1.A.79. The Cholesterol Uptake Protein (ChUP) or Double Stranded RNA Uptake Family

When dsRNA is injected into C. elegans, it spreads to silence gene expression throughout the animal and in its progeny. This phenomenon is termed RNA interference (RNAi) and has been observed in plants and nematodes. SID-1 is a 776 aa residue integral membrane C. elegans protein with a 400 aa extracelular N-terminal domain and a C-terminal domain of 11 putative TMSs that mediates passive dsRNA transport into cells. However, export of RNA silencing from C. elegans tissues does not require SID-1 (Jose et al., 2009). A 9 TMS model with two regions that dip into the membrane from the external side has been proposed (Feinberg and Hunter, 2003). Several distant but probable paralogues of SID-1 are found in C. elegans, and mammals contain SID-1 homologues. It has been shown that a human SID-1 homologue enhances siRNA uptake and gene silencing (Duxbury et al., 2005). A homologue could not be identified encoded within the genome of Drosophila melanogaster or in other organisms.

The human SID-1 homologue FLJ20174 localizes to the cell plasma membrane and enhances uptake of small interfering RNA (siRNA). This results in increased siRNA-mediated gene silencing efficacy. Thus, overexpression enhances siRNA internalization in mammalian cells. The N-terminal extracellular domain of human SID-1 has been characterized (Pratt et al., 2012). It is glycosylated and forms a compact, globular tetramer. It may control access of dsRNA to the transmembrane pore. SID-1 is a dsRNA-selective dsRNA-gated channel (Shih and Hunter, 2011). Both single- and double-stranded dsRNA, such as hairpin RNA and pre-microRNA, can be transported by SID-1.

Survival of C. elegans depends on the dietary absorption of sterols present in the environment. Valdes et al. (2012) provided evidence that Cholesterol Uptake Protein-1 (ChUP-1) (ZK721; tag-130) is involved in dietary cholesterol uptake in C. elegans. Animals lacking ChUP-1 showed hypersensitivity to cholesterol limitation and were unable to uptake cholesterol. A ChUP-1-GFP fusion protein colocalized with cholesterol-rich vesicles, endosomes and lysosomes as well as the plasma membrane. A direct interaction was found between the cholesterol analog DHE and the transmembrane 'cholesterol recognition/interaction amino acid consensus' (CRAC) motif present in C. elegans ChUP-1. In-silico analysis identified two mammalian homologues of ChUP-1. CRAC motifs are conserved in mammalian ChUP-1 homologues (Valdes et al., 2012). 

Single-stranded oligonucleotides (ssOligos) are efficiently taken up by living cells without the use of transfection reagents. This phenomenon, called 'gymnosis', enables the sequence-specific silencing of target genes. Several antisense ssOligos are used for the treatment of human diseases. Systemic RNA interference deficient-1 (SID-1) transmembrane family 2 (SIDT2), a mammalian ortholog of the Caenorhabditis elegans double-stranded RNA channel SID-1, mediates gymnosis. Takahashi et al. 2017 showed that the uptake of naked ssOligos into cells is downregulated by knockdown of SIDT2, and it inhibited the effect of antisense RNA mediated by gymnosis. Overexpression of SIDT2 enhanced the uptake of naked ssOligos into cells, while a single amino acid mutation in SIDT2 abolished this effect. Thus, SIDT2 mediates extra- and intracellular RNA transport. 

Sidt2, is a highly glycosylated multichannel lysosomal transmembrane protein. Sidt2 can maintain the normal morphology of lysosomes and help stabilize them from the acidic pH environment within (Song et al. 2022). As a receptor/transporter, it binds and transports nucleic acids and mediates the uptake and degradation of RNA and DNA by the lysosome. During glucose metabolism, deletion of Sidt2 can cause an increase in fasting blood glucose and the impairment of grape tolerance, which is closely related to the secretion of insulin. During lipid metabolism, the loss of Sidt2 causes hepatic steatosis and lipid metabolism disorders and can also play a role in signal regulation and transport. Song et al. 2022 reviewed the function of Sidt2, focusing on its role in glucose and lipid metabolism, autophagy and nucleotide (DNA/RNA) transport.


