SisCsx1 is a hexameric RNase

SisCsx1, as other members of the Csm6/Csx1 family5,12, is composed of an N-terminal CRISPR-Cas-associated Rossmann Fold (CARF) domain connected to an α-helical higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain by a helix-turn-helix (HTH) domain (Fig. 1a). We expressed SisCsx1 in S. islandicus, and the protein was purified as previously described24. A SEC-MALLS analysis revealed that SisCsx1 is an oligomer of 300 kDa corresponding to a hexameric assembly (Supplementary Fig. 2). A cryoEM map of the complex at medium resolution (Fig. 1b, Supplementary Fig. 3, Supplementary Table 1) was used as a model to solve the phase problem by molecular replacement in an apo SisCsx1 crystal belonging to the P2 1 space group (Methods). The Cα backbone built in this map was later combined with an iodine derivative of a better diffracting crystal of SisCsx1 in the space group I2 1 2 1 2 1 to determine the structure of the apo SisCsx1 to 2.9 Å. Subsequently the apo SisCsx1 structure was used two determine by molecular replacement two SisCsx1 conformations in complex with cOA 4 at 3.1 and 2.7 Å resolution respectively (Supplementary Table 2, Methods), thus providing the molecular details of the RNase activation by the secondary messenger.

Fig. 1 Structure of SisCsx1 endoribonuclease. a Domain organization of SisCsx1. b Cartoon model of the apo SisCsx1 crystal structure superimposed on the cryoEM map used to solve the phase problem. Each monomer is colored differently. Asterisks indicate the cOA 4 binding sites and the black circles the catalytic pockets. c The monomers of SisCsx1 are twisted around the dimer axis. Detailed view of one of the dimers with the different domains showing the cOA 4 -binding and catalytic pockets. The monomers are shown following the color scheme in panel a. A pale tone of the same color is used to differentiate each monomer. d Top view of the cOA 4 -binding pocket between the CARF domains colored in magenta. (e) Bottom view of the SisCsx1 dimer showing the RNase catalytic site built by the HEPN domains (colored in blue) Full size image

The SisCsx1 monomers are curled around the two-fold axis to form a dimer (Fig. 1c), and three of these dimers oligomerize through their HEPN domains into an equilateral trimer, thus assembling into a hexamer (Fig. 1b). Each dimer contains a cOA 4 binding site at the vertex of the triangular assembly (Fig. 1d), and a catalytic pocket located 70 Å away inside the triangular oligomer along the two-fold dimer axis (Fig. 1e). The two-fold axis bisects the cOA 4 and catalytic pockets in the dimer. Hence, the triangular assembly forms two equilateral triangles, one inside the other, with the vertexes formed by the CARF and HEPN domains, building the cOA 4 binding and catalytic sites in each dimer (Fig. 1b, c).

Despite the conservation of the domain architecture between Csm6 and Csx1 RNases, a comparison of SisCsx1 with ToCsm6, TtCsm6, PfuCsx1 and SsoCsx1 reveals large differences in protein sequence (Supplementary Fig. 1) and topology, especially in the HTH and HEPN domains (Fig. 2a, b). Only the CARF domain displays structural conservation between these proteins. These differences determine that in SisCsx1, the monomers are curled around the dimer axis (Fig. 1c, Fig. 2b), while the dimers of the other Csx1 and Csm6 RNases interact along the axis from the CARF to the HEPN domains (Fig. 2b). In addition, the sequence alignment and the superposition reveal a unique insertion region in the HEPN domain of SisCsx1, which is the responsible for the protein hexamerization (Supplementary Fig. 1, Fig. 2a, b). Collectively, these observations suggest that while cOA 4 binding is conserved, the mode of RNase regulation may be different.

