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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 overexpression on RNA uptake and degradation by lysosomes. (A and B) Outlines of RNA uptake assays (A) and RNA degradation assays (B) using isolated lysosomes. (C) SIDT2 was overexpressed in Neuro2a cells. Protein levels were analyzed by immunoblotting using a goat anti-SIDT2 antibody. (D) The RNA uptake assay I indicated in (A) was performed using 5 μg of total RNA derived from mouse brains and isolated lysosomes derived from cells overexpressing SIDT2, or from control cells transfected with empty vector. Relative RNA levels in the solution outside lysosomes were quantified, and levels of RNA uptake were measured by subtracting RNA levels remaining in solution outside lysosomes from RNA input levels. Mean values are shown with SEM (n = 3). ***, P < 0.001. (E) RNA uptake assay II was performed as indicated in (A). Relative levels of RNA resistant to exogenous RNase A were analyzed. Mean ± SEM (n = 3). *, P < 0.05. (F) Isolated lysosomes were incubated with RNA and ATP as indicated in (A). Post-embedding immunoelectron microscopy was performed using an anti-rRNA antibody followed by anti-mouse IgG coupled with 10-nm gold particles. Gold particles were observed in the lysosomes. The numbers of gold particles per lysosome were counted. Mean ± SD (n = 25). ***, P < 0.001. Scale bars: 200 nm. (G) RNA degradation assays were performed as indicated in (B). Total RNA levels in samples were quantified, and levels of RNA degradation were measured by subtracting the RNA levels remaining in samples from the levels of input RNA. Mean ± SEM (n = 3). ***, P < 0.001. (H) Degradation of various RNAs by isolated lysosomes. RNA degradation assays were performed as described in Fig. 2B. Relative levels of RNAs in samples were measured by qPCR analyses. Mean values are shown with SEM (n = 3). Actb, β-actin. *, P < 0.05; **, P < 0.01; ***, P < 0.001, n.s., not significant (Tukey test or Fisher LSD test). (I) Degradation of 28S and 18S rRNAs by isolated lysosomes. RNA degradation assays were performed using total RNA that does not contain small RNAs (under 200 bases). Undegraded and partially degraded RNAs were visualized using ethidium bromide staining (left). Relative levels of rRNAs (28S and 18S) were quantified, and levels of RNA degradation were measured by subtracting the RNA remaining in samples from the levels of input RNA (middle). Relative levels of partially degraded RNAs were quantified (right). Mean values are shown with SEM (n = 3). **, P < 0.01. (J) RNAs were not degraded in the solution outside of lysosomes. Isolated lysosomes were incubated with ATP for 5 min at 37°C. The lysosomes were removed by centrifugation, and the solution outside lysosomes was incubated with 5 μg of total RNA for 5 min at 37°C. Mean values are shown with SEM (n = 3). n.s., not significant. RNAs were visualized by ethidium bromide staining. (K) RNA degradation by lysed lysosomes. Isolated lysosomes were lysed in citrate-phosphate buffer (pH 5.0) containing 1% Triton X-100, mixed with 5 μg of total RNA, and incubated for 5 min at 37°C. Mean values are shown with SEM (n = 3). n.s., not significant. (L) Absence of RNA in isolated lysosomes incubated without exogenous RNA. Isolated lysosomes were incubated without exogenous RNA in the presence of ATP for 5 min at 37°C. (M) ATP requirement of RNautophagy. RNA degradation assays were performed in the absence of ATP. Total RNA levels in samples were quantified. Mean values are shown with SEM (n = 3). n.s., not significant.