Transport reactions believed to be catalyzed by SID-1 and ChUP1 are:


dsRNA (out) ⇌ dsRNA (in)

Cholesterol (out)  ⇌  Cholesterol (in)

A transport reaction believed to be catalyzed by SIDT2 of mammals is:

ssRNAout (oligonucleoties) ⇌ ssRNAin (oligonucleotides)


 

References:

Aizawa, S., V.R. Contu, Y. Fujiwara, K. Hase, H. Kikuchi, C. Kabuta, K. Wada, and T. Kabuta. (2016). Lysosomal membrane protein SIDT2 mediates the direct uptake of DNA by lysosomes. Autophagy 0. [Epub: Ahead of Print]

Aizawa, S., Y. Fujiwara, V.R. Contu, K. Hase, M. Takahashi, H. Kikuchi, C. Kabuta, K. Wada, and T. Kabuta. (2016). Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes. Autophagy 12: 565-578.

Cappelle, K., C.F. de Oliveira, B. Van Eynde, O. Christiaens, and G. Smagghe. (2016). The involvement of clathrin-mediated endocytosis and two Sid-1-like transmembrane proteins in double-stranded RNA uptake in the Colorado potato beetle midgut. Insect Mol Biol. [Epub: Ahead of Print]

Chen, Q., F. Zhang, L. Dong, H. Wu, J. Xu, H. Li, J. Wang, Z. Zhou, C. Liu, Y. Wang, Y. Liu, L. Lu, C. Wang, M. Liu, X. Chen, C. Wang, C. Zhang, D. Li, K. Zen, F. Wang, Q. Zhang, and C.Y. Zhang. (2021). SIDT1-dependent absorption in the stomach mediates host uptake of dietary and orally administered microRNAs. Cell Res 31: 247-258.

Contu, V.R., K. Hase, H. Kozuka-Hata, M. Oyama, Y. Fujiwara, C. Kabuta, M. Takahashi, F. Hakuno, S.I. Takahashi, K. Wada, and T. Kabuta. (2017). Lysosomal targeting of SIDT2 via multiple YXXΦ motifs is required for SIDT2 function in the process of RNautophagy. J Cell Sci. [Epub: Ahead of Print]

Dickson, E.J., J.B. Jensen, O. Vivas, M. Kruse, A.E. Traynor-Kaplan, and B. Hille. (2016). Dynamic formation of ER-PM junctions presents a lipid phosphatase to regulate phosphoinositides. J. Cell Biol. [Epub: Ahead of Print]

Duxbury, M.S., S.W. Ashley, and E.E. Whang. (2005). RNA interference: a mammalian SID-1 homologue enhances siRNA uptake and gene silencing efficacy in human cells. Biochem. Biophys. Res. Commun. 331: 459-463.

Feinberg, E.H. and C.P. Hunter. (2003). Transport of dsRNA into cells by the transmembrane protein SID-1. Science 301: 1545-1547.

Hase, K., V.R. Contu, C. Kabuta, R. Sakai, M. Takahashi, N. Kataoka, F. Hakuno, S.I. Takahashi, Y. Fujiwara, K. Wada, and T. Kabuta. (2020). Cytosolic domain of SIDT2 carries an arginine-rich motif that binds to RNA/DNA and is important for the direct transport of nucleic acids into lysosomes. Autophagy 1-15. [Epub: Ahead of Print]

Herdman, C. and T. Moss. (2016). Extended-Synaptotagmins (E-Syts); the extended story. Pharmacol Res 107: 48-56. [Epub: Ahead of Print]

Jose, A.M., J.J. Smith, and C.P. Hunter. (2009). Export of RNA silencing from C. elegans tissues does not require the RNA channel SID-1. Proc. Natl. Acad. Sci. USA 106: 2283-2288.