Fig. 2 Comparison between type III CRISPR-associated ribonucleases. a Structural comparison between SisCsx1 and the dimeric SsoCsx1, PfuCsx1, ToCsm6, TtCsm6. Ribbon representation of the SisCsx1 (green) and PfuCsx1, SsoCsx1 and TtCsm6 (orange) monomers which have been superposed using the DALI server. SsoCsx1 (PDB 2I71) r.m.s.d. to SisCsx1 is 5.1 Å for 184 residues out of 376 with an identity of 20%, for PfuCsx1 (PDB 4EOG) r.m.s.d. to SisCsx1 is 6.1 Å for 200 residues out of the 466 of the protein with an identity of 12%, for ToCsm6 (PDB 6O6S) r.m.s.d. to SisCsx1 is 6.0 Å for 194 residues out of the 433 of the protein with an identity of 15%, while for TtCsm6 (PDB 5FSH) the r.m.s.d. is 5.0 Å for 224 out of the 448 of the protein with an identity of 11%. b Comparison of the dimeric arrangements of SsoCsx1, PfuCsx1, ToCsm6, TtCsm6 with SisCsx1 Full size image

SisCsx1-cOA 4 complex displays different conformations

The SisCsx1-cOA 4 complex structure provides evidence of how the cOA 4 activator binding to the CARF domain triggers RNA degradation. We synthesized cOA 4 using purified Type III-B SisCmr−α complex (Supplementary Fig. 4), and observed that the binding of cOA 4 at room temperature, barely activates SisCsx1, while heating the reaction mixture triggered full RNase activity (Supplementary Fig. 5a). As with other HEPN domain nucleases, SisCsx1 does not require metal cofactors for its activity (Supplementary Fig. 5b). To explore these differences, we crystallized the SisCsx1-cOA 4 complex by soaking the compound in crystals of the apo SisCsx1 at 25 °C, and we also co-crystallized the complex after heating up the mixture of cOA 4 with the isolated SisCsx1 to monitor possible conformational changes that could elicit the RNase activity by the presence of cOA 4 . The different crystallization procedures yielded the same type of crystals (Supplementary Table 2), which displayed two different conformations of cOA 4 (conf1 and conf2) in the binding pocket, along with rearrangements of the protein moiety (Fig. 3, Supplementary Fig. 6, Supplementary movie 1, 2).

Fig. 3 cOA 4 configurations in the binding pockets. a Detailed view of the cOA 4 binding site in conf1 including its interactions with key side chains. This configuration is observed in two of the binding pockets of the hexamer (indicated with an asterisk). b The third pocket in conf1 exhibits a different configuration of cOA 4 (indicated with a black circle). c Detailed view of cOA 4 binding site in conf2 including key side chain. This configuration is visualized in two of the binding pockets of the hexamer (indicated with a black square). d The conformation of cOA 4 is also different in the third binding pocket in conf2 (indicated with a black triangle). The 2mFo-DFc electron density maps in panels A-D are displayed at 1.1 σ contour. Views of the omit maps of a–d panels are provided in Supplementary Fig. 7B Full size image

The dimeric assembly of SisCsx1 is essential for the formation of the cOA 4 binding and catalytic pockets by the CARF and HEPN domains. The curled arrangement of the monomers positions the CARF domain of one monomer on top of the HTH domain of the other, while the regions spanning residues 405–427 in the HEPN domains form two “pins” that clip each monomer between the HTH and the HEPN domains (Fig. 1c–e, Fig. 2b). The cOA 4 binding cleft is formed by the loops connecting several secondary structure elements of the CARF domains, including residues in loops spanning residues 6–18, 28–30, 47–54, 92–100, 153–158 and 178–188. The conformational changes upon cOA 4 binding induce a reshaping of the loops, so that the oval cleft observed in the apo structure shifts to form a cruciform pocket, in which the cOA 4 is bound (Fig. 3, Supplementary movie 1). This reconfiguration of the cOA 4 binding pocket promotes a change in the electrostatic potential of the cavity turning the neutral potential observed in the apo structure to the polar cleft observed in conf1 and conf2 (Supplementary Fig. 7A).

