Cryo-EM analysis reveals human SID-1 transmembrane family member 1 dynamics underlying lipid hydrolytic activity

Two mammalian homologs of systemic RNA interference defective protein 1 (SID-1) (SIDT1/2) are suggested to function as double-stranded RNA (dsRNA) transporters for extracellular dsRNA uptake or for release of incorporated dsRNA from lysosome to cytoplasm. SIDT1/2 is also suggested to be involved in cholesterol transport and lipid metabolism. Here, we determine the cryo-electron microscopy structures of human SIDT1, homodimer in a side-by-side arrangement, with two distinct conformations, the cholesterol-bound form and the unbound form. Our structures reveal that the membrane-spanning region of SIDT1 harbors conserved histidine and aspartate residues coordinating to putative zinc ion, in a structurally similar manner to alkaline ceramidases or adiponectin receptors that require zinc for ceramidase activity. We identify that SIDT1 has a ceramidase activity that is attenuated by cholesterol binding. Observations from two structures suggest that cholesterol molecules serve as allosteric regulator that binds the transmembrane region of SIDT1 and induces the conformation change and the reorientation of the catalytic residues. This study represents a contribution to the elucidation of the cholesterol-mediated mechanisms of lipid hydrolytic activity and RNA transport in the SID-1 family proteins.


Introduction
The extracellular RNAs in mammalian body play important roles in cell-cell communication, virus-host interactions and potent RNA therapeutics 1,2 .Viral double-stranded RNA (dsRNA) triggers an antiviral defense system in vertebrates through an activation of innate immunity.Synthetic small interfering (si)RNAs and micro RNAs (miRNAs) induce evolutionary conserved RNA interference (RNAi), which results in posttranscriptional gene silencing.These RNAs has been suggested to exert their functions in a noncell autonomous manner through secretion from host cells or exogenous supply and uptake to recipient cells 1,2 .However, dsRNA uptake mechanism is largely unknown.
RNAi initiated by injection of double-stranded RNA (dsRNA) is initially found in Caenorhabditis elegans (C.elegans) 3 .In C. elegans, the effect of RNAi spreads from the introduced cells or tissues throughout the organism and its progeny.The C. elegans systemic RNA interference defective protein 1 (SID-1) has been identi ed as a gene required for systemic but not cell-autonomous RNA 4 .SID-1 serves as a dsRNA transporter or channel for uptake into cells in an energy independent manner [5][6][7] .SID-1 protein is evolutionary conserved although systemic RNAi phenomena are not apparent in mammals, which implies the functional divergence.In mammals, SID-1 homologues SIDT1 and SIDT2 have an ability to transport dsRNA [8][9][10][11][12] .SIDT2 is expressed on endolysosomal membranes to release internalized poly(I:C), a synthetic analog of viral dsRNA, to cytoplasm, which is known as endosomal escape.The released dsRNA activates innate immunity through RIG-I like receptor pathway.Sidt2 −/− knockout mice exposed to Encephalomyocarditis virus (EMCV), or herpes simplex virus 1 (HSV-1) show impaired production of in ammatory cytokines and reduced survival 10 .SIDT1 can also transport incorporated internalized poly(I:C) from endolysosome to cytoplasm in the cell-based assay, but Sidt1 knock out mice exposed to EMCV or HSV-1 survive normally, suggesting that SIDT1 less contributes to the activation of innate immunity 11 .SIDT2 is ubiquitously expressed but SIDT1 is mainly expressed in the lymphoid lineage and seems to have a role in the anti-viral IFN-I responses in vivo.Recent report suggests that SIDT1 plays a role in type I interferon response to nucleic acids in plasmacytoid dendritic cells 13 .SIDT1 is also required for cellular uptake of cholesterol conjugated siRNAs in liver HepG2 cells 14 .Moreover, SIDT1 transport miRNAs miR-21 of which is an oncogenic miRNA to mediate chemoresistance in human adenocarcinoma 9 .SIDT1 expressed in gastric pit cells of the stomach is required for the absorption of dietary miRNAs.The stomach is the primary site for dietary miRNA absorption, which is dramatically attenuated in the stomachs of SIDT1-de cient mice 12 .The uptake of miRNAs or double-stranded miRNA mimics by stomach cells are increased in acidic condition, which is not affected in sidt1 −/− cells.
