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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 SIDT2 mutation on RNautophagy. (A) Neuro2a cells expressing GFP-tagged SIDT2S564A 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 and D) Lysosomes were isolated from Neuro2a cells overexpressing WT or mutant SIDT2S564A or control transfectants. SIDT2 levels in lysosomes were analyzed by immunoblotting (B). The RNA uptake assay I indicated in Fig. 2A was performed (D). Relative levels of RNA uptake were quantified. Mean ± SEM (n = 3). ***, P < 0.001. (C) LAMP2C or LAMP2C-FLAG and WT or mutant SIDT2S564A were overexpressed in Neuro2a cells. Cell lysates were prepared and immunoprecipitated with an anti-FLAG antibody. Cell lysates and the resulting immunoprecipitant were analyzed by immunoblotting.
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f0004: Effect of SIDT2 mutation on RNautophagy. (A) Neuro2a cells expressing GFP-tagged SIDT2S564A 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 and D) Lysosomes were isolated from Neuro2a cells overexpressing WT or mutant SIDT2S564A or control transfectants. SIDT2 levels in lysosomes were analyzed by immunoblotting (B). The RNA uptake assay I indicated in Fig. 2A was performed (D). Relative levels of RNA uptake were quantified. Mean ± SEM (n = 3). ***, P < 0.001. (C) LAMP2C or LAMP2C-FLAG and WT or mutant SIDT2S564A were overexpressed in Neuro2a cells. Cell lysates were prepared and immunoprecipitated with an anti-FLAG antibody. Cell lysates and the resulting immunoprecipitant were analyzed by immunoblotting.

Mentions: Shih and Hunter report that the S536I mutant SID-1, which is found in a systemic RNA interference-deficient mutant in Caenorhabditis elegans,19 lacks RNA translocation function,8,9 and the Ser536 of SID-1 corresponds to Ser564 of mouse SIDT2 (Fig. S6). Therefore, we speculated that a mutation at Ser564 inhibits lysosomal RNA uptake by SIDT2. We constructed a S564A mutant SIDT2, and tested the effects of this mutation on RNA uptake by lysosomes. We observed that SIDT2S564A localizes to lysosomes as readily as WT SIDT2 (Fig. 4A–B), and that the mutation does not affect the interaction with LAMP2C (Fig. 4C). In contrast to WT SIDT2, however, overexpression of SIDT2S564A did not increase RNautophagy (Fig. 4D), indicating that functional SIDT2 protein is required to import RNAs into lysosomes. Taken together, our results support the notion that SIDT2 mediates RNA translocation during RNautophagy.Figure 4.


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 SIDT2 mutation on RNautophagy. (A) Neuro2a cells expressing GFP-tagged SIDT2S564A 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 and D) Lysosomes were isolated from Neuro2a cells overexpressing WT or mutant SIDT2S564A or control transfectants. SIDT2 levels in lysosomes were analyzed by immunoblotting (B). The RNA uptake assay I indicated in Fig. 2A was performed (D). Relative levels of RNA uptake were quantified. Mean ± SEM (n = 3). ***, P < 0.001. (C) LAMP2C or LAMP2C-FLAG and WT or mutant SIDT2S564A were overexpressed in Neuro2a cells. Cell lysates were prepared and immunoprecipitated with an anti-FLAG antibody. Cell lysates and the resulting immunoprecipitant were analyzed by immunoblotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f0004: Effect of SIDT2 mutation on RNautophagy. (A) Neuro2a cells expressing GFP-tagged SIDT2S564A 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 and D) Lysosomes were isolated from Neuro2a cells overexpressing WT or mutant SIDT2S564A or control transfectants. SIDT2 levels in lysosomes were analyzed by immunoblotting (B). The RNA uptake assay I indicated in Fig. 2A was performed (D). Relative levels of RNA uptake were quantified. Mean ± SEM (n = 3). ***, P < 0.001. (C) LAMP2C or LAMP2C-FLAG and WT or mutant SIDT2S564A were overexpressed in Neuro2a cells. Cell lysates were prepared and immunoprecipitated with an anti-FLAG antibody. Cell lysates and the resulting immunoprecipitant were analyzed by immunoblotting.
Mentions: Shih and Hunter report that the S536I mutant SID-1, which is found in a systemic RNA interference-deficient mutant in Caenorhabditis elegans,19 lacks RNA translocation function,8,9 and the Ser536 of SID-1 corresponds to Ser564 of mouse SIDT2 (Fig. S6). Therefore, we speculated that a mutation at Ser564 inhibits lysosomal RNA uptake by SIDT2. We constructed a S564A mutant SIDT2, and tested the effects of this mutation on RNA uptake by lysosomes. We observed that SIDT2S564A localizes to lysosomes as readily as WT SIDT2 (Fig. 4A–B), and that the mutation does not affect the interaction with LAMP2C (Fig. 4C). In contrast to WT SIDT2, however, overexpression of SIDT2S564A did not increase RNautophagy (Fig. 4D), indicating that functional SIDT2 protein is required to import RNAs into lysosomes. Taken together, our results support the notion that SIDT2 mediates RNA translocation during RNautophagy.Figure 4.

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