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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

Effects of Sidt2 knockdown on cellular RNA degradation. (A) Experimental paradigm for monitoring the degradation of cellular RNA. CQ, chloroquine. (B, F, H and K) Decreased levels of SIDT2 proteins in atg5 KO MEFs and in WT MEFs transfected with Sidt2-siRNA were confirmed by immunoblotting. Mean ± SEM (n = 4). ***, P < 0.001. * indicates nonspecific bands which are not decreased by Sidt2 knockdown. (C) RNA turnover in atg5 KO MEFs cells, transfected as indicated, was measured as described in (A, upper panel) and Materials and Methods. Results are expressed as mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant, compared with 0 h. §§§P < 0.001, compared with time-matched control. In control and Sidt2-knockdown cells, 37.3 ± 0.6 and 21.1 ± 0.6 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively, and 20.9 ± 3.1 and 5.5 ± 1.3 (mean ± SEM) % during 6 h, respectively. (D) No conversion of MAP1LC3A/B-I to MAP1LC3A/B-II in atg5 KO MEFs was confirmed by immunoblotting. (E) RNA turnover in atg5 KO MEFs cells, transfected as indicated, with or without CQ was measured as described in (A, lower panel) and Materials and Methods. Mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant. In control and Sidt2-knockdown cells without CQ treatment, 40.1 ± 2.0 and 26.2 ± 0.6 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. In control and Sidt2-knockdown cells with CQ treatment, 24.7 ± 2.0 and 20.9 ± 0.9 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. (G) RNA turnover in WT MEFs, transfected as indicated, with or without CQ were measured as described in (E). Mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant. In control and Sidt2-knockdown cells without CQ treatment, 43.8 ± 2.6 and 21.8 ± 1.9 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. Contribution of SIDT2 for total cellular RNA degradation was calculated to be 50.2%. In control and Sidt2-knockdown cells with CQ treatment, 23.7 ± 2.1 and 16.6 ± 1.8 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. (I) RNA turnover in atg5 KO MEFs cells, transfected as indicated, was measured as described in (C). Results are expressed as mean ± SEM (n = 4). ***, P < 0.001; n.s., not significant, compared with 0 h. §§§, P < 0.001, compared with time-matched control. (J) RNA turnover in atg5 KO MEFs cells, transfected as indicated, with or without CQ was measured as described in (E). Mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant. (L) RNA turnover in WT MEFs, transfected as indicated, with or without CQ were measured as described in (E). Mean ± SEM (n = 4). ***, P < 0.001; n.s., not significant. (M and N) Macroautophagic flux assay was performed as described in Materials and Methods. Mean ± SEM (n = 3). n.s., not significant. (O) WT MEFs were transfected with siRNAs as indicated, and labeled with [3H]-uridine for 24 h. Then, acid-soluble radioactivity of cells was measured as described in Materials and Methods. Mean ± SEM (n = 4). ***, P < 0.001.
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f0007: Effects of Sidt2 knockdown on cellular RNA degradation. (A) Experimental paradigm for monitoring the degradation of cellular RNA. CQ, chloroquine. (B, F, H and K) Decreased levels of SIDT2 proteins in atg5 KO MEFs and in WT MEFs transfected with Sidt2-siRNA were confirmed by immunoblotting. Mean ± SEM (n = 4). ***, P < 0.001. * indicates nonspecific bands which are not decreased by Sidt2 knockdown. (C) RNA turnover in atg5 KO MEFs cells, transfected as indicated, was measured as described in (A, upper panel) and Materials and Methods. Results are expressed as mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant, compared with 0 h. §§§P < 0.001, compared with time-matched control. In control and Sidt2-knockdown cells, 37.3 ± 0.6 and 21.1 ± 0.6 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively, and 20.9 ± 3.1 and 5.5 ± 1.3 (mean ± SEM) % during 6 h, respectively. (D) No conversion of MAP1LC3A/B-I to MAP1LC3A/B-II in atg5 KO MEFs was confirmed by immunoblotting. (E) RNA turnover in atg5 KO MEFs cells, transfected as indicated, with or without CQ was measured as described in (A, lower panel) and Materials and Methods. Mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant. In