Studies from another perspective that SIDT1/SIDT2 shares sequence similarity with C. elegans CHUP-1 mediating dietary cholesterol uptake 15 suggest that mammalian SIDT1/SIDT2 is involved in cholesterol transport.CRAC motif, an α-helical cholesterol binding motif observed in transmembrane proteins, is found in SIDT1/SIDT2 and binds to cholesterol and co-localize with cholesterol in intracellular compartment.Cells overexpressing SIDT1 enhances cholesterol uptake while the cholesterol reduction in cells induces the relocalization of SIDT1 to the plasma membrane in clathrin dependent manner 16 .Additionally, CHUP-1 is suggested to play a role in protective immune response by linking cholesterol metabolism and the immune response 17 .
The structure of SIDT2 has been recently reported 18 , providing the architecture of SID-1 family protein.
But cholesterol-mediated regulation mechanism remains unknown.Our structural work revealed two distinct conformations, the cholesterol-bound closed-form and the unbound open-form.Cholesterol molecules binds the transmembrane region of SIDT1 and reorient catalytic residues accompanied with a large conformational change of transmembrane helices, suggesting that cholesterol molecule serve as allosteric regulator.Moreover, we identi ed dsRNA binding site by structure-based mutational studies.

Structure determination of SIDT1 in the absence and presence of dsRNA
The full length of human SIDT1 (hSIDT1) was puri ed from mammalian cells and subjected to cryoelectron microscopy (cryo-EM) single particle analysis for structure determination.SIDT1 embedded in a GDN micelle exhibited the 2D averages of relatively resolved transmembrane helices and we successfully generated a reconstruction of hSIDT1 to an overall resolution of 2.77 Å (Supplementary Fig. 2).
To clarify the dsRNA recognition by SIDT1, cryo-EM analysis was performed in the presence of dsRNA.
Although clear densities corresponding to dsRNA were not observed, the cryo-EM structures of hSIDT1 in the presence of 25 bp dsRNA at pH5.0 were interestingly converged to 2 classes; one class is essentially same as the structure in the absence of dsRNA, while the other class has different conformation of the transmembrane (TM) region (Supplementary Fig. 3).Hereafter, these structures are referred as the closedform and open-from, respectively based on the conformation of TM region.
The lipid like densities were inserted at the cytoplasmic side of TM region in the closed-form but not in the open-form.
Overall structure of the closed-form of human SIDT1 SIDT1 is assembled as a homodimer (Fig. 1b).The N-terminal extracellular region of hSIDT1 contains a disordered region (residues 1-43) and following two extracellular domains (ECD1 and ECD2) arranged in β-sandwich folds, consisting of two four-stranded β-sheets packed against each other.ECD1 contains an extra β-hairpin between β5-and β8-strands, which contacts with ECD2 (Fig. 1b, Supplementary Fig. 7).The N-glycan densities are observed to be attached to four N-glycosylation sites of ECD1 (Asn57, Asn67, Asn83 and Asn136).The inter-domain disul de bond (Cys130-Cys222) and the intra-domain disul de bond in ECD2 (Cys212-Cys271) are formed, of which cysteine residues are conserved in vertebrate SID-1 family proteins, suggesting the evolutionary conserved contribution of disul de bonds for the stabilization of inter-domain orientation and structural rigidity (Supplementary Fig. 1, 7).
The membrane spanning region is clearly resolved su cient to model all 11 TM helices de novo (Fig. 1b, 1c, Supplementary Fig. 4).The cytoplasmic long TM1-TM2 and TM7-TM8 loops are largely invisible in the cryo-EM map.The extracellular loops, TM4-TM5 loop and TM10-TM11 loop, are linked to TM2-TM3 loop by disul de bonds (Cys479-Cys565 and Cys485-Cys782) (Supplementary Fig. 7).The short helix H1 located in TM10-TM11 loop packs with ECD2 along with TM2-TM3 loop.On the extracellular side of the TM helices, we detected density that would be assignable to Zn 2+ , being tetrahedrally coordinated with His563, Asp574, His791 and His795 (Fig. 1d, Supplementary Fig. 4).Residues coordinating Zn 2+ ion and forming disul de bonds are completely conserved in SID-1 family proteins (Supplementary Fig. 1).Interestingly, mutations introduced in Cys464 and His740 of C. elegans SID-1 (equivalent to Cys479 and His791 of hSIDT1, respectively) have been shown to strongly attenuate environmental RNAi by ingested dsRNA 19 , suggesting the functional importance of those conserved residues beyond species.This "closed-form" of hSIDT1 has no tunnels for nucleotides to pass through within a single protomer or at the dimer-interface (Fig. 1b).
