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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

Characterization of SIDT2. (A) Lysosomes (Lys) were isolated from mouse brain homogenates (Hom), and analyzed by immunoblotting using polyclonal goat anti-SIDT2 antibody and antibodies against LAMP2 (lysosomal marker), RAB7A (late endosome and lysosome), RAB5A (early endosome), CANX (endoplasmic reticulum), COX4I1 (mitochondria), GOLGA1 (Golgi apparatus), GAPDH (cytosol), LMNA/lamin A (nuclei), and MAP1LC3A/B (autophagosome). (B) Neuro2a cells expressing GFP-tagged SIDT2 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 μm. Colocalization rate was quantified using ImageJ software (right panel, n = 3). (C) Neuro2a cells expressing GFP-tagged SIDT2 were fixed, and immunostained using anti-RAB7A, anti-EEA1 (early endosomal marker) or anti-MAP1LC3A/B antibodies. Fluorescent images were obtained using confocal microscopy. Scale bars: 5 μm. Colocalization rate was quantified (right panels, n = 3). (D) Lysosomes were isolated from HeLa cells expressing SIDT2-FLAG or CTSB-FLAG. Isolated lysosomes (4 μg protein) were incubated with the indicated concentrations of trypsin at 37°C for 5 min. Proteins in the samples were analyzed by immunoblotting using an anti-FLAG antibody. (E) LAMP2C and SIDT2 or SIDT2-FLAG were overexpressed in HeLa cells. Cell lysates were prepared and immunoprecipitated with an anti-FLAG antibody. Cell lysates and the resulting immunoprecipitant were analyzed by immunoblotting. (F) Lysates were prepared from HeLa cells overexpressing SIDT2 and LAMP2C or LAMP2C-FLAG and coimmunoprecipitation assays performed. (G) Endogenous interaction of SIDT2 with LAMP2C. Coimmunoprecipitation assays were performed using mouse brain lysates. (H) Neuro2a cells coexpressing FLAG-tagged LAMP2C and GFP-tagged SIDT2 were fixed, and immunostained using anti-FLAG antibody. Scale bars: 10 μm. Colocalization rate was quantified (right panel, n = 3).
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f0001: Characterization of SIDT2. (A) Lysosomes (Lys) were isolated from mouse brain homogenates (Hom), and analyzed by immunoblotting using polyclonal goat anti-SIDT2 antibody and antibodies against LAMP2 (lysosomal marker), RAB7A (late endosome and lysosome), RAB5A (early endosome), CANX (endoplasmic reticulum), COX4I1 (mitochondria), GOLGA1 (Golgi apparatus), GAPDH (cytosol), LMNA/lamin A (nuclei), and MAP1LC3A/B (autophagosome). (B) Neuro2a cells expressing GFP-tagged SIDT2 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 μm. Colocalization rate was quantified using ImageJ software (right panel, n = 3). (C) Neuro2a cells expressing GFP-tagged SIDT2 were fixed, and immunostained using anti-RAB7A, anti-EEA1 (early endosomal marker) or anti-MAP1LC3A/B antibodies. Fluorescent images were obtained using confocal microscopy. Scale bars: 5 μm. Colocalization rate was quantified (right panels, n = 3). (D) Lysosomes were isolated from HeLa cells expressing SIDT2-FLAG or CTSB-FLAG. Isolated lysosomes (4 μg protein) were incubated with the indicated concentrations of trypsin at 37°C for 5 min. Proteins in the samples were analyzed by immunoblotting using an anti-FLAG antibody. (E) LAMP2C and SIDT2 or SIDT2-FLAG were overexpressed in HeLa cells. Cell lysates were prepared and immunoprecipitated with an anti-FLAG antibody. Cell lysates and the resulting immunoprecipitant were analyzed by immunoblotting. (F) Lysates were prepared from HeLa cells overexpressing SIDT2 and LAMP2C or LAMP2C-FLAG and coimmunoprecipitation assays performed. (G) Endogenous interaction of SIDT2 with LAMP2C. Coimmunoprecipitation assays were performed using mouse brain lysates. (H) Neuro2a cells coexpressing FLAG-tagged LAMP2C and GFP-tagged SIDT2 were fixed, and immunostained using anti-FLAG antibody. Scale bars: 10 μm. Colocalization rate was quantified (right panel, n = 3).

