in RNA [64,65].…”