Structural similarity analysis using the DALI server 20 suggested that the membrane spanning region of SIDT1 has no similarities to known structures.However, a deep sequence similarity search previously suggested that SID-1 family is a member of a diverse superfamily of putative metal-dependent transmembrane hydrolases including 7 TM protein alkaline ceramidases (ACERs) and adiponectin receptors (ADIPORs) before their structure determination, mainly based on the conservation of metal-ion coordinating residues 21 .Although SIDT1 contains 11 TMs which is more than 7 TMs observed in the structures of ACERs and ADIPORs [22][23][24] , structure superposition focused on helices containing the conserved metal-ion coordinating residues surprisingly revealed that membrane spanning region of SIDT1 shares structural similarity with ACER3 and ADIPORs (Supplementary Fig. 8).Out of 11 TMs of SIDT1, 7 TMs except for TM1, TM2, TM7 and TM8 are moderately tted to ACER3 and ADIPOR2 with a root mean squared deviation (RMSD) of 3 to 4 Å.These structural features raise the possibility that SIDT1 has a ceramidase activity, which will be described later together with the structure of open-form of SIDT1.
Dimer interface of the closed-form SIDT1 forms a side-by-side homodimer along the C2 symmetry axis, which is mediated by extensive interactions via several surfaces (Fig. 2).On the extracellular side, ECD1 contains salt bridges or hydrogen bonds, while in ECD2, the β14-β15 loops of both protomers contact each other primarily through non-polar interactions (Fig. 2b).In the TM region, TM2 and TM6 of both protomers pack with each other, incorporating hydrophobic interactions in the center of the membrane region and hydrogen bonds at the top and bottom of the membrane region (Fig. 2c).These residues are highly conserved in mammalian SID-1 family proteins although it is less conserved in C. elegans SID-1 (Supplementary Fig. 1).
Several lipid-like elongated densities were observed in the transmembrane region of the closed-form.The two densities run parallel to each other in a small concave formed by TM5, TM6 and TM7 at the intracellular side, which can be modeled as cholesterol molecules (Fig. 2d).Since we did not add cholesterol during puri cation, endogenous cholesterol is assumed to be co-puri ed with SIDT1.The putative cholesterol density which is better resolved than the other (CLR1) is located on the opposite of the homo-dimer interface of TM5 and lls the space between TM5 and TM6.Another cholesterol molecule (CLR2) is packed with TM5 and TM7.These cholesterol molecules were surrounded by hydrophobic residues, most of which are conserved in mammalian SID-1 family proteins, suggesting that the cholesterols serve as molecular glue that stabilizes the conformation of TM5, TM6 and TM7.SIDT1 is previously reported to bind cholesterol via CRAC motif 16 characterized by the sequence 25 .Two CRAC motif identi ed in SIDT1by the previous in silico analysis; one locates at the extracellular region and the other locates at the TM region, of which the one in the TM region (residues 633-659) is indicated to be important for cholesterol binding 16 .In our cryo-EM map, the rst part of the residues 633-659 in SIDT1 forming TM7 interacts with one of two observed cholesterol molecules (CLR2), and the rest of the region is disordered.However, we found that another CRAC motif in TM5 which is not indicated previously interacts with cholesterol molecules more extensively than TM7 (Fig. 2d, Supplementary Fig. 1).