control and Sidt2-knockdown cells without CQ treatment, 40.1 ± 2.0 and 26.2 ± 0.6 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. In control and Sidt2-knockdown cells with CQ treatment, 24.7 ± 2.0 and 20.9 ± 0.9 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. (G) RNA turnover in WT MEFs, transfected as indicated, with or without CQ were measured as described in (E). Mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant. In control and Sidt2-knockdown cells without CQ treatment, 43.8 ± 2.6 and 21.8 ± 1.9 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. Contribution of SIDT2 for total cellular RNA degradation was calculated to be 50.2%. In control and Sidt2-knockdown cells with CQ treatment, 23.7 ± 2.1 and 16.6 ± 1.8 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. (I) RNA turnover in atg5 KO MEFs cells, transfected as indicated, was measured as described in (C). Results are expressed as mean ± SEM (n = 4). ***, P < 0.001; n.s., not significant, compared with 0 h. §§§, P < 0.001, compared with time-matched control. (J) RNA turnover in atg5 KO MEFs cells, transfected as indicated, with or without CQ was measured as described in (E). Mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant. (L) RNA turnover in WT MEFs, transfected as indicated, with or without CQ were measured as described in (E). Mean ± SEM (n = 4). ***, P < 0.001; n.s., not significant. (M and N) Macroautophagic flux assay was performed as described in Materials and Methods. Mean ± SEM (n = 3). n.s., not significant. (O) WT MEFs were transfected with siRNAs as indicated, and labeled with [3H]-uridine for 24 h. Then, acid-soluble radioactivity of cells was measured as described in Materials and Methods. Mean ± SEM (n = 4). ***, P < 0.001.

Mentions: A constitutive degradation of cellular components or proteins is one of the most important physiological roles of both macroautophagy and CMA.1,2 To determine whether endogenous SIDT2 plays a role in RNA degradation at the cellular level, endogenous RNA was labeled with [3H]-uridine in Sidt2 knockdown cells or control siRNA-transfected cells. The levels of labeled RNA in cells were measured at 0, 6 and 24 h (Fig. 7A, upper panel), with labeled RNA levels normalized to total protein levels. To exclude the involvement of macroautophagy, we performed pulse-chase experiments using the macroautophagy deficient atg5 (autophagy-related 5)-knockout (KO) MEFs. Our results show that RNA degradation was impaired in Sidt2 knockdown cells compared with that in control cells (Fig. 7B–C). We confirmed that the conversion of MAP1LC3A/B-I to MAP1LC3A/B-II was not observed in atg5 KO MEFs (Fig. D). To confirm that the effects of Sidt2-knockdown on radioactive uridine are due to impaired lysosomal degradation of RNA, we used chloroquine (CQ), which is an established inhibitor of lysosomal enzymes,20 in pulse-chase experiments (Fig. 7A, lower panel). If knockdown of Sidt2 impairs lysosomal degradation of RNA, effect of CQ on RNA degradation should be decreased in Sidt2-knockdown cells. CQ treatment inhibited RNA degradation in control knockdown cells, whereas it did not inhibit it in Sidt2-knockdown cells (Fig. 7E). This result supports the notion that Sidt2 knockdown impairs the lysosomal degradation of RNA in cells. In WT MEFs, knockdown of Sidt2 inhibited ˜50% of total RNA degradation (Fig. 7F–G), and pulse-chase experiments using CQ confirmed that knockdown of Sidt2 inhibits lysosomal degradation of RNA in these cells (Fig. 7G), suggesting that SIDT2-mediated RNA degradation, presumably RNautophagy, is a main pathway for constitutive lysosomal degradation of RNA in MEFs. Similar results were obtained when another siRNA against Sidt2 (Sidt2 siRNA-B) was used for knockdown (Fig. 7H–L). We confirmed that the macroautophagic flux, an indicator of activity of macroautophagy,20 is not significantly changed by knockdown of Sidt2 in WT MEFs (Fig. 7M–N). Thus, the effect of Sidt2 knockdown is independent of macroautophagy, and Sidt2 knockdown does not affect lysosomal enzymatic activity. Taken together, our findings indicate that SIDT2 is essential for normal levels of RNA degradation in cells, and strongly suggest that one of the physiological roles of RNautophagy is a constitutive degradation of cellular RNA.Figure 7.


