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.


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)



This family belongs to the .

 

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]

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.

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.

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.

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

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.

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.

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]

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; Xu et al. 2013).  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 SID1 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.  SIDT1 localizes to endolysosomes and mediates double-stranded RNA transport into the cytoplasm (Nguyen et al. 2019).

Animals

SID1 of Homo sapiens (Q9NXL6)

 
1.A.79.1.3

Lysosomal systemic RNA interference defective protein-2, SidT2 of 832 aas and 12 TMSs. 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).

Animals

SidT2 of Homo sapiens (Q8NBJ9)

 
1.A.79.1.4

SidT2 dsRNA uptake channel

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