Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes

Abstract

Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ˜50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy.

Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes

“…The primary and secondary antibodies used are: polyclonal goat anti-SIDT2 (N-20; Santa Cruz Biotechnology, sc-19991) to detect murine SIDT2, monoclonal rabbit anti-SCARB2/LIMP2 (EPR12080; Abcam, ab176317), horseradish peroxidase-conjugated anti-goat IgG (Jackson ImmunoResearch, 305-036-003) and horseradish peroxidase-conjugated anti-rabbit IgG (Thermo Scientific, 31460). The rabbit polyclonal anti-SIDT2 antibody used to detect human SIDT2 was prepared as described previously 9 . The Can Get Signal Immunoreaction Enhancer Solution (Toyobo, NKB101) was used for blots against murine SIDT2.…”

“…We have reported that constitutive degradation of cellular RNA is one of the physiological functions of RNautophagy 9 . Currently, the physiological roles of DNautophagy are unclear and warrant further examination.…”

“…We have previously reported that S564A mutant SIDT2 lacks the ability to mediate translocation of RNA 9 . SIDT2 has been suggested to possess hydrolase activity and S564 seems to be important for this function 14 .…”

“…RNautophagy and DNautophagy possess some selectivity for RNA and DNA substrates in vitro 8 . Most recently, we found that SIDT2, an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1, is a lysosomal membrane protein that mediates RNA translocation during RNautophagy 9 . Following a recent study showing that plasmid DNA can be imported into cultured silkworm cells by SID-1, 10 we hypothesized that SIDT2 also mediates the translocation of DNA during DNautophagy.…”

“…1D). We have previously reported that a serine 564 to alanine mutation (S564A) inhibits RNA uptake activity of SIDT2 9 . We tested the effect of this mutation on DNA uptake by performing DNA uptake assay using lysosomes derived from mutant SIDT2-overexpressing cells.…”