Jose, A.M., Y.A. Kim, S. Leal-Ekman, and C.P. Hunter. (2012). Conserved tyrosine kinase promotes the import of silencing RNA into Caenorhabditis elegans cells. Proc. Natl. Acad. Sci. USA 109: 14520-14525.

León-Reyes, G., B. Rivera-Paredez, J.C.F. López, E.G. Ramírez-Salazar, A. Aquino-Gálvez, K. Gallegos-Carrillo, E. Denova-Gutiérrez, J. Salmerón, and R. Velázquez-Cruz. (2020). The Variant rs1784042 of the Gene is Associated with Metabolic Syndrome through Low HDL-c Levels in a Mexican Population. Genes (Basel) 11:.

McEwan, D.L., A.S. Weisman, and C.P. Hunter. (2012). Uptake of extracellular double-stranded RNA by SID-2. Mol. Cell 47: 746-754.

Méndez-Acevedo, K.M., V.J. Valdes, A. Asanov, and L. Vaca. (2017). A novel family of mammalian transmembrane proteins involved in cholesterol transport. Sci Rep 7: 7450.

Morell, M., N. Varela, C. Castillejo-López, C. Coppard, M.J. Luque, Y.Y. Wu, N. Martín-Morales, F. Pérez-Cózar, G. Gómez-Hernández, R. Kumar, F. O''Valle, M.E. Alarcón-Riquelme, and C. Marañón. (2022). SIDT1 plays a key role in type I IFN responses to nucleic acids in plasmacytoid dendritic cells and mediates the pathogenesis of an imiquimod-induced psoriasis model. EBioMedicine 76: 103808.

Navratna, V., A. Kumar, J.K. Rana, and S. Mosalaganti. (2023). Structure of the human systemic RNAi defective transmembrane protein 1 (hSIDT1) reveals the conformational flexibility of its lipid binding domain. bioRxiv.

Nguyen, T.A., B.R.C. Smith, K.D. Elgass, S.J. Creed, S. Cheung, M.D. Tate, G.T. Belz, I.P. Wicks, S.L. Masters, and K.C. Pang. (2019). SIDT1 Localizes to Endolysosomes and Mediates Double-Stranded RNA Transport into the Cytoplasm. J Immunol. [Epub: Ahead of Print]

Nguyen, T.A., K.T. Bieging-Rolett, T.L. Putoczki, I.P. Wicks, L.D. Attardi, and K.C. Pang. (2019). SIDT2 RNA Transporter Promotes Lung and Gastrointestinal Tumor Development. iScience 20: 14-24. [Epub: Ahead of Print]

Pratt, A.J., R.P. Rambo, P.W. Lau, and I.J. MacRae. (2012). Preparation and characterization of the extracellular domain of human Sid-1. PLoS One 7: e33607.

Qian, D., Y. Cong, R. Wang, Q. Chen, C. Yan, and D. Gong. (2023). Structural insight into the human SID1 transmembrane family member 2 reveals its lipid hydrolytic activity. Nat Commun 14: 3568.

Shih, J.D. and C.P. Hunter. (2011). SID-1 is a dsRNA-selective dsRNA-gated channel. RNA 17: 1057-1065.

Song, Y., J. Gu, J. You, Y. Tao, Y. Zhang, L. Wang, and J. Gao. (2022). The functions of SID1 transmembrane family, member 2 (Sidt2). FEBS J. [Epub: Ahead of Print]

Sun, H., J.M. Ding, H.H. Zheng, K.J. Lv, Y.F. Hu, Y.H. Luo, X. Wu, W.J. Pei, L.Z. Wang, M.C. Wu, Y. Zhang, and J.L. Gao. (2020). The Effects of Sidt2 on the Inflammatory Pathway in Mouse Mesangial Cells. Mediators Inflamm 2020: 3560793.

