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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 overexpression on RNautophagy in the absence of LAMP2. (A) LAMP2 levels in LAMP2-deficient HeLa cells and parental HeLa cells (control HeLa) were analyzed by immunoblotting. (B and C) RNA uptake assays were performed using isolated lysosomes derived from LAMP2-deficient HeLa cells (B) or parental HeLa cells (C). Relative levels of RNA uptake were quantified. Results are expressed as mean ± SEM (n = 3). **, P < 0.01. In the absence of LAMP2, SIDT2 increased RNautophagy at similar levels to in the presence of LAMP2.
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f0003: Effect of SIDT2 overexpression on RNautophagy in the absence of LAMP2. (A) LAMP2 levels in LAMP2-deficient HeLa cells and parental HeLa cells (control HeLa) were analyzed by immunoblotting. (B and C) RNA uptake assays were performed using isolated lysosomes derived from LAMP2-deficient HeLa cells (B) or parental HeLa cells (C). Relative levels of RNA uptake were quantified. Results are expressed as mean ± SEM (n = 3). **, P < 0.01. In the absence of LAMP2, SIDT2 increased RNautophagy at similar levels to in the presence of LAMP2.

Mentions: We investigated the relationship between SIDT2 and LAMP2C during RNautophagy. We tested the effect of SIDT2 overexpression on RNA transport activity in lysosomes derived from LAMP2-deficient cells. Overexpression of SIDT2 increased RNautophagy even in LAMP2-deficient lysosomes (Fig. 3A–C), indicating that SIDT2 is able to function independently of LAMP2C. This result is consistent with our previous report showing that LAMP2 is not indispensable for RNautophagy.3Figure 3.


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 overexpression on RNautophagy in the absence of LAMP2. (A) LAMP2 levels in LAMP2-deficient HeLa cells and parental HeLa cells (control HeLa) were analyzed by immunoblotting. (B and C) RNA uptake assays were performed using isolated lysosomes derived from LAMP2-deficient HeLa cells (B) or parental HeLa cells (C). Relative levels of RNA uptake were quantified. Results are expressed as mean ± SEM (n = 3). **, P < 0.01. In the absence of LAMP2, SIDT2 increased RNautophagy at similar levels to in the presence of LAMP2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836006&req=5

f0003: Effect of SIDT2 overexpression on RNautophagy in the absence of LAMP2. (A) LAMP2 levels in LAMP2-deficient HeLa cells and parental HeLa cells (control HeLa) were analyzed by immunoblotting. (B and C) RNA uptake assays were performed using isolated lysosomes derived from LAMP2-deficient HeLa cells (B) or parental HeLa cells (C). Relative levels of RNA uptake were quantified. Results are expressed as mean ± SEM (n = 3). **, P < 0.01. In the absence of LAMP2, SIDT2 increased RNautophagy at similar levels to in the presence of LAMP2.
Mentions: We investigated the relationship between SIDT2 and LAMP2C during RNautophagy. We tested the effect of SIDT2 overexpression on RNA transport activity in lysosomes derived from LAMP2-deficient cells. Overexpression of SIDT2 increased RNautophagy even in LAMP2-deficient lysosomes (Fig. 3A–C), indicating that SIDT2 is able to function independently of LAMP2C. This result is consistent with our previous report showing that LAMP2 is not indispensable for RNautophagy.3Figure 3.

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