A conf1-conf2 transition triggers RNase activity

Each Csx1 monomer hosts a pair of adenines of cOA 4 , A1-A2 and A3-A4 (Fig. 3). A comparison of the cOA 4 conformations in conf1 and conf2 revealed that the molecule undergoes a conformational change in the binding pockets of the hexamer (Fig. 3, Supplementary movie 1). In the conf1 configuration, cOA 4 displays a “wrinkled” conformation of the cyclic compound with all bases in a syn or near to syn arrangement in two of the second messenger binding pockets (Fig. 3a, Supplementary Fig. 6). The A1 base makes polar interactions with the main chain of S51 and A1 ribose 2-OH interacts with the main chain of D10. A2 is stabilized in the syn configuration by polar contacts with the main chains of S15 and F29. All contacts in the A3, A4 pair are made by residues in the second monomer where these nucleotides are located. The A3 base is stabilized in a near syn configuration by interactions with the side chain of S51′ as well as with the main chains of S51′ and S98′ while A3 ribose 2-OH contacts with the side chain of Y14′. A3 phosphate interacts with G182. The A4 phosphate contacts the G156′ amide thanks to N158, which arranges the loop by contacting with the main chain of T154′ and G156′ to promote that interaction. Finally, the A4 base is stabilized in the syn configuration by polar contacts with the side chain of Y19′ and the main chain of S15′ and F29′. Interestingly, A4 phosphate is stabilized by contacting with the side chains of N158 and N158′ through a water molecule. Multiple polar and hydrophobic contacts with the main and side chains of several residues in the cleft contribute to accommodate the cOA 4 molecule. However, in the third binding site, cOA 4 displays an alternate configuration syn and anti, due to a conformational change to the anti configuration in A2 and A4 by stabilizing their contacts with Y19 and Y19′, respectively (Fig. 3a, b, Supplementary Fig. 6).

The disposition of cOA 4 in conf2 reveals a rearrangement of the cyclic compound, whose configuration coincides again in two of the three binding pockets (Fig. 3c, d, Supplementary Fig. 6). An inversion of the phosphate groups forces the P = O bonds of A1 and A3 towards the bottom of the pocket, inducing the changes from syn to the anti conformation of the A2 and A4 nucleotides, thus transiting from the “wrinkled” conformation observed in conf1 to a more symmetrical arrangement in conf2. This change in the phosphate backbone and base configuration is supported by the Fo-Fc omit maps of the cyclic compound and is accompanied by new polar and hydrophobic interactions in the binding site (Supplementary Fig. 7b-c, Supplementary movie 1). The phosphate inversion in cOA 4 is stabilized in both cases by hydrogen bonds of the phosphate groups of A1 and A3 with N158 and N158′ through a water molecule. A rotamer change of H155 in both monomers allows the polar interaction of the imidazole group with the A2 and A4 bases in each monomer, thus positioning the cyclic compound into the cruciform pocket. In addition, A2 and A4 phosphates are stabilized by the side chains of Y14 and Y14′. Finally, the anti conformation of A2 and A4 is stabilized by interactions of the base with the main chain of S15, S15′, F29′, and V180′ and the OH of Y19′, while the syn configurations observed for A1 and A3 are stabilized by polar interactions with the main chain of D10, D10′, S51, S51′, and S98 and hydrophobic contacts with M181 side chain in both monomers. The alternate configuration observed in the third binding pocket is accompanied by a reshaping of the polar and hydrophobic interactions in the binding site (Fig. 3c, d, Fig. 4b–d, Supplementary Fig. 6).