Mentions: Candidate lysosomal proteins that might translocate RNA or DNA were identified using the AmiGO gene ontology database,7 which was designed for the standardization of gene product attributes across species. According to AmiGO, the SID-1 family proteins (SID-1, SIDT1 [SID1 transmembrane family, member 1] and SIDT2) are the only group of putative RNA-specific transporters. Caenorhabditis elegans SID-1 protein has been reported to transport extracellular double-stranded RNA (dsRNA) into cells.8 SID-1 is a multipass transmembrane protein and partly localizes to the plasma membrane.8 An electrophysiological study suggested that SID-1 functions as a bidirectional channel for dsRNA.9 Mammals have 2 SID-1 orthologs, SIDT1 and SIDT2. SIDT1 has been reported to localize to the plasma membrane of human cells and to mediate bidirectional transport of RNA.10,11 Human SIDT1 is predominantly expressed in dendritic cells and lymphocytes,12 whereas SIDT2 is almost ubiquitously expressed.12,13 Although multiple studies have reported that SIDT2 is a membrane protein which mainly localizes to lysosomes,13-15 its role on the lysosomal membrane remains unclear. To confirm the lysosomal localization of SIDT2, we isolated lysosomes from the mouse brain using a method described previously.3 Analysis of lysosome content showed that SIDT2 is enriched in the lysosomal fraction that is positive for LAMP2, a lysosomal marker (Fig. 1A), indicating that endogenous SIDT2 localizes to lysosomes. We confirmed that other organelle markers are not enriched in the lysosomal fraction (Fig. 1A). In addition, electron microscopy revealed that the lysosomal fraction is rich in electron-dense lysosomes (primary lysosomes), and we could not find any other intact organelles, such as late endosomes, in the lysosomal fraction (Fig. S1). For further confirmation of the lysosomal localization of SIDT2, we examined its localization using a C-terminal GFP-tag in Neuro2a murine cells, where lysosomal compartments are clearly observable with LysoTracker Red. Fluorescent signals for SIDT2 were detected in lysosomes, which were stained with LysoTracker Red (Fig. 1B). In contrast, colocalization rates of SIDT2 with RAB7A (RAB7A, member RAS oncogene family; a late endosomal and lysosomal marker), EEA1 (early endosome antigen 1; an early endosomal marker) or MAP1LC3A/B (microtubule associated protein 1 light chain 3 α/β an autophagosomal marker) were very low (Fig. 1C), indicating that SIDT2 is not primarily localized to early and late endosomes, or autophagosomes. Considering that there are RAB7A-positive and -negative lysosomes,16-18 our results also indicate that SIDT2 does not mainly localize to RAB7-positive lysosomes. By using a transmembrane protein topology prediction method based on a hidden Markov model (TMHMM 2.0), SIDT2 was predicted to possess 9 transmembrane domains (Fig. S2). To test for the localization of SIDT2 on the lysosomal membrane, we performed a trypsin digestion of membrane proteins of lysosomes isolated from cells expressing FLAG-tagged SIDT2. As shown in Fig. 1D, SIDT2-FLAG was digested by trypsin in a dose-dependent manner. We confirmed that FLAG-tagged CTSB (cathepsin B), which is a known lysosomal lumenal protein, was not affected by trypsin treatment (Fig. 1D). Our results, taken together with the previous reports,13-15 indicate that SIDT2 is a lysosomal membrane protein.Figure 1.


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)

