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. 2024 Jan;21(1):1-9.
doi: 10.1080/15476286.2023.2291610. Epub 2024 Jan 10.

Expression of RNautophagy/DNautophagy-related genes is regulated under control of an innate immune receptor

Yuuki Fujiwara  1   2 , Kazuki Oroku  3 , Yinping Zhou  2 , Masayuki Takahashi  1 , Taiichi Katayama  2 , Keiji Wada  1 , Nobuyuki Tsutsumi  3 , Tetsuo Sato  3 , Tomohiro Kabuta  1
Affiliations

Expression of RNautophagy/DNautophagy-related genes is regulated under control of an innate immune receptor

Yuuki Fujiwara et al. RNA Biol. 2024 Jan.

Abstract

Double-stranded RNA (dsRNA) is a molecular pattern uniquely produced in cells infected with various viruses as a product or byproduct of replication. Cells detect such molecules, which indicate non-self invasion, and induce diverse immune responses to eliminate them. The degradation of virus-derived molecules can also play a role in the removal of pathogens and suppression of their replication. RNautophagy and DNautophagy are cellular degradative pathways in which RNA and DNA are directly imported into a hydrolytic organelle, the lysosome. Two lysosomal membrane proteins, SIDT2 and LAMP2C, mediate nucleic acid uptake via this pathway. Here, we showed that the expression of both SIDT2 and LAMP2C is selectively upregulated during the intracellular detection of poly(I:C), a synthetic analog of dsRNA that mimics viral infection. The upregulation of these two gene products upon poly(I:C) introduction was transient and synchronized. We also observed that the induction of SIDT2 and LAMP2C expression by poly(I:C) was dependent on MDA5, a cytoplasmic innate immune receptor that directly recognizes poly(I:C) and induces various antiviral responses. Finally, we showed that lysosomes can target viral RNA for degradation via RNautophagy and may suppress viral replication. Our results revealed a novel degradative pathway in cells as a downstream component of the innate immune response and provided evidence suggesting that the degradation of viral nucleic acids via RNautophagy/DNautophagy contributes to the suppression of viral replication.

Keywords: DNautophagy; Double-stranded RNA; Lysosome; PAMPs; PRRs; RNautophagy; immune response; innate immunity; virus infection; virus replication.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Transient increase in expression of RNautophagy/DNautophagy-related genes by poly(I:C). A, B. relative levels of SIDT2 (A) or LAMP2C (B) mRNA in cells transfected with either poly(I:C) or mouse total RNA. C, D. relative levels of SIDT2 (C) or LAMP2C (D) mRNA in cells with poly(I:C) added directly into culture media. E, F. temporal change in expression levels of SIDT2 (E) or LAMP2C (F) mRNA in cells transfected with poly(I:C). (n = 3). ** p < 0.01, N.S., not significant.
Figure 2.
Figure 2.
Poly(i:c) selectively upregulates the expression of RNautophagy/DNautophagy-related genes. A, B. dose-dependency in the expression levels of SIDT2 (A) or LAMP2C (B) mRNA in cells transfected with poly(I:C). C–F. relative levels of LAMP2A (C), LAMP2B (D), all LAMP2 isoforms (E), or LAMP1 (F) mRNA in cells transfected with poly(I:C). G. immunoblotting of SIDT2, LAMP2C, all LAMP2 isoforms, and LAMP1 protein in cells transfected with poly(I:C). H–K. quantification of respective gene products shown in G. (n = 3). * p < 0.05, ** p < 0.01, N.S., not significant.
Figure 3.
Figure 3.
MDA5 mediates the upregulation of RNautophagy/DNautophagy-related genes expression by poly(I:C). A, B. relative levels of SIDT2 (A) or LAMP2C (B) mRNA in cells transfected with respective siRNA, followed by mock- or poly(I:C)-transfection. C, D. relative levels of SIDT2 (C) or LAMP2C (D) mRNA in cells transfected with respective expression vectors, followed by mock- or poly(I:C)-transfection. (n = 3). ** p < 0.01, N.S., not significant.
Figure 4.
Figure 4.
Viral RNA can be targeted for degradation via RNautophagy. A, B. relative levels of extracellular (A) and intracellular (B) viral RNA produced in WT or Sidt2 KO cells infected with JEV at MOI = 1. C. immunoblotting of extracellular viral protein produced by WT or Sidt2 KO cells infected with JEV at MOI = 1. D. a representative image of plaque assays using cultured media derived from WT or Sidt2 KO cells infected with JEV at MOI = 1. Plaques are surrounded by pen strokes. E. virus titre of cultured media derived from WT or Sidt2 KO cells infected with JEV at MOI = 1. F. relative levels of viral RNA in extralysosomal solution or lysosomal pellet following the incubation with isolated lysosomes in the absence or presence of ATP. G. relative levels of total viral RNA following the incubation with isolated lysosomes in the absence or presence of ATP. (n = 3). ** p < 0.01.
Figure 5.
Figure 5.
A schematic of our current model. Pattern recognition receptors (PRRs), such as MDA5, recognize pathogen-associated molecular patterns (PAMPs) such as dsRnas and upregulate RNautophagy/DNautophagy-related genes, to activate RNautophagy/DNautophagy. RNautophagy/DNautophagy contributes to clearance of viral nucleic acids, thereby suppressing virus replication. RNautophagy/DNautophagy may also contribute to the detection of PAMPs via endolysosomal TLRs by taking up viral nucleic acids into their recognition sites, which leads to induction of various anti-viral responses that may also include the activation of RNautophagy/DNautophagy as a positive feedback loop.
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Grants and funding

This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (14J08223, 17J10610, 21K15367, and 23K14453 to Y.F.; 16H05146, 16H01211, and 19H05710 to T.K.), ACT-X from Japan Science and Technology Agency (JPMJAX222H to Y.F.), and research grants from the Takeda Science Foundation (to Y.F.).

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