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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 RNA uptake and degradation by lysosomes. (A and D) Decreased levels of SIDT2 proteins in HeLa cells transfected with SIDT2-siRNA were confirmed by immunoblotting. Relative levels of SIDT2 were quantified. Results are expressed as mean ± SEM (n = 3). (B and E) RNA uptake assay I (Fig. 2A) was performed using isolated lysosomes derived from SIDT2 knockdown or control siRNA-transfected cells. Relative levels of RNA uptake were quantified. Mean ± SEM (n = 3). ***, P < 0.001. (C and F) RNA degradation assay using lysosomes isolated from SIDT2-knockdown cells or from control cells. Relative levels of RNA degradation were quantified. Mean ± SEM (n = 3). ***, P < 0.001; **, P < 0.01.
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f0005: Effects of SIDT2 knockdown on RNA uptake and degradation by lysosomes. (A and D) Decreased levels of SIDT2 proteins in HeLa cells transfected with SIDT2-siRNA were confirmed by immunoblotting. Relative levels of SIDT2 were quantified. Results are expressed as mean ± SEM (n = 3). (B and E) RNA uptake assay I (Fig. 2A) was performed using isolated lysosomes derived from SIDT2 knockdown or control siRNA-transfected cells. Relative levels of RNA uptake were quantified. Mean ± SEM (n = 3). ***, P < 0.001. (C and F) RNA degradation assay using lysosomes isolated from SIDT2-knockdown cells or from control cells. Relative levels of RNA degradation were quantified. Mean ± SEM (n = 3). ***, P < 0.001; **, P < 0.01.

Mentions: To investigate the effects of SIDT2 knockdown on RNautophagy activity, HeLa cells were transfected with siRNAs targeting SIDT2. Lysosomes were isolated and RNA uptake assays were performed. Lysosomes isolated from SIDT2 knockdown cells showed significantly lower RNA uptake than lysosomes from control siRNA-transfected cells (Fig. 5A–B). A significant reduction of RNA degradation was observed in lysosomes isolated from SIDT2 knockdown cells (Fig. 5C). Similar results were obtained when we used another siRNA against SIDT2 (Fig. 5D–F). We confirmed that knockdown of SIDT2 did not affect lysosomal pH (Fig. S4). The impairment of RNA uptake and degradation by SIDT2 knockdown was also confirmed in the 293FT cell line (Fig. S7). Collectively, these results demonstrate that SIDT2 is a critical determinant of RNautophagy activity.Figure 5.


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 RNA uptake and degradation by lysosomes. (A and D) Decreased levels of SIDT2 proteins in HeLa cells transfected with SIDT2-siRNA were confirmed by immunoblotting. Relative levels of SIDT2 were quantified. Results are expressed as mean ± SEM (n = 3). (B and E) RNA uptake assay I (Fig. 2A) was performed using isolated lysosomes derived from SIDT2 knockdown or control siRNA-transfected cells. Relative levels of RNA uptake were quantified. Mean ± SEM (n = 3). ***, P < 0.001. (C and F) RNA degradation assay using lysosomes isolated from SIDT2-knockdown cells or from control cells. Relative levels of RNA degradation were quantified. Mean ± SEM (n = 3). ***, P < 0.001; **, P < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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f0005: Effects of SIDT2 knockdown on RNA uptake and degradation by lysosomes. (A and D) Decreased levels of SIDT2 proteins in HeLa cells transfected with SIDT2-siRNA were confirmed by immunoblotting. Relative levels of SIDT2 were quantified. Results are expressed as mean ± SEM (n = 3). (B and E) RNA uptake assay I (Fig. 2A) was performed using isolated lysosomes derived from SIDT2 knockdown or control siRNA-transfected cells. Relative levels of RNA uptake were quantified. Mean ± SEM (n = 3). ***, P < 0.001. (C and F) RNA degradation assay using lysosomes isolated from SIDT2-knockdown cells or from control cells. Relative levels of RNA degradation were quantified. Mean ± SEM (n = 3). ***, P < 0.001; **, P < 0.01.
Mentions: To investigate the effects of SIDT2 knockdown on RNautophagy activity, HeLa cells were transfected with siRNAs targeting SIDT2. Lysosomes were isolated and RNA uptake assays were performed. Lysosomes isolated from SIDT2 knockdown cells showed significantly lower RNA uptake than lysosomes from control siRNA-transfected cells (Fig. 5A–B). A significant reduction of RNA degradation was observed in lysosomes isolated from SIDT2 knockdown cells (Fig. 5C). Similar results were obtained when we used another siRNA against SIDT2 (Fig. 5D–F). We confirmed that knockdown of SIDT2 did not affect lysosomal pH (Fig. S4). The impairment of RNA uptake and degradation by SIDT2 knockdown was also confirmed in the 293FT cell line (Fig. S7). Collectively, these results demonstrate that SIDT2 is a critical determinant of RNautophagy activity.Figure 5.

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