Conformational exibility associated with cholesterolbinding
In the open-form, the transmembrane region undergoes a large conformational change while the extracellular region is well tted to that of the closed-form structure (RMSD of 0.3 Å for Cα atoms) (Fig. 2a, Supplementary Fig. 9).In the open-form, TM5 rotates about 25° and its cytoplasmic side signi cantly moves from inside the membrane spanning region to the dimer interface (14 Å shift of the Cα atom of Thr592), which resulted in the interaction with TM2 of the other protomer (Fig. 2e).TM6 which interacts with TM2 in the closed-form is pushed out (15.6 Å shift of the Cα atom of Tyr605) by the TM5 rotational motion and its N-terminal region is partially disordered.These conformational changes of transmembrane region are primarily caused by helix rotation, resulting in shifts of cytoplasmic side with less changes in the extracellular side.The exception is TM7, where the extracellular side is shifted (5.9 Å shift of the Cα atom of Trp627) and TM8 is almost disordered (Fig. 2e).Of note, despite the transmembrane region undergoes a large conformational change, the homodimer is maintained in the open-form by the alternative interface in which TM5 displaces TM6 in the closed-form.The displaced interface is mainly formed by non-polar interactions, which brings the dimer interface of 2,622 Å 2 as large as that of 2,656 Å 2 in the closed-form (Fig. 2c).In the open-form, no lipid like density was observed between TM5 and TM6, which suggests that cholesterol stabilize the closed-form structure through binding to the concave between TM5 and TM6.
Interestingly, a tunnel is found to be formed in the transmembrane region in the open-form structure by the conformational change in the transmembrane region (Fig. 3a).This tunnel is surrounded by TM4, TM5, TM9, TM10 and TM11 and penetrates the transmembrane region by passing through beside the zinc ion.TM8 is supposed to be located outside TM7 according to the weak density although TM8 is disordered in the cryo-EM map of the open-form, which strongly indicates that TM8 do not intercept the tunnel.This tunnel has a maximum diameter of ~ 5.0 Å and is ~ 2.2 Å at its narrowest area, which indicates that the pore size is not acceptable for transport of dsRNA.
The tunnel of the open-form is partly overlaps with the substrate binding pocket in ACER3.In the catalytic mechanism of ACER3, Zn 2+ is directly coordinated by three His residues and a water molecule that forms hydrogen bonding with Asp, in which the activated water molecule undergoes nucleophilic attack on the ceramide amide bond.Additionally, Ser residue is predicted to form hydrogen bonding with carbonyl group from the amide bond of ceramide and stabilize the oxyanion hole in the transition state (Fig. 3a).
As mentioned above, in the closed-form of SIDT1, Zn 2+ is coordinated by the conserved three His residues and Asp residue, in which there is no space for a water molecule which is activated through the coordination with Zn 2+ .On the other hand, in the open-form, the carboxyl group of Asp574 is 6.3 Å away from Zn 2+ , which is suitable to place a water molecule between them.These structural features raise the possibility that SIDT1 has a ceramidase activity in a conformation dependent manner.We performed ceramidase assay using uorescent labeled ceramide or dihydroceramide.Interestingly, SIDT1 exhibited a ceramidase activity only in the presence of methyl β-cyclodextrin, which is often used for the depletion of cholesterol from membranes (Fig. 3b).Overall, our results suggest that SIDT1 endows a hydrolase activity which is allosterically inhibited by cholesterol although no endogenous substrates have been identi ed.

SIDT1 binds dsRNA in a pH-dependent manner
The extracellular region of SID-1 family proteins has been shown to bind dsRNA 26 .We performed electro mobility shift assay (EMSA) at pH 7.0 using full length hSIDT1 and observed a concentration-dependent dsRNA shift, con rming the interaction between hSIDT1 and dsRNA (Fig. 4b).Structural analysis of hSIDT1 revealed that the extracellular region of hSIDT1 homodimer exhibits a positively charged surface on the lateral side, but a strongly negatively charged surface in the anterior side (Fig. 4a).Since this negative charge might be unfavorable for dsRNA-binding due to its electrostatic repulsion with the negatively charged phosphate group of dsRNA, we speculated that the dsRNA interaction might be pHdependent and performed EMSA at different pH (4.0-7.0) to verify the pH dependence of dsRNA binding.Unshifted band corresponding to unbound dsRNA was monitored to estimate dsRNA binding with SIDT1.