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f0002: Effects of SIDT2 overexpression on RNA uptake and degradation by lysosomes. (A and B) Outlines of RNA uptake assays (A) and RNA degradation assays (B) using isolated lysosomes. (C) SIDT2 was overexpressed in Neuro2a cells. Protein levels were analyzed by immunoblotting using a goat anti-SIDT2 antibody. (D) The RNA uptake assay I indicated in (A) was performed using 5 μg of total RNA derived from mouse brains and isolated lysosomes derived from cells overexpressing SIDT2, or from control cells transfected with empty vector. Relative RNA levels in the solution outside lysosomes were quantified, and levels of RNA uptake were measured by subtracting RNA levels remaining in solution outside lysosomes from RNA input levels. Mean values are shown with SEM (n = 3). ***, P < 0.001. (E) RNA uptake assay II was performed as indicated in (A). Relative levels of RNA resistant to exogenous RNase A were analyzed. Mean ± SEM (n = 3). *, P < 0.05. (F) Isolated lysosomes were incubated with RNA and ATP as indicated in (A). Post-embedding immunoelectron microscopy was performed using an anti-rRNA antibody followed by anti-mouse IgG coupled with 10-nm gold particles. Gold particles were observed in the lysosomes. The numbers of gold particles per lysosome were counted. Mean ± SD (n = 25). ***, P < 0.001. Scale bars: 200 nm. (G) RNA degradation assays were performed as indicated in (B). Total RNA levels in samples were quantified, and levels of RNA degradation were measured by subtracting the RNA levels remaining in samples from the levels of input RNA. Mean ± SEM (n = 3). ***, P < 0.001. (H) Degradation of various RNAs by isolated lysosomes. RNA degradation assays were performed as described in Fig. 2B. Relative levels of RNAs in samples were measured by qPCR analyses. Mean values are shown with SEM (n = 3). Actb, β-actin. *, P < 0.05; **, P < 0.01; ***, P < 0.001, n.s., not significant (Tukey test or Fisher LSD test). (I) Degradation of 28S and 18S rRNAs by isolated lysosomes. RNA degradation assays were performed using total RNA that does not contain small RNAs (under 200 bases). Undegraded and partially degraded RNAs were visualized using ethidium bromide staining (left). Relative levels of rRNAs (28S and 18S) were quantified, and levels of RNA degradation were measured by subtracting the RNA remaining in samples from the levels of input RNA (middle). Relative levels of partially degraded RNAs were quantified (right). Mean values are shown with SEM (n = 3). **, P < 0.01. (J) RNAs were not degraded in the solution outside of lysosomes. Isolated lysosomes were incubated with ATP for 5 min at 37°C. The lysosomes were removed by centrifugation, and the solution outside lysosomes was incubated with 5 μg of total RNA for 5 min at 37°C. Mean values are shown with SEM (n = 3). n.s., not significant. RNAs were visualized by ethidium bromide staining. (K) RNA degradation by lysed lysosomes. Isolated lysosomes were lysed in citrate-phosphate buffer (pH 5.0) containing 1% Triton X-100, mixed with 5 μg of total RNA, and incubated for 5 min at 37°C. Mean values are shown with SEM (n = 3). n.s., not significant. (L) Absence of RNA in isolated lysosomes incubated without exogenous RNA. Isolated lysosomes were incubated without exogenous RNA in the presence of ATP for 5 min at 37°C. (M) ATP requirement of RNautophagy. RNA degradation assays were performed in the absence of ATP. Total RNA levels in samples were quantified. Mean values are shown with SEM (n = 3). n.s., not significant.

Mentions: To investigate whether SIDT2 mediates translocation of RNA in the process of RNautophagy, we performed gain- and loss-of-function studies combined with RNA uptake and degradation assays. Uptake and degradation of RNA was assessed in isolated lysosomes using a method reported previously.3 Isolated lysosomes and RNA were incubated with ATP, lysosomes were precipitated by centrifugation, and RNA levels remaining in the solution outside of lysosomes were analyzed as an indicator of RNA uptake activity3 (Fig. 2A). RNA uptake activity was also assessed by incubating lysosomes with RNA and ATP, degrading RNA outside of lysosomes with exogenous ribonuclease A (RNase A), and then analyzing the levels of RNase A-resistant RNA, which corresponds to RNA inside lysosomes (Fig. 2A). Furthermore, RNA uptake activity was confirmed using postembedding immunoelectron microscopy.Figure 2.


