A ribosomal RNA fragment with 2′,3′-cyclic phosphate and GTP-binding activity acts as RIG-I ligand

Abstract

The RNA helicase RIG-I plays a key role in sensing pathogen-derived RNA. Double-stranded RNA structures bearing 5′-tri- or diphosphates are commonly referred to as activating RIG-I ligands. However, endogenous RNA fragments generated during viral infection via RNase L also activate RIG-I. Of note, RNase-digested RNA fragments bear a 5′-hydroxyl group and a 2′,3′-cyclic phosphate. How endogenous RNA fragments activate RIG-I despite the lack of 5′-phosphorylation has not been elucidated. Here we describe an endogenous RIG-I ligand (eRL) that is derived from the internal transcribed spacer 2 region (ITS2) of the 45S ribosomal RNA after partial RNase A digestion in vitro, RNase A protein transfection or RNase L activation. The immunostimulatory property of the eRL is dependent on 2′,3′-cyclic phosphate and its sequence is characterized by a G-quadruplex containing sequence motif mediating guanosine-5′-triphosphate (GTP) binding. In summary, RNase generated self-RNA fragments with 2′,3′-cyclic phosphate function as nucleotide-5′-triphosphate binding aptamers activating RIG-I.

The Endogenous RIG-I Ligand Is Generated in Influenza A-Virus Infected Cells

“…In order to monitor eRL generation in virus-infected cells, we employed a previously established PCR-strategy distinguishing between eRL and uncut ITS2-RNA ( Figure 1 a and [ 10 ]). Applying this method, two forward (fw)-primers are used: if the longer, uncut RNA sequence is present and the fw-uncut primer can bind, this leads to displacement of the fw-eRL primer, resulting in preferential amplification of the uncut template ( Figure 1 a, right panel, mock sample).…”

“…E2040S, Ipswich, MA, USA). eRL was produced as described [ 10 ]. Human IFN-α was quantified using the IFN alpha Human Matched Antibody Pair of eBiosciences (Cat.…”

“…4716728001, Basel, Switzerland). The ratio of eRL to uncut ITS2 was determined as described previously [ 10 ] using QuantiTect Reverse Transcription Kit (Cat. No.…”

“…In this case, the recognition of double-stranded RNA by the oligoadenylate synthetase leads to downstream activation of RNase L. The latter cleaves cellular RNA, which in turn functions as an endogenous RIG-I ligand (eRL). We identified the eRL as a specific sequence derived from the internal transcribed spacer 2 (ITS2)-region of 45S ribosomal RNA (rRNA) [ 10 ]. Unlike the classical RIG-I ligand, the eRL is not phosphorylated at the 5′ end, but its immunostimulatory activity depends on a 2′3′ cyclic phosphate and GTP binding by internal G-quadruplex structures [ 10 ].…”

“…We identified the eRL as a specific sequence derived from the internal transcribed spacer 2 (ITS2)-region of 45S ribosomal RNA (rRNA) [