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:
The dsRNA transporter, SID-1 (Systematic RNA interference defective-1). Sid1 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. In Caenorhabditis elegans, inter-cellular transport of the small non-coding RNA causing systemic RNAi is mediated by the transmembrane protein SID1, encoded by the sid1 gene in the systemic RNAi defective (sid) loci. SID1 shares structural and sequence similarity with cholesterol uptake protein 1 (CHUP1) and is classified as a member of the ChUP family. Although systemic RNAi is not an evolutionarily conserved process, the sid gene products are found across the animal kingdom, suggesting the existence of other novel gene regulatory mechanisms mediated by small non-coding RNAs (Navratna et al. 2024).
Animals, plants
SID-1 of Caenorhabditis elegans (AAF98593)
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). Several subgroups of the family have been identified as cognate endopeptidases for four protein-sorting signals processed by a previously unknown machinery. Sorting signals with newly identified processing enzymes include MYXO-CTERM and three novel ones (Haft 2024). N-glycosylation is required for its functional role in SIDT1-mediated RNA uptake (Yang et al. 2024). The structure of recombinant human SIDT1 has been solved revealing that the extra-cytosolic domain of hSIDT1 adopts a double jelly roll fold, and the transmembrane domain exists as two modules - a flexible lipid binding domain and a rigid transmembrane domain core. These structural analyses provide insights into the inherent conformational dynamics within the lipid binding domain in ChUP family members (Navratna et al. 2024).
Animals
SIDT1 of Homo sapiens (Q9NXL6)
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). SIDT2 is a player in cholesterol and lipoprotein metabolism in humans (León-Mimila et al. 2021).
Animals
SidT2 of Homo sapiens (Q8NBJ9)
SidT2 dsRNA uptake channel of 856 aas and 12 or 13 TMSs.
Animals
SidT2 of Siniperca chuatsi
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)
The ChUP-1 homologue, Sid1
Slime Molds
ChUP-1 homologue of Dictyostelium discoideum (B0G177)
Insect Sid-1 of 766 aas (Xu and Han 2008).
Animals
Sid-1 of Aphis gossypii
Sid-1 homologue of 718 aas
Animals
Sid-1 homologue of Caenorhabditis elegans
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)
Prokaryotic Sid-1 homologue of 258 aas
Proteobacteria
Sid-1 homologue of Nitrosococcus watsoni