Lysosomal endonuclease RNase T2 and PLD exonucleases cooperatively generate RNA ligands for TLR7 activation

Highlights

• •RNase T2 and PLD exonucleases process RNA upstream of TLR7
• •PLD exonucleases release terminal 2′,3′-cyclic GMPs to engage TLR7 pocket 1
• •PLD enzymes are also critical to generate RNA fragments for TLR7 pocket 2
• •PLDs dimer formation is needed for RNA substrate processing

Summary

Toll-like receptor 7 (TLR7) is essential for recognition of RNA viruses and initiation of antiviral immunity. TLR7 contains two ligand-binding pockets that recognize different RNA degradation products: pocket 1 recognizes guanosine, while pocket 2 coordinates pyrimidine-rich RNA fragments. We found that the endonuclease RNase T2, along with 5′ exonucleases PLD3 and PLD4, collaboratively generate the ligands for TLR7. Specifically, RNase T2 generated guanosine 2′,3′-cyclic monophosphate-terminated RNA fragments. PLD exonuclease activity further released the terminal 2′,3′-cyclic guanosine monophosphate (2′,3′-cGMP) to engage pocket 1 and was also needed to generate RNA fragments for pocket 2. Loss-of-function studies in cell lines and primary cells confirmed the critical requirement for PLD activity. Biochemical and structural studies showed that PLD enzymes form homodimers with two ligand-binding sites important for activity. Previously identified disease-associated PLD mutants failed to form stable dimers. Together, our data provide a mechanistic basis for the detection of RNA fragments by TLR7.

Introduction

Toll-like receptor 7 (TLR7) is a key sentinel of the innate immune system that plays a critical role in detecting non-self RNA,^1,2 primarily from viral sources.^3,4 At the same time, TLR7 can also be erroneously activated by endogenous RNA, which has been implicated in the pathogenesis of several autoimmune diseases.⁵ Indeed, TLR7 activation must be tightly balanced, and much progress has been made in understanding the regulation of TLR7 responses at the level of the receptor and also its subcellular compartment, which plays an important role in regulating its activity.^6,7 However, the exact process by which RNA is made “visible” to TLR7 remains unclear. TLR7 is highly expressed in plasmacytoid dendritic cells (pDCs), positioning these cells as central players in RNA-mediated immune surveillance and response.⁸ TLR7 and its homolog TLR8 are positioned as homodimers in the endolysosomal compartment according to a rotational symmetry axis, while their leucine-rich repeat (LRR) ligand-binding domains face the lumen. Structural studies have identified two distinct bindings pockets that engage with distinct types of RNA molecules. TLR7 binds to guanosine with its first binding pocket, whereas the second binding pocket engages with pyrimidine-rich oligoribonucleotides (ORNs) that preferably contain two consecutive uridine nucleotides.^9,10 TLR8, on the other hand, binds to uridine with the first binding pocket, yet detects purine-terminated ORN fragments with the second binding pocket.¹¹ The engagement of the second binding pocket allosterically increases the affinity of the first binding pocket toward its ligand. The first binding pocket lies within the dimerization interface of these TLRs, and agonistic ligands within this pocket bridge the two TLR molecules. In the case of TLR7, this event results in the stable dimerization of this receptor and thereby results in the formation of a signaling-competent state. Interestingly, biochemical studies have shown that guanosine 2′,3′-cyclic monophosphate (2′,3′-cGMP) is a high-affinity ligand for the first pocket of TLR7,¹⁰ suggesting that it may be an endogenous agonist for TLR7.

We and others have found that the endolysosomal nuclease RNase T2 is indispensable for TLR8 activation.^12,13 RNase T2 cleaves single-stranded RNA (ssRNA) with a preference for purine-uridine motifs, thereby generating fragments that are terminated with a purine 2′,3′-cyclic phosphate and initiated with a 5′ hydroxyl uridine. Thereby, RNase T2 activity contributes to two critical steps: on the one hand, RNase T2 generates purine 2′,3′-cyclic phosphate-terminated fragments that engage pocket 2 and on the other hand it results in the increase of uridine, while the latter mechanism is not fully explored. Interestingly, loss-of-function studies have shown that RNase T2 also plays a role upstream of TLR7.¹⁴ However, despite its genetically proven involvement, the precise mechanistic role of RNase T2 in relation to TLR7 remains unclear, which prompted the initiation of this study.

Results

RNase T2 is required for TLR7 activation

To study TLR7 signaling in a physiologically relevant setting, we used the CAL-1 cell line, a human pDC line derived from a male patient with a blastic plasmacytoid DC neoplasm.¹⁵ CAL-1 cells express a similar repertoire of PRRs as primary pDCs, in particular TLR7 and TLR9, and respond to ssRNA and CpG DNA with the production of antiviral and pro-inflammatory cytokines. We stimulated wild-type (WT) or TLR7^−/y CAL-1 cells with the RNA-based TLR7 agonists RNA40² and RNA9.2s.¹⁶ RNA40 was delivered as a phosphodiester version (RNA40^O) as well as a stabilized phosphorothioate version (RNA40^S) (Figures 1A and 1B). The small molecule TLR7 agonists, R848 and 2′,3′-cGMP, and CpG DNA—either phosphodiester (CpG^O) or phosphorothioate (PTO)-stabilized (CpG^S) engaging TLR9—were used as controls. The ORNs triggered a robust type I interferon (IFN) response, albeit at a lower magnitude compared with R848, 2′,3′-cGMP, and CpG DNA (Figure 1A). At the concentration tested, the phosphorothioate-stabilized version RNA40^S was completely inactive in CAL-1 cells. As expected, ablation of TLR7 resulted in a complete loss of cytokine production for ORNs as well as for the small molecule TLR7 agonists. CpG DNA triggered IFN responses in both WT and TLR7-deficient CAL-1 cells, yet only when the phosphorothioate-stabilized ODNs were used (Figure 1A). Similar results were obtained in primary pDCs that responded to phosphodiester RNA40 as well as RNA9.2s, but not to the phosphorothioate-stabilized version of RNA40. R848 and 2′,3′-cGMP also triggered TLR7 activation, with R848 being a more potent activator compared with 2′,3′-cGMP (Figure 1C). Addressing the role of RNase T2 in this context revealed that the ORNs were indeed completely dependent on this enzyme for their immune-stimulatory activity, while its requirement could be bypassed by using R848 or 2′,3′-cGMP as direct pocket 1 agonists (Figure 1D). In light of the notion that RNase T2 generates 2′,3′-cGMP-terminated RNAs and the fact that 2′,3′-cGMP levels are strongly decreased in RNASET2^−/− cells (Greulich et al.¹² and below), we addressed whether RNase T2 could liberate 2′,3′-cGMP from ssRNA in vitro. To address this, we designed an ORN in which an RNase T2 cleavage site (GU) was positioned directly at the 5′ end, while we also generated ORNs in which this dinucleotide motif was stepwise moved to the 3′ end of the ORN (Figure 1E). Testing these ORNs using recombinant RNase T2 revealed that RNase T2 required at least two nucleotides 5′ to its recognition motif to cleave. These results suggested that RNase T2 on its own was not able to release 2′,3′-cGMP within the endolysosomal compartment, but rather additional enzyme activities were required for this.