Characterization of SIDT2. (A) Lysosomes (Lys) were isolated from mouse brain homogenates (Hom), and analyzed by immunoblotting using polyclonal goat anti-SIDT2 antibody and antibodies against LAMP2 (lysosomal marker), RAB7A (late endosome and lysosome), RAB5A (early endosome), CANX (endoplasmic reticulum), COX4I1 (mitochondria), GOLGA1 (Golgi apparatus), GAPDH (cytosol), LMNA/lamin A (nuclei), and MAP1LC3A/B (autophagosome). (B) Neuro2a cells expressing GFP-tagged SIDT2 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 μm. Colocalization rate was quantified using ImageJ software (right panel, n = 3). (C) Neuro2a cells expressing GFP-tagged SIDT2 were fixed, and immunostained using anti-RAB7A, anti-EEA1 (early endosomal marker) or anti-MAP1LC3A/B antibodies. Fluorescent images were obtained using confocal microscopy. Scale bars: 5 μm. Colocalization rate was quantified (right panels, n = 3). (D) Lysosomes were isolated from HeLa cells expressing SIDT2-FLAG or CTSB-FLAG. Isolated lysosomes (4 μg protein) were incubated with the indicated concentrations of trypsin at 37°C for 5 min. Proteins in the samples were analyzed by immunoblotting using an anti-FLAG antibody. (E) LAMP2C and SIDT2 or SIDT2-FLAG were overexpressed in HeLa cells. Cell lysates were prepared and immunoprecipitated with an anti-FLAG antibody. Cell lysates and the resulting immunoprecipitant were analyzed by immunoblotting. (F) Lysates were prepared from HeLa cells overexpressing SIDT2 and LAMP2C or LAMP2C-FLAG and coimmunoprecipitation assays performed. (G) Endogenous interaction of SIDT2 with LAMP2C. Coimmunoprecipitation assays were performed using mouse brain lysates. (H) Neuro2a cells coexpressing FLAG-tagged LAMP2C and GFP-tagged SIDT2 were fixed, and immunostained using anti-FLAG antibody. Scale bars: 10 μm. Colocalization rate was quantified (right panel, n = 3).
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f0001: Characterization of SIDT2. (A) Lysosomes (Lys) were isolated from mouse brain homogenates (Hom), and analyzed by immunoblotting using polyclonal goat anti-SIDT2 antibody and antibodies against LAMP2 (lysosomal marker), RAB7A (late endosome and lysosome), RAB5A (early endosome), CANX (endoplasmic reticulum), COX4I1 (mitochondria), GOLGA1 (Golgi apparatus), GAPDH (cytosol), LMNA/lamin A (nuclei), and MAP1LC3A/B (autophagosome). (B) Neuro2a cells expressing GFP-tagged SIDT2 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 μm. Colocalization rate was quantified using ImageJ software (right panel, n = 3). (C) Neuro2a cells expressing GFP-tagged SIDT2 were fixed, and immunostained using anti-RAB7A, anti-EEA1 (early endosomal marker) or anti-MAP1LC3A/B antibodies. Fluorescent images were obtained using confocal microscopy. Scale bars: 5 μm. Colocalization rate was quantified (right panels, n = 3). (D) Lysosomes were isolated from HeLa cells expressing SIDT2-FLAG or CTSB-FLAG. Isolated lysosomes (4 μg protein) were incubated with the indicated concentrations of trypsin at 37°C for 5 min. Proteins in the samples were analyzed by immunoblotting using an anti-FLAG antibody. (E) LAMP2C and SIDT2 or SIDT2-FLAG were overexpressed in HeLa cells. Cell lysates were prepared and immunoprecipitated with an anti-FLAG antibody. Cell lysates and the resulting immunoprecipitant were analyzed by immunoblotting. (F) Lysates were prepared from HeLa cells overexpressing SIDT2 and LAMP2C or LAMP2C-FLAG and coimmunoprecipitation assays performed. (G) Endogenous interaction of SIDT2 with LAMP2C. Coimmunoprecipitation assays were performed using mouse brain lysates. (H) Neuro2a cells coexpressing FLAG-tagged LAMP2C and GFP-tagged SIDT2 were fixed, and immunostained using anti-FLAG antibody. Scale bars: 10 μm. Colocalization rate was quantified (right panel, n = 3).
Mentions: Candidate lysosomal proteins that might translocate RNA or DNA were identified using the AmiGO gene ontology database,7 which was designed for the standardization of gene product attributes across species. According to AmiGO, the SID-1 family proteins (SID-1, SIDT1 [SID1 transmembrane family, member 1] and SIDT2) are the only group of putative RNA-specific transporters. Caenorhabditis elegans SID-1 protein has been reported to transport extracellular double-stranded RNA (dsRNA) into cells.8 SID-1 is a multipass transmembrane protein and partly localizes to the plasma membrane.8 An electrophysiological study suggested that SID-1 functions as a bidirectional channel for dsRNA.9 Mammals have 2 SID-1 orthologs, SIDT1 and SIDT2. SIDT1 has been reported to localize to the plasma membrane of human cells and to mediate bidirectional transport of RNA.10,11 Human SIDT1 is predominantly expressed in dendritic cells and lymphocytes,12 whereas SIDT2 is almost ubiquitously expressed.12,13 Although multiple studies have reported that SIDT2 is a membrane protein which mainly localizes to lysosomes,13-15 its role on the lysosomal membrane remains unclear. To confirm the lysosomal localization of SIDT2, we isolated lysosomes from the mouse brain using a method described previously.3 Analysis of lysosome content showed that SIDT2 is enriched in the lysosomal fraction that is positive for LAMP2, a lysosomal marker (Fig. 1A), indicating that endogenous SIDT2 localizes to lysosomes. We confirmed that other organelle markers are not enriched in the lysosomal fraction (Fig. 1A). In addition, electron microscopy revealed that the lysosomal fraction is rich in electron-dense lysosomes (primary lysosomes), and we could not find any other intact organelles, such as late endosomes, in the lysosomal fraction (Fig. S1). For further confirmation of the lysosomal localization of SIDT2, we examined its localization using a C-terminal GFP-tag in Neuro2a murine cells, where lysosomal compartments are clearly observable with LysoTracker Red. Fluorescent signals for SIDT2 were detected in lysosomes, which were stained with LysoTracker Red (Fig. 1B). In contrast, colocalization rates of SIDT2 with RAB7A (RAB7A, member RAS oncogene family; a late endosomal and lysosomal marker), EEA1 (early endosome antigen 1; an early endosomal marker) or MAP1LC3A/B (microtubule associated protein 1 light chain 3 α/β an autophagosomal marker) were very low (Fig. 1C), indicating that SIDT2 is not primarily localized to early and late endosomes, or autophagosomes. Considering that there are RAB7A-positive and -negative lysosomes,16-18 our results also indicate that SIDT2 does not mainly localize to RAB7-positive lysosomes. By using a transmembrane protein topology prediction method based on a hidden Markov model (TMHMM 2.0), SIDT2 was predicted to possess 9 transmembrane domains (Fig. S2). To test for the localization of SIDT2 on the lysosomal membrane, we performed a trypsin digestion of membrane proteins of lysosomes isolated from cells expressing FLAG-tagged SIDT2. As shown in Fig. 1D, SIDT2-FLAG was digested by trypsin in a dose-dependent manner. We confirmed that FLAG-tagged CTSB (cathepsin B), which is a known lysosomal lumenal protein, was not affected by trypsin treatment (Fig. 1D). Our results, taken together with the previous reports,13-15 indicate that SIDT2 is a lysosomal membrane protein.Figure 1.

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