Fig. 4 Comparison of SisCsx1:cOA 4 and ToCsm6:cOA 4 CARF domains and RNase deactivation. Detailed view superimposition at the CARF domains of SiScsx1:cOA 4 complex in Conf1 asterisk (a) or Conf1 solid circle (b) vs. ToCsm6:cOA 4 . Detailed view superimposition at the CARF domains of SiScsx1:cOA 4 complex in Conf2 solid square (c) or Conformation 2 solid triangle (d) vs. ToCsm6:cOA 4 . S. islandicus Ring nucleases 0455 (e) and 0811 (f) deactivate the RNase activity of SisCsx1. Increasing concentrations of both Ring nucleases from 10 nM to 150 nM were incubated with 25 nM of cOA 4 at 70 °C for 30 min followed by addition of 18 nM of SisCsx1 and 2.5 μΜ of RNA1 and further incubation at 70 °C for 5 min. The reactions were then separated using 15% Novex TBE-urea gel (Invitrogen). g Cleavage of cOA 4 by Ring nucleases 0455 and 0811. On the left side of the gel cOA 4 is incubated with Ring nucleases 0455 and 0811. The cyclic compound migrates faster than the linear product generated by the ring nucleases. On the right side of the gel SisCsx1 has been included in the reaction showing that it does not cleave cOA 4 and protects its degradation by the ring nucleases. Each experiment for the e, f, g panels has been repeated at least three times. The error bars represent the s.d. Source data are provided as a Source Data file for 4E-G Full size image

Comparison of cOA 4 binding by ToCsm6 and SisCsx1

The amino acids involved in the cOA 4 binding are well-conserved between the CARF domains of Csx1 and Csm6 ribonucleases. These include SisCsx1 residues Y14, Y19, S51, T154, H155, G156, N158, while the hydrophobic V180 and M181 could be substituted by other non-polar residues (Supplementary Fig. 1). A recent analysis of the ToCsm6-cOA 4 complex shows that upon binding, the cyclic molecule is cleaved by the CARF domain, thus deactivating its RNase activity22. However, W14 and H132, which have been identified as important residues in the CARF domain of ToCsm6 for cOA 4 cleavage, are not well conserved among the Csm6/Csx1 proteins. A genomic analysis of 294 prokaryotic genomes shows that these two residues are present in less than 10% of the Csm6 proteins and they are frequently replaced by Y and S respectively (Supplementary Data 1). In the case of the Csx1 family only 5% of the proteins encode these residues in the equivalent positions, the H residue is commonly found while W is substituted by other amino acids, generally by Y, as observed in SisCsx1 (Supplementary Fig. 1, Supplementary Data 1). A superposition of the ToCsm6 and SisCsx1 structures shows that the conformations of the CARF domain and the cyclic molecule are different (Fig. 4a–d), specially because cOA 4 is not cleaved in the SisCsx1 structures (Fig. 3, Supplementary Fig. 7B-C). ToCsm6 cOA 4 degradation involves W14, H132 and N135 residues, corresponding to Y14, H155 and N158 in SisCsx1 (Fig. 4a–d), consequently, the key W14 is substituted by Y14 in SisCsx1 altering the possible catalytic configuration. In addition, the configuration of N135 side chain, which has been linked to the cleavage of cOA 4 22, can be found pointing inwards (non-cleaved cOA 4 ) or outwards (cleaved cOA 4 ) of the cyclic oligoadenylate ring. In contrast, we observed that N158 in SisCsx1, is pointing inwards in the apo, conf1 and conf2 structures (Fig. 4a–d). Overall, these observations indicate that the CARF domain of SisCsx1 cannot cleave cOA 4 .

