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)
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.
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).
SID1 of Homo sapiens (Q9NXL6)
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). Sidt2 influences the three inflammatory signal pathways, eventually leading to the 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).
SidT2 of Homo sapiens (Q8NBJ9)
SidT2 dsRNA uptake channel of 856 aas and 12 or 13 TMSs.
SidT2 of Siniperca chuatsi
The Cholesterol Uptake Protein ChUP-1 of 756 aas and 12 or 13 TMSs (Valdes et al., 2012).
ChUP-1 of Caenorhabditis elegans (Q9GYF0)
The ChUP-1 homologue, Sid1
ChUP-1 homologue of Dictyostelium discoideum (B0G177)
Insect Sid-1 of 766 aas (Xu and Han 2008).
Sid-1 of Aphis gossypii
Sid-1 homologue of 718 aas
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
Sid-1 homologue of Nitrosococcus watsoni