Interestingly, unbound dsRNA was diminished with lower concentration of SIDT1 as pH decreased, strongly suggesting that SIDT1 binds dsRNA in a low-pH dependent manner (Fig. 4b).The low-pH dependent dsRNA binding of SIDT1 is consistent with the previous observations that microRNA or double-stranded microRNA mimic uptake in mouse primary gastric epithelial cells (PGECs) is SIDT1 and low-pH dependent 12 .
The previous genetic screens in C. elegans have identi ed several loss-of-function mutations in SID-1 14,19 .As the result of mapping these mutations on our hSIDT1 structure, most residues are buried inside SIDT1 to support the protein folding while Arg172 (corresponding to His168 in hSIDT1) is exposed to solvent (Supplementary Fig. 10).Moreover, low sequence identities between SID-1 and mammalian SIDTs hampered the identi cation of dsRNA binding site.To estimate the dsRNA binding site of SIDT1, we have applied alanine scanning mutagenesis to mutate residues with polar groups in the extracellular region to alanine and monitored their effects on dsRNA binding by EMSAs (Fig. 4c).The wild type SIDT1 required 3-4 equimolar amounts to saturate dsRNA binding.The nine mutants (site1-site9), containing a total of 22 mutations, were classi ed into three classes based on how much they attenuated dsRNA binding of wild type SIDT1; signi cant, modest or less effect.The mutants site3, site4 and site9 have signi cant effects and the mutants site1, site2, site7 and site8 have modest effects while the mutants site5 and site6 have less effects.Those mutants are mapped on the structure of SIDT1 (Fig. 4d).Among mutations that reduce dsRNA binding of SIDT1, site1 (Arg79, Tyr81), site2 (Glu87, Asp90), site4 (Asn121, Gln123) and site9 (Gln288, Lys290, Asn292) are located in close proximity to the lateral side of the extracellular region, suggesting that these residues are involved in a dsRNA binding.Site7 (Lys192, Lys195, Asp196) and site8 (Lys250, Lys251, Asp252) are distant from those sites and near the transmembrane region.Placing a liner dsRNA on a line connecting these two regions allows a dsRNA to be placed across the lateral side of SIDT1 ECD with only a slight steric clash (Fig. 4e).
Additionally, in SIDT2, arginine-rich motif mostly conserved in SIDT1 in the TM1-TM2 loop, which is largely disordered in our cryo-EM map (Supplementary Fig. 6a), is reported to bind RNA, DNA and oligonucleotide 27,28,29 .Hence an EMSA assay was performed to verify the contribution of the TM1-TM2 loop to dsRNA binding of SIDT1.The deletion of residues of the TM1-TM2 loop (residues 391-442 and 381-442) decreased dsRNA binding of SIDT1, suggesting that the TM1-TM2 loop of SIDT1binds to dsRNA (Supplementary Fig. 6b).

Discussion
Our biochemical and structural analyses have identi ed the putative dsRNA binding site at the lateral side of extracellular region (Fig. 4d), the surface of which is positively charged, but the anterior side is strongly negatively charged (Fig. 4a).Unlike hSIDT1, the predicted structure of C. elegans SID-1 by alphafold2 has a strong positive charge on the lateral side and no negative charge on the anterior side (Supplementary Fig. 11.This difference in surface charge may be one of the reasons for the difference in a nity, as it was previously reported that the a nity of SIDT1 to dsRNA (500 bp or 700 bp long chain) is about 1/5 that of SID-1 in EMSA using extracellular domains 26 .Moreover, we have revealed that SIDT1 binds dsRNAs in a low pH dependent manner.The low-pH-dependent dsRNA binding is consistent with a physiological function of SIDT1, since the extracellular domain of SIDT1 is exposed to an acidic environment outside the cell membrane of gastric pit cells of the stomach or inside lysosomal membranes 12 .Bound dsRNA can spontaneously dissociate from SIDT1 in a neutral pH of the cytoplasm, which might work in the releasing process.