SisCsx1 RNase activation by cOA 4 is regulated by Ring nucleases

To elucidate whether SisCsx1 can cleave cOA 4 , we performed a cleavage assay of the cyclic molecule in the presence and absence of Ring nucleases, which have been shown to deactivate the cOA 4 -stimulated RNAse activity of the Csx1 family members via degradation of the cyclic molecule23. We tested whether SisCsx1 could cleave cOA 4 or whether its activation by cOA 4 was affected by the presence of Ring nucleases (Fig. 4e–g). No cleavage of the cyclic molecule by SisCsx1 could be observed in the absence of Ring nucleases, as it was equally observed for the dimeric SsoCsx123. However, a substantial decrease of SisCsx1 RNase activity could be observed in the presence of Ring nucleases, thereby endorsing the deactivating effect caused by the action of these enzymes on the cyclic compound23. Consequently, the lack of conservation of key amino acids for catalysis (ToCsm6 W14 and H132) in the Csm6 family and the absence of cOA 4 cleavage in SisCsx1, suggest that different RNase regulatory mechanisms are employed by other members of the Csm6/Csx1 families.

SisCsx1 activation by cOA 4 displays cooperativity

SisCsx1 binding of cOA 4 is cooperative with a Hill coefficient n, of 2 and a K d of ~18 nM (Supplementary Fig. 8A). Single mutants in the CARF domain such as N158A, H155 A and S51A are still able to display detectable RNase activity and a double mutation H155D/N158D is needed to abrogate RNase activity (Supplementary Fig. 8B). The shape of the pocket and the conformational change upon second messenger binding is reminiscent of a “tippy tippy tap” paper toy, i.e., the configuration of the binding pocket is coupled with conformational changes in the cOA 4 molecule to accommodate the compound. Collectively, these findings indicate that the configuration of cOA 4 undergoes conformational changes between conf1 and conf2, which are transmitted to the HTH domain for RNase activation (Supplementary movie 2).

The structure of SisCsx1 and cOA 4 binding data strongly suggested that the endoribonuclease activation by the cyclic compound is cooperative. The SisCsx1 hexamer is built because of a split helical insertion (V365-N400) and the last helix at the C-terminus (D445-A454) in the HEPN domain (Fig. 5, Supplementary Fig. 1). The α-helix spanning residues 375-391, establishes a series of contacts with the same segment in the adjacent monomer with the helices inversely oriented (Fig. 5a), and a well-conserved cysteine bridge (C361-C380) positions the hexamerization helix. The helices fit into each other and the central interaction surface is mainly hydrophobic. The hydrophobic interface is flanked by several polar interactions (Fig. 5b). To understand how catalysis works upon cOA 4 binding, we performed activity experiments, which demonstrated that SisCsx1 displays cooperative catalysis (Fig. 5c, Supplementary Fig. 8d). The cleavage of a ssRNA substrate was quantified in the presence of increasing amounts of cOA 4 . The sigmoidal curve displayed a Hill coefficient n, of 2.5, indicating a cOA 4 dependent cooperativity to catalyze phosphodiester hydrolysis of the ssRNA substrate. The hydrogen bond pattern of the interaction surfaces between the HEPN domains of the hexamer is different in the apo, conf1 and conf2 structures (Fig. 5d), revealing that cOA 4 binding induces a remodeling of the interactions in the catalytic domains. While the central hydrophobic interfaces are very similar in the three conformations, an interchangeability of the hydrogen bonding scheme of R391, Y387, K446, A453 and K364 could be observed between the apo, conf1 and conf2 structures (Fig. 5d, Supplementary Table 3, Supplementary movie 3). These interactions occurring at the C-terminus of each monomer appear to provide a signaling pathway within the HEPN domains of the hexamer.

Fig. 5 Molecular basis of SisCsx1 cooperativity in ssRNA degradation. a Detailed view of the SisCsx1 helices building the hexamer interface. The sketch shows the domain organization of one of the SisCsx1 dimers in the hexameric arrangement. b Electrostatic potential of the interaction surface between the HEPN domains. c SisCsx1 RNase activity displays a cooperative behavior in the presence of increasing concentrations of cOA 4 . The experiment was repeated 3 times, the error bars represent the s.d. (Supplementary Fig. 8D). d Detailed view of the oligomerization interface in the apo, conf1 and conf2 structures showing the interplay of the hydrogen bond network between R391, Y387, K446, A453, and K364. e Detailed view of the pin of one of the HEPN domains (residues 405–427), which inserts in the neighbouring monomer. This feature is conserved in all the catalytic sites of the assembly forming a staggered arrangement (See also Supplementary movie 3-4) Full size image