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)

Effects of Sidt2 knockdown on cellular RNA degradation. (A) Experimental paradigm for monitoring the degradation of cellular RNA. CQ, chloroquine. (B, F, H and K) Decreased levels of SIDT2 proteins in atg5 KO MEFs and in WT MEFs transfected with Sidt2-siRNA were confirmed by immunoblotting. Mean ± SEM (n = 4). ***, P < 0.001. * indicates nonspecific bands which are not decreased by Sidt2 knockdown. (C) RNA turnover in atg5 KO MEFs cells, transfected as indicated, was measured as described in (A, upper panel) and Materials and Methods. Results are expressed as mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant, compared with 0 h. §§§P < 0.001, compared with time-matched control. In control and Sidt2-knockdown cells, 37.3 ± 0.6 and 21.1 ± 0.6 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively, and 20.9 ± 3.1 and 5.5 ± 1.3 (mean ± SEM) % during 6 h, respectively. (D) No conversion of MAP1LC3A/B-I to MAP1LC3A/B-II in atg5 KO MEFs was confirmed by immunoblotting. (E) RNA turnover in atg5 KO MEFs cells, transfected as indicated, with or without CQ was measured as described in (A, lower panel) and Materials and Methods. Mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant. In control and Sidt2-knockdown cells without CQ treatment, 40.1 ± 2.0 and 26.2 ± 0.6 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. In control and Sidt2-knockdown cells with CQ treatment, 24.7 ± 2.0 and 20.9 ± 0.9 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. (G) RNA turnover in WT MEFs, transfected as indicated, with or without CQ were measured as described in (E). Mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant. In control and Sidt2-knockdown cells without CQ treatment, 43.8 ± 2.6 and 21.8 ± 1.9 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. Contribution of SIDT2 for total cellular RNA degradation was calculated to be 50.2%. In control and Sidt2-knockdown cells with CQ treatment, 23.7 ± 2.1 and 16.6 ± 1.8 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. (I) RNA turnover in atg5 KO MEFs cells, transfected as indicated, was measured as described in (C). Results are expressed as mean ± SEM (n = 4). ***, P < 0.001; n.s., not significant, compared with 0 h. §§§, P < 0.001, compared with time-matched control. (J) RNA turnover in atg5 KO MEFs cells, transfected as indicated, with or without CQ was measured as described in (E). Mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant. (L) RNA turnover in WT MEFs, transfected as indicated, with or without CQ were measured as described in (E). Mean ± SEM (n = 4). ***, P < 0.001; n.s., not significant. (M and N) Macroautophagic flux assay was performed as described in Materials and Methods. Mean ± SEM (n = 3). n.s., not significant. (O) WT MEFs were transfected with siRNAs as indicated, and labeled with [3H]-uridine for 24 h. Then, acid-soluble radioactivity of cells was measured as described in Materials and Methods. Mean ± SEM (n = 4). ***, P < 0.001.
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f0007: Effects of Sidt2 knockdown on cellular RNA degradation. (A) Experimental paradigm for monitoring the degradation of cellular RNA. CQ, chloroquine. (B, F, H and K) Decreased levels of SIDT2 proteins in atg5 KO MEFs and in WT MEFs transfected with Sidt2-siRNA were confirmed by immunoblotting. Mean ± SEM (n = 4). ***, P < 0.001. * indicates nonspecific bands which are not decreased by Sidt2 knockdown. (C) RNA turnover in atg5 KO MEFs cells, transfected as indicated, was measured as described in (A, upper panel) and Materials and Methods. Results are expressed as mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant, compared with 0 h. §§§P < 0.001, compared with time-matched control. In control and Sidt2-knockdown cells, 37.3 ± 0.6 and 21.1 ± 0.6 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively, and 20.9 ± 3.1 and 5.5 ± 1.3 (mean ± SEM) % during 6 h, respectively. (D) No conversion of MAP1LC3A/B-I to MAP1LC3A/B-II in atg5 KO MEFs was confirmed by immunoblotting. (E) RNA turnover in atg5 KO MEFs cells, transfected as indicated, with or without CQ was measured as described in (A, lower panel) and Materials and Methods. Mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant. In control and Sidt2-knockdown cells without CQ treatment, 40.1 ± 2.0 and 26.2 ± 0.6 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. In control and Sidt2-knockdown cells with CQ treatment, 24.7 ± 2.0 and 20.9 ± 0.9 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. (G) RNA turnover in WT MEFs, transfected as indicated, with or without CQ were measured as described in (E). Mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant. In control and Sidt2-knockdown cells without CQ treatment, 43.8 ± 2.6 and 21.8 ± 1.9 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. Contribution of SIDT2 for total cellular RNA degradation was calculated to be 50.2%. In control and Sidt2-knockdown cells with CQ treatment, 23.7 ± 2.1 and 16.6 ± 1.8 (mean ± SEM) % of RNA was calculated to be degraded during 24 h, respectively. (I) RNA turnover in atg5 KO MEFs cells, transfected as indicated, was measured as described in (C). Results are expressed as mean ± SEM (n = 4). ***, P < 0.001; n.s., not significant, compared with 0 h. §§§, P < 0.001, compared with time-matched control. (J) RNA turnover in atg5 KO MEFs cells, transfected as indicated, with or without CQ was measured as described in (E). Mean ± SEM (n = 4). ***, P < 0.001, n.s., not significant. (L) RNA turnover in WT MEFs, transfected as indicated, with or without CQ were measured as described in (E). Mean ± SEM (n = 4). ***, P < 0.001; n.s., not significant. (M and N) Macroautophagic flux assay was performed as described in Materials and Methods. Mean ± SEM (n = 3). n.s., not significant. (O) WT MEFs were transfected with siRNAs as indicated, and labeled with [3H]-uridine for 24 h. Then, acid-soluble radioactivity of cells was measured as described in Materials and Methods. Mean ± SEM (n = 4). ***, P < 0.001.
Mentions: A constitutive degradation of cellular components or proteins is one of the most important physiological roles of both macroautophagy and CMA.1,2 To determine whether endogenous SIDT2 plays a role in RNA degradation at the cellular level, endogenous RNA was labeled with [3H]-uridine in Sidt2 knockdown cells or control siRNA-transfected cells. The levels of labeled RNA in cells were measured at 0, 6 and 24 h (Fig. 7A, upper panel), with labeled RNA levels normalized to total protein levels. To exclude the involvement of macroautophagy, we performed pulse-chase experiments using the macroautophagy deficient atg5 (autophagy-related 5)-knockout (KO) MEFs. Our results show that RNA degradation was impaired in Sidt2 knockdown cells compared with that in control cells (Fig. 7B–C). We confirmed that the conversion of MAP1LC3A/B-I to MAP1LC3A/B-II was not observed in atg5 KO MEFs (Fig. D). To confirm that the effects of Sidt2-knockdown on radioactive uridine are due to impaired lysosomal degradation of RNA, we used chloroquine (CQ), which is an established inhibitor of lysosomal enzymes,20 in pulse-chase experiments (Fig. 7A, lower panel). If knockdown of Sidt2 impairs lysosomal degradation of RNA, effect of CQ on RNA degradation should be decreased in Sidt2-knockdown cells. CQ treatment inhibited RNA degradation in control knockdown cells, whereas it did not inhibit it in Sidt2-knockdown cells (Fig. 7E). This result supports the notion that Sidt2 knockdown impairs the lysosomal degradation of RNA in cells. In WT MEFs, knockdown of Sidt2 inhibited ˜50% of total RNA degradation (Fig. 7F–G), and pulse-chase experiments using CQ confirmed that knockdown of Sidt2 inhibits lysosomal degradation of RNA in these cells (Fig. 7G), suggesting that SIDT2-mediated RNA degradation, presumably RNautophagy, is a main pathway for constitutive lysosomal degradation of RNA in MEFs. Similar results were obtained when another siRNA against Sidt2 (Sidt2 siRNA-B) was used for knockdown (Fig. 7H–L). We confirmed that the macroautophagic flux, an indicator of activity of macroautophagy,20 is not significantly changed by knockdown of Sidt2 in WT MEFs (Fig. 7M–N). Thus, the effect of Sidt2 knockdown is independent of macroautophagy, and Sidt2 knockdown does not affect lysosomal enzymatic activity. Taken together, our findings indicate that SIDT2 is essential for normal levels of RNA degradation in cells, and strongly suggest that one of the physiological roles of RNautophagy is a constitutive degradation of cellular RNA.Figure 7.

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