Takahashi, M., V.R. Contu, C. Kabuta, K. Hase, Y. Fujiwara, K. Wada, and T. Kabuta. (2017). SIDT2 mediates gymnosis, the uptake of naked single-stranded oligonucleotides into living cells. RNA Biol 0. [Epub: Ahead of Print]

Valdes, V.J., A. Athie, L.S. Salinas, R.E. Navarro, and L. Vaca. (2012). CUP-1 Is a Novel Protein Involved in Dietary Cholesterol Uptake in Caenorhabditis elegans. PLoS One 7: e33962.

Xiong, Q.Y., C.Q. Xiong, L.Z. Wang, and J.L. Gao. (2020). Effect of sidt2 Gene on Cell Insulin Resistance and Its Molecular Mechanism. J Diabetes Res 2020: 4217607.

Xu J., Yoshimura K., Mon H., Li Z., Zhu L., Iiyama K., Kusakabe T. and Lee JM. (2014). Establishment of Caenorhabditis elegans SID-1-dependent DNA delivery system in cultured silkworm cells. Mol Biotechnol. 56(3):193-8.

Xu, W. and Z. Han. (2008). Cloning and phylogenetic analysis of sid-1-like genes from aphids. J Insect Sci 8: 1-6.

Yi, D., D. Zhang, and J. He. (2021). Long non-coding RNA LIFR-AS1 suppressed the proliferation, angiogenesis, migration and invasion of papillary thyroid cancer cells via the miR-31-5p/SIDT2 axis. Cell Cycle 20: 2619-2637.

Yu, H., Y. Liu, D.R. Gulbranson, A. Paine, S.S. Rathore, and J. Shen. (2016). Extended synaptotagmins are Ca2+-dependent lipid transfer proteins at membrane contact sites. Proc. Natl. Acad. Sci. USA. [Epub: Ahead of Print]

Zhao, J.C., A. Saleh, and S.T. Crooke. (2023). SIDT2 Inhibits Phosphorothioate Antisense Oligonucleotide Activity by Regulating Cellular Localization of Lysosomes. Nucleic Acid Ther 33: 108-116.

Examples:

TC#NameOrganismal TypeExample
1.A.79.1.1

The dsRNA transporter, SID-1 (Systematic RNA interference defective-1).  Forms a gated transmembrane channel (Shih and Hunter 2011).  It may function together with or be regulated by Sid-2, a metal-dependent nucleic acid binding protein (Q9GZC9) (McEwan et al. 2012), Sid-3, a tyrosyl protein kinase (Q10925), named Cdc-42-associated kinase, Ack, in mammals (Jose et al. 2012) and Sid-5 (Q19443) which co-localizes with RAB-7 (Q23146) and RLP-1 (Q11117).  Endocytosis may play a role in dsRNA uptake.

Animals, plants

SID-1 of Caenorhabditis elegans (AAF98593)

 
1.A.79.1.2

The human SIDT1 protein (Duxbury et al. 2005; Pratt et al. 2012). This protein as well as SidT2 may be cholesterol transporters (Méndez-Acevedo et al. 2017), although they are annotated as RNA transporters, in accordance with several earlier publications. Morreover, SIDT1 localizes to endolysosomes and mediates double-stranded RNA transport into the cytoplasm (Nguyen et al. 2019). SIDT1 plays a key role in type I IFN responses to nucleic acids in plasmacytoid dendritic cells and mediates the pathogenesis of an imiquimod-induced psoriasis model (Morell et al. 2022). SIDT1-dependent absorption in the stomach mediates host uptake of dietary and orally administered microRNAs (Chen et al. 2021). The structure of the human systemic RNAi defective transmembrane protein 1 (hSIDT1) has revealed the conformational flexibility of its lipid binding domain (Navratna et al. 2023).

 

Animals

SIDT1 of Homo sapiens (Q9NXL6)

 
1.A.79.1.3

Lysosomal systemic RNA interference defective protein-2, (systemic RNAi-defective (SID)) SidT2 of 832 aas and 12 TMSs in a 1 (N-terminal) + 1 (at residue 300) + 10 TMS arrangement. It increases the uptake of exogenous dsRNA and DNA (Aizawa et al. 2016).  RNA and DNA are directly taken up by lysosomes in an ATP-dependent manner and degraded. SIDT2 has been reported to mediate RNA translocation during RNA autophagy and DNA translocation during DNA autophagy. Knockdown of Sidt2 inhibited, up to ~50%, total RNA degradation at the cellular level, independently of macroautophagy (Aizawa et al. 2016).  RNA autophagy plays a role in constitutive cellular RNA degradation. SIDT2 also takes up single stranded oligonucleotides into cells (Takahashi et al. 2017). Contu et al. 2017 showed that three cytosolic YXXPhi motifs in SIDT2 are required for the lysosomal localization of SIDT2, and that SIDT2 interacts with adaptor protein complexes AP-1 and AP-2.  On the other hand, Méndez-Acevedo et al. 2017 reported that this protein and SIDT1 transport cholesterol and not RNA. SIDT2 and RNautophagy promote tumor development (Nguyen et al. 2019). The cytosolic domain of SIDT2 carries an arginine-rich motif that binds to RNA/DNA and is important for the direct transport of nucleic acids into lysosomes (Hase et al. 2020). SIDT2 influences the three inflammatory signal pathways, eventually leading to damage of glomerular mesangial cells in mice (Sun et al. 2020). The variant rs1784042 of the SIDT2 gene is associated with the metabolic syndrome through Low HDL-c levels (León-Reyes et al. 2020). SidT2 enhances glucose uptake in peripheral tissues upon insulin stimulation (Xiong et al. 2020). The LIFR-AS1/miR-31-5p/SIDT2 axis modulated the development of papillary thyroid carcinoma (PTC) (Yi et al. 2021).  The cryo-EM structures of human SIDT2 forms a tightly packed dimer with extensive interactions mediated by two previously uncharacterized extracellular/luminal beta-strand-rich domains and the unique transmembrane domain (TMD) (Qian et al. 2023). The TMD of each SIDT2 protomer contains eleven TMSs), and no discernible nucleic acid conduction pathway within the TMD, suggesting that it may act as a transporter. TM3-6 and TM9-11 form a large cavity with a putative catalytic zinc atom coordinated by three conserved histidine residues and one aspartate residue lying approximately 6 Å from the extracellular/luminal surface of the membrane. SIDT2 can hydrolyze C18 ceramide into sphingosine and fatty acid with a slow rate (Qian et al. 2023). SIDT2 inhibits phosphorothioate Aantisense oligonucleotide activity by regulating cellular localization of lysosomes (Zhao et al. 2023).

Animals

SidT2 of Homo sapiens (Q8NBJ9)

 
1.A.79.1.4

SidT2 dsRNA uptake channel of 856 aas and 12 or 13 TMSs.

Animals

SidT2 of Siniperca chuatsi

 
1.A.79.1.5

The Cholesterol Uptake Protein ChUP-1 of 756 aas and 12 or 13 TMSs (Valdes et al., 2012).

Animals

ChUP-1 of Caenorhabditis elegans (Q9GYF0)

 
1.A.79.1.6

The ChUP-1 homologue, Sid1

Slime Molds

ChUP-1 homologue of Dictyostelium discoideum (B0G177)

 
1.A.79.1.7

Insect Sid-1 of 766 aas (Xu and Han 2008).

Animals

Sid-1 of Aphis gossypii

 
1.A.79.1.8

Sid-1 homologue of 718 aas

Animals

Sid-1 homologue of Caenorhabditis elegans

 
1.A.79.1.9

Systemic RNA interference deficient-1 (Sid-1) transmembrane channel for the uptake of dsRNA, involving Sid-1-like proteins A and C, SilA and SilC (Cappelle et al. 2016).

SilA/C of Leptinotarsa decemlineata (Colorado potato beetle) (Doryphora decemlineata)

 
Examples:

TC#NameOrganismal TypeExample
1.A.79.2.1

Prokaryotic Sid-1 homologue of 258 aas

Proteobacteria

Sid-1 homologue of Nitrosococcus watsoni