Interestingly, this region is in the neighborhood of the pin extension from another monomer, which fits snugly between the HEPN and HTH domains, providing a staggered interaction of the catalytic sites in the hexamer (Fig. 1e). This arrangement of the hexamer provides the scaffolding supporting a sequential cooperativity mechanism transmitting the cOA 4 binding signal from the CARF through the HTH (Supplementary movie 2) domains to the catalytic sites in the different HEPN domains (Supplementary movie 4) through the hexamer oligomerization interface (Supplementary movie 3). To test this observation, we designed the I383A/L390D/R391A mutant, which disrupts key interactions for oligomerization in the helical insertion (residues 375-391) without compromising dimer assembly (Supplementary Fig. 2). The I383A/L390D/R391A mutant displayed cOA 4 binding (Supplementary Fig. 8a); however, its RNase activity is severely affected indicating that the hexamer is essential for ssRNA decay (Supplementary Fig. 8c).

The high RNase activity (Supplementary Fig. 5a) and the changes observed in the HEPN interfaces, suggest that upon cOA 4 binding, the change of conformation between conf1 and conf2 (Fig. 2) would be coupled with the full activation of the RNase activity by signaling the conformational change through the HTH and the HEPN pins (Supplementary movie 4), thus remodeling the contacts between the HEPN hexamer interfaces (Fig. 5, Supplementary movie 3). Collectively, the analysis indicates that the interplay of the interactions in the HEPN domain interfaces is a consequence of the intramolecular signaling of cOA 4 binding, which is propagated through the hexamer to induce RNase activity in the catalytic sites.

SisCsx1 catalytic center

The active center cleft comprises R354, D372, R389, R399, N400, M403, H404, T409, and D410 residues (Supplementary Fig. 1, Fig. 6a, b). The SisCsx1 dimeric arrangement results in a symmetric placement of these amino acids, creating a diagonal electropositive stripe flanked by two electronegative regions on each side (Fig. 6a). Changes in the interfaces of HEPN domains observed in the apo, conf1, and conf2 structures induce subtle conformational differences in the side chains of these residues (Supplementary movie 5). In fact, these rearrangements appear to be responsible for the RNase activation. We attempted to co-crystallize SisCsx1 with an excess of cOA 4 or ssRNA, but we were unable to visualize the cyclic compound or the oligonucleotide in the active site in the HEPN domain. However, we observed two additional densities from sulfate ions present in the crystallization solution associated with R354 and R354′ in the catalytic pocket (Supplementary Fig. 9A). This finding, together with our mutational analysis (Supplementary Fig. 8C) and the electrostatic potential of the catalytic site, offered some hints on orientation of the ssRNA phosphate backbone in the active center. To understand the mechanism of phosphodiester hydrolysis, we utilized the identified key residues in an information-driven flexible docking modeling approach25,26 of a polyC oligonucleotide in the catalytic pocket (Fig. 6a). As expected, the docking results revealed a ssRNA backbone located on the diagonal electropositive stripe, with R354, R399, R389, N400 in possible contact distances of the phosphate groups (Fig. 6a, b). In the model, the bases of the ssRNA can interact with the electronegative patches in the upper left and lower right corners of the catalytic cleft where T385, D410, S408, T409, and D372 are located (Fig. 6a, b). The position of the phosphate backbone places one of the phosphodiester bonds between the H404 and H404′ in the center of the cavity, suggesting that these residues are directly involved in catalysis. This conclusion is supported by the observation that the H404A mutation abolishes cleavage (Supplementary Fig. 8C) and the abrogation of SisCsx1 activity at high pH (Supplementary Fig. 5C). Histidine is a residue conducive for phosphodiester catalysis as it can accept and donate protons since the pKa value of histidine is close to neutral. Because of the dual role of histidine in catalysis and depending on whether the ssRNA enters the symmetric catalytic site with a 5′-3′ or 3′-5′ polarity, one of the H404 residues would be positioned to activate the 2′-OH of the ribose, thus initiating the cleavage reaction. The transition state could be stabilized by R399 or N400, which are positioned in the vicinity of the H404.