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 overexpression on RNA uptake and degradation by lysosomes. (A and B) Outlines of RNA uptake assays (A) and RNA degradation assays (B) using isolated lysosomes. (C) SIDT2 was overexpressed in Neuro2a cells. Protein levels were analyzed by immunoblotting using a goat anti-SIDT2 antibody. (D) The RNA uptake assay I indicated in (A) was performed using 5 μg of total RNA derived from mouse brains and isolated lysosomes derived from cells overexpressing SIDT2, or from control cells transfected with empty vector. Relative RNA levels in the solution outside lysosomes were quantified, and levels of RNA uptake were measured by subtracting RNA levels remaining in solution outside lysosomes from RNA input levels. Mean values are shown with SEM (n = 3). ***, P < 0.001. (E) RNA uptake assay II was performed as indicated in (A). Relative levels of RNA resistant to exogenous RNase A were analyzed. Mean ± SEM (n = 3). *, P < 0.05. (F) Isolated lysosomes were incubated with RNA and ATP as indicated in (A). Post-embedding immunoelectron microscopy was performed using an anti-rRNA antibody followed by anti-mouse IgG coupled with 10-nm gold particles. Gold particles were observed in the lysosomes. The numbers of gold particles per lysosome were counted. Mean ± SD (n = 25). ***, P < 0.001. Scale bars: 200 nm. (G) RNA degradation assays were performed as indicated in (B). Total RNA levels in samples were quantified, and levels of RNA degradation were measured by subtracting the RNA levels remaining in samples from the levels of input RNA. Mean ± SEM (n = 3). ***, P < 0.001. (H) Degradation of various RNAs by isolated lysosomes. RNA degradation assays were performed as described in Fig. 2B. Relative levels of RNAs in samples were measured by qPCR analyses. Mean values are shown with SEM (n = 3). Actb, β-actin. *, P < 0.05; **, P < 0.01; ***, P < 0.001, n.s., not significant (Tukey test or Fisher LSD test). (I) Degradation of 28S and 18S rRNAs by isolated lysosomes. RNA degradation assays were performed using total RNA that does not contain small RNAs (under 200 bases). Undegraded and partially degraded RNAs were visualized using ethidium bromide staining (left). Relative levels of rRNAs (28S and 18S) were quantified, and levels of RNA degradation were measured by subtracting the RNA remaining in samples from the levels of input RNA (middle). Relative levels of partially degraded RNAs were quantified (right). Mean values are shown with SEM (n = 3). **, P < 0.01. (J) RNAs were not degraded in the solution outside of lysosomes. Isolated lysosomes were incubated with ATP for 5 min at 37°C. The lysosomes were removed by centrifugation, and the solution outside lysosomes was incubated with 5 μg of total RNA for 5 min at 37°C. Mean values are shown with SEM (n = 3). n.s., not significant. RNAs were visualized by ethidium bromide staining. (K) RNA degradation by lysed lysosomes. Isolated lysosomes were lysed in citrate-phosphate buffer (pH 5.0) containing 1% Triton X-100, mixed with 5 μg of total RNA, and incubated for 5 min at 37°C. Mean values are shown with SEM (n = 3). n.s., not significant. (L) Absence of RNA in isolated lysosomes incubated without exogenous RNA. Isolated lysosomes were incubated without exogenous RNA in the presence of ATP for 5 min at 37°C. (M) ATP requirement of RNautophagy. RNA degradation assays were performed in the absence of ATP. Total RNA levels in samples were quantified. Mean values are shown with SEM (n = 3). n.s., not significant.
