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

Aizawa S, Fujiwara Y, Contu VR, Hase K, Takahashi M, Kikuchi H, Kabuta C, Wada K, Kabuta T - Autophagy (2016)

Bottom Line: 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.Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy.

View Article: PubMed Central - PubMed

Affiliation: a Department of Degenerative Neurological Diseases , National Institute of Neuroscience, National Center of Neurology and Psychiatry , Kodaira , Tokyo , Japan.

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.

No MeSH data available.


Related in: MedlinePlus

Effect of SIDT1 overexpression on RNautophagy. (A) Neuro2a cells expressing GFP-tagged SIDT1 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 μm. Colocalization rate was quantified (right panel, n=3). (B) SIDT1 was overexpressed in Neuro2a cells. Protein levels were analyzed by immunoblotting using an anti-SIDT1 antibody. (C) Lysosomes were isolated from Neuro2a cells overexpressing SIDT1 or control transfectants. The RNA uptake assay I indicated in Fig. 2A was performed. Relative levels of RNA uptake were quantified. Mean ± SEM (n = 3). n.s., not significant.
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f0006: Effect of SIDT1 overexpression on RNautophagy. (A) Neuro2a cells expressing GFP-tagged SIDT1 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 μm. Colocalization rate was quantified (right panel, n=3). (B) SIDT1 was overexpressed in Neuro2a cells. Protein levels were analyzed by immunoblotting using an anti-SIDT1 antibody. (C) Lysosomes were isolated from Neuro2a cells overexpressing SIDT1 or control transfectants. The RNA uptake assay I indicated in Fig. 2A was performed. Relative levels of RNA uptake were quantified. Mean ± SEM (n = 3). n.s., not significant.

Mentions: We investigated whether or not SIDT1 is involved in RNautophagy. We examined intracellular localization of SIDT1 using a C-terminal GFP-tag. Fluorescent signals for SIDT1 scarcely colocalized with LysoTracker Red (Fig. 6A). In addition, overexpression of SIDT1 did not affect RNautophagy in RNA uptake assays (Fig. 6B–C). Thus, in contrast to SIDT2, SIDT1 does not function in RNautophagy.Figure 6.


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

Aizawa S, Fujiwara Y, Contu VR, Hase K, Takahashi M, Kikuchi H, Kabuta C, Wada K, Kabuta T - Autophagy (2016)

Effect of SIDT1 overexpression on RNautophagy. (A) Neuro2a cells expressing GFP-tagged SIDT1 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 μm. Colocalization rate was quantified (right panel, n=3). (B) SIDT1 was overexpressed in Neuro2a cells. Protein levels were analyzed by immunoblotting using an anti-SIDT1 antibody. (C) Lysosomes were isolated from Neuro2a cells overexpressing SIDT1 or control transfectants. The RNA uptake assay I indicated in Fig. 2A was performed. Relative levels of RNA uptake were quantified. Mean ± SEM (n = 3). n.s., not significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836006&req=5

f0006: Effect of SIDT1 overexpression on RNautophagy. (A) Neuro2a cells expressing GFP-tagged SIDT1 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 μm. Colocalization rate was quantified (right panel, n=3). (B) SIDT1 was overexpressed in Neuro2a cells. Protein levels were analyzed by immunoblotting using an anti-SIDT1 antibody. (C) Lysosomes were isolated from Neuro2a cells overexpressing SIDT1 or control transfectants. The RNA uptake assay I indicated in Fig. 2A was performed. Relative levels of RNA uptake were quantified. Mean ± SEM (n = 3). n.s., not significant.
Mentions: We investigated whether or not SIDT1 is involved in RNautophagy. We examined intracellular localization of SIDT1 using a C-terminal GFP-tag. Fluorescent signals for SIDT1 scarcely colocalized with LysoTracker Red (Fig. 6A). In addition, overexpression of SIDT1 did not affect RNautophagy in RNA uptake assays (Fig. 6B–C). Thus, in contrast to SIDT2, SIDT1 does not function in RNautophagy.Figure 6.

Bottom Line: 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.Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy.

View Article: PubMed Central - PubMed

Affiliation: a Department of Degenerative Neurological Diseases , National Institute of Neuroscience, National Center of Neurology and Psychiatry , Kodaira , Tokyo , Japan.

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.

No MeSH data available.


Related in: MedlinePlus