Fig. 6 Catalytic pocket of SisCsx1 in the HEPN domains and cleavage specificity. a Docking of polyC ssRNA (stick model) into the catalytic pocket of a SisCsx1 dimer. b The docked polyC molecule and the positioning of the 2′-OH of ribose with respect to one of the H404 residues for the initiation of phosphodiester hydrolysis. Residues from each monomer are displayed in cyan and blue respectively. The residues in the later one is labelled with ´. c SisCsx1 shows a strong preference for the hydrolysis of phosphodiester bonds between 5′-C–C-3′ located in the central section of ssRNA, as determined by activity assays using RNA1–RNA4. The substrates are at least 9–12-nt-long, as determined by activity assays with RNA5–RNA9. Red slashes in the gel and ssRNA substrates (below the gel), the cleaved bond. The experiment has been repeated three times. Source data are provided as a Source Data file for Fig. 6c Full size image

Some similitudes between SisCsx1 and another RNases with histidines in their catalytic centers can be found. An example is the dimeric RNase L, an interferon activated RNase, which contains two histidines in the active center. However, phosphodiester catalysis by RNase L supposedly involves only one of them27. In SisCsx1, the two histidines are close and the movements of the HEPN domains observed in the different structures do not suggest large rearrangements in the active site after cOA 4 binding (Supplementary movie 5) as observed in RNase L, suggesting that both histidines are involved in catalysis. The proposed mechanism more closely resembles that of RNase A, a classical ssRNA endonuclease, which also contains two histidines in the catalytic site but lacks the two-fold symmetry observed in the SisCsx1 dimer. These histidines alternate their role as proton donor and acceptor during catalysis28. Although, further analysis will be needed to fully decipher SisCsx1 ssRNA phosphodiester hydrolysis, the current evidence suggests that SisCsx1 catalytic center would allow phosphodiester hydrolysis independently of the ssRNA substrate polarity in the pocket.

SisCsx1 cleaves phosphodiester bonds between cytidines

Some endoribonucleases can catalyze reactions involving RNA molecules containing specific sequences, structures or sequences within a specific structure providing tools for RNA manipulation. However, a possible SisCsx1 cleavage specificity was ambiguous24. In contrast with previous suppositions, we observed that SisCsx1 exhibits a strong preference for cleaving substrates with phosphodiester bonds between 5′-C–C-3′, and with a minor and very low efficiency ones with phosphodiester bonds between 5′-U-C-3′ and 5′-U-A-3′ (Fig. 6c). Such cleavage preference likely arises from the putative contacts between the cytosine base and the acidic regions of the pocket (Fig. 6a, b). Further activity experiments with different ssRNA substrates revealed that the cleavage occurs in the central part of the oligonucleotide, with the efficiency of the otherwise preferential 5′-C–C-3′ cleavage greatly diminished when the phosphodiester bond between cytidines is located at the 5′- or 3′-end of the substrate molecule (Fig. 6c). The cleavage assay also reveals that the ssRNA must be at least 12nt long to observe cleavage. As the distance between the catalytic centres is around 35 Å, a ssRNA substrate between 10-12 nucleotides could interact with two catalytic sites, suggesting that they may work on the same RNA molecule simultaneously, which would agree with the observed cooperativity. Based on the data presented in this manuscript, we propose a sequential cooperativity model for SisCsx1 RNase. In this model, cOA 4 binding subsequently signals to the monomers in the other two dimers of the hexamer to facilitate binding of other cOA 4 molecules promoting full activation of the RNase activity (Fig. 7).