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f0002: Effects of SIDT2 overexpression on RNA uptake and degradation by lysosomes. (A and B) Outlines of RNA uptake assays (A) and RNA degradation assays (B) using isolated lysosomes. (C) SIDT2 was overexpressed in Neuro2a cells. Protein levels were analyzed by immunoblotting using a goat anti-SIDT2 antibody. (D) The RNA uptake assay I indicated in (A) was performed using 5 μg of total RNA derived from mouse brains and isolated lysosomes derived from cells overexpressing SIDT2, or from control cells transfected with empty vector. Relative RNA levels in the solution outside lysosomes were quantified, and levels of RNA uptake were measured by subtracting RNA levels remaining in solution outside lysosomes from RNA input levels. Mean values are shown with SEM (n = 3). ***, P < 0.001. (E) RNA uptake assay II was performed as indicated in (A). Relative levels of RNA resistant to exogenous RNase A were analyzed. Mean ± SEM (n = 3). *, P < 0.05. (F) Isolated lysosomes were incubated with RNA and ATP as indicated in (A). Post-embedding immunoelectron microscopy was performed using an anti-rRNA antibody followed by anti-mouse IgG coupled with 10-nm gold particles. Gold particles were observed in the lysosomes. The numbers of gold particles per lysosome were counted. Mean ± SD (n = 25). ***, P < 0.001. Scale bars: 200 nm. (G) RNA degradation assays were performed as indicated in (B). Total RNA levels in samples were quantified, and levels of RNA degradation were measured by subtracting the RNA levels remaining in samples from the levels of input RNA. Mean ± SEM (n = 3). ***, P < 0.001. (H) Degradation of various RNAs by isolated lysosomes. RNA degradation assays were performed as described in Fig. 2B. Relative levels of RNAs in samples were measured by qPCR analyses. Mean values are shown with SEM (n = 3). Actb, β-actin. *, P < 0.05; **, P < 0.01; ***, P < 0.001, n.s., not significant (Tukey test or Fisher LSD test). (I) Degradation of 28S and 18S rRNAs by isolated lysosomes. RNA degradation assays were performed using total RNA that does not contain small RNAs (under 200 bases). Undegraded and partially degraded RNAs were visualized using ethidium bromide staining (left). Relative levels of rRNAs (28S and 18S) were quantified, and levels of RNA degradation were measured by subtracting the RNA remaining in samples from the levels of input RNA (middle). Relative levels of partially degraded RNAs were quantified (right). Mean values are shown with SEM (n = 3). **, P < 0.01. (J) RNAs were not degraded in the solution outside of lysosomes. Isolated lysosomes were incubated with ATP for 5 min at 37°C. The lysosomes were removed by centrifugation, and the solution outside lysosomes was incubated with 5 μg of total RNA for 5 min at 37°C. Mean values are shown with SEM (n = 3). n.s., not significant. RNAs were visualized by ethidium bromide staining. (K) RNA degradation by lysed lysosomes. Isolated lysosomes were lysed in citrate-phosphate buffer (pH 5.0) containing 1% Triton X-100, mixed with 5 μg of total RNA, and incubated for 5 min at 37°C. Mean values are shown with SEM (n = 3). n.s., not significant. (L) Absence of RNA in isolated lysosomes incubated without exogenous RNA. Isolated lysosomes were incubated without exogenous RNA in the presence of ATP for 5 min at 37°C. (M) ATP requirement of RNautophagy. RNA degradation assays were performed in the absence of ATP. Total RNA levels in samples were quantified. Mean values are shown with SEM (n = 3). n.s., not significant.
Mentions: To investigate whether SIDT2 mediates translocation of RNA in the process of RNautophagy, we performed gain- and loss-of-function studies combined with RNA uptake and degradation assays. Uptake and degradation of RNA was assessed in isolated lysosomes using a method reported previously.3 Isolated lysosomes and RNA were incubated with ATP, lysosomes were precipitated by centrifugation, and RNA levels remaining in the solution outside of lysosomes were analyzed as an indicator of RNA uptake activity3 (Fig. 2A). RNA uptake activity was also assessed by incubating lysosomes with RNA and ATP, degrading RNA outside of lysosomes with exogenous ribonuclease A (RNase A), and then analyzing the levels of RNase A-resistant RNA, which corresponds to RNA inside lysosomes (Fig. 2A). Furthermore, RNA uptake activity was confirmed using postembedding immunoelectron microscopy.Figure 2.

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