Recently, the structure of human SIDT2 has been reported 29 .The dimer architecture of human SIDT2 mediated by the interaction between TM2 and TM6 in the TM region is similar to the closed-form of SIDT1 although the ECD1 of SIDT1 lacks an N-terminal β-strand observed in SIDT2 (Supplementary Fig. 12a).Strikingly, the structure of SIDT2 did not contain cholesterol molecules.Compared with SIDT2, the concave between TM6 and TM7 of SIDT1 is forced to expand by cholesterol binding, which brings the TM6 longer and closer to dimer-interface.Thus, the interfacing TM2 of the other protomer adopts a longer helix to extensively interact with the TM6.Additionally, the putative Zn 2+ coordination by SIDT2 is signi cantly different from that of the closed-form of SIDT1.As described above, SIDT1 Asp574 coordinates Zn 2+ together with the conserved triad of histidine residues, while in SIDT2, the corresponding residues Asp579 is as far from Zn 2+ as the corresponding aspartate residues in ACERs, ADIPORs or the open-form of SIDT1 (Fig. 3a Supplementary Fig. 12b).Consistent with the structural insight, it is revealed that SIDT2 has a lipid hydrolase activity for ceramide 18 , which is rst evidence that SID-1 family proteins have a hydrolase activity.We here showed that SIDT1 also harbors a ceramidase activity to ceramide and dihydroceramide although the physiological substrate is not identi ed.
Importantly, the cholesterol depletion by MβCD enhanced the hydrolase activity of SIDT1 (Fig. 3b), which suggests that cholesterol inhibits SIDT1 hydrolase activity.Furthermore, open-closed interconversion of the TM region likely associated with cholesterol binding observed in our cryo-EM study provides the structural insight into the action of cholesterol as an allosteric regulator, which induces the rearrangement of TM region mainly in the TM5-TM8 segment to lock Asp574 and excludes a water molecule from zinc ion coordination, probably resulting in the inhibition of the hydrolase activity.In the crystal structure of ADIPOR1, closed-and open-form are observed, mainly due to the difference of the TM5 conformation (corresponding to the TM9 in SIDT1) 24,30 .The dynamic equilibrium between open and closed conformation in ADIPORs hypothesized to regulate the substrate binding and the product release 24 .Thus, the open-closed conformational change of ADIPORs is extremely different from SIDT1, indicating that SIDT1 develops a unique catalytic regulation mechanism.Further study is required to verify whether this mechanism is conserved in other SID-1 family proteins.
The structures of SIDT1 presented here provide insight into the architecture of SID-1 family proteins, homo-dimer state, and the conformational change.The membrane spanning region of SIDT1 has a conformation-dependent tunnel which partially shares the substrate binding pocket of AdipoRs and ACERs.However, the pore size of the tunnel is not acceptable for dsRNA transport.Our structures revealed that cholesterol molecules bind to TM region of SIDT1 and likely induce the conformation change to interrupt the tunnel.Previous study has reported that sterol molecules bind to SIDT1 and SIDT2, which control the cellular localization 16 .Of note, this study also reported that mixing dsRNA with cholesterol resulted in increased uptake of dsRNA in HEK293 cells overexpressing SIDT1.Taken together, cholesterol might regulate dsRNA transport by SIDT1 although it is not clear how dsRNA transport is linked to the cholesterol-binding induced conformational change.Further structures that capture the progressive states of RNA binding and cytoplasmic release may enable the mechanism of the RNA transport to be determined.

Protein expression and puri cation.
The cDNAs of human SIDT1 were provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.
DNA fragments were ampli ed by the polymerase chain reaction (PCR) and cloned into the pEZT-BM vector.These proteins were fused with a C-terminal TEV protease cleavage site, FLAG tag and decahistidine tag.We designated the pEZT-BM vector to incorporate TAR-Tat element derived from pHEK293 ultra vector (TaKaRa) to enhance protein expression.All plasmids were veri ed by DNA sequencing.Baculoviruses were generated in Spodoptera frugiperda Sf9 cells using the Bac-to-Bac system (Invitrogen).For protein expression, Expi293F cells (Invitrogen) were cultured in Expi293 expression medium at 37°C under 8% CO 2 in a CO 2 incubator (PHC).When the cell density reached 3.0-6.0×10 6 cells per mL, cells were diluted to 3.0 ×10 6 cells per mL and P4 virus was added at a nal concentration of 7% (v/v).Cells were cultured at 30°C for 4 days in the presence of 10 mM sodium butyrate or 5 mM sodium valproate to enhance protein expression.
Harvested cells were suspended in buffer A (20 mM Tris-HCl buffer (pH 7.0) containing 150 mM NaCl) and protease inhibitor cocktail, and then disrupted by sonication on ice.After sonication, lysates were solubilized by 1.7% (w/v) LMNG or digitonin at 4°C for 1-2 h.After centrifugation at 18,000 rpm for 20 m, the supernatant was collected and loaded on to Anti DYKDDDDK tag Antibody Beads (Fuji lm).After extensively washing with buffer A containing 0.01% LMNG or 0.01% GDN proteins were eluted with buffer B (20 mM MES-NaOH (pH6.0)containing 150 mM NaCl and 0.01% LMNG or GDN101) supplemented with 5 M LiCl.The eluted proteins were further puri ed by gel ltration chromatography (Superdex 200 increase, GE Healthcare) in buffer B. The peak fractions were concentrated using Amicon Ultra centrifugal lter (100-kDa MW cut-off), frozen in liquid nitrogen and stored at -80°C until use.
Cryo-EM sample preparation and data acquisition.
Protein samples were adjusted to 2.5 to 5.0 mg ml -1 .For sample preparation of SIDT1 in the presence of dsRNA, 40 µM of 25 bp dsRNA (5'-CUGCGGACUAUUUGGCAAAGGAAGC) was mixed with protein samples in a buffer 100 mM sodium acetate (pH5.0), 10 mM MES-NaOH, 75 mM NaCl and 0.005% GDN.Threemicroliter aliquots of samples were placed onto a freshly glow-discharged Quantifoil holey carbon grids (R1.2/1.3,Cu, 300 mesh).After 4 s of blotting in 100% humidity at 6°C, the grid was plunged into liquid ethane using a Vitrobot MkIV (Thermo Fisher Scienti c).Cryo-EM data collection was performed using a Titan Krios G4 microscope (Thermo Fisher Scienti c), running at 300 kV and equipped with a Gatan Quantum-LS Energy lter (GIF) and a Gatan K3 camera in electron-counting mode, at the Cryo-EM facility of the University of Tokyo, Japan.Imaging was performed at a nominal magni cation of ×105,000, which corresponded to a calibrated pixel size of 0.83 Å px -1 .Each movie was recorded in CDS mode for 5.0 s and subdivided into 64 frames with an accumulated exposure of 65.7 e -per Å 2 at the specimen.The data were acquired by the image-shift method using the SerialEM software 31 .Cryo-EM data were analyzed using RELION 3.1 32 .Raw movie stacks were motion-corrected using MotionCor2 33 .The CTF parameters were determined using the CTFFIND4 program 34 .The data processing work ow is summarized in Supplementary Figs. 2 and 3.The nal resolution was estimated by gold-standard Fourier shell correlation (FSC) between the two independently re ned half maps (FSC = 0.143) 35 .The atomic model of hSIDT1 was manually accomplished using the Coot program 36 .The built model was re ned through alternating cycles using the Coot and PHENIX programs 37 .The re nement statistics are summarized in Supplementary Table 1.

Structure and sequence comparison.
Multiple sequence alignments of the SID-1 family proteins were performed using CLUSTAL Omega.Structure gures were prepared using the PyMOL Molecular Graphics System, Schrödinger, L., & DeLano, W or UCSF Chimera 38 .

Ceramidase assay.
C12 NBD Ceramide (d18:1/12:0) or NBD dihydro Ceramide (d18:0/12:0) was dissolved in chloroform and dried by speed-vac.1 nmol of NBD ceramide were suspended in the buffer containing 20 mM Tris-HCl (pH8.0), 150 mM NaCl and 0.01% GDN and sonicated.8 µg of SIDT1 was added to start the reaction in the presence or absence of methyl β-cyclodextrin (10 mM) and incubated at 37°C for 3 hr.The extraction solvent (chloroform: methanol, 1:1) was added with 3 volumes of reaction mixture to quench the reactions.After centrifugation, the organic phase was collected and dried by speed-vac.5 µl of each reaction mixture was spotted onto a TLC plate, developed in a solvent system consisting of chloroform, methanol, and 25% ammonium hydroxide (90:30:0.5).The TLC plate was dried and scanned by an imaging system.EMSA assay.

Figure 1 Structure
Figure 1

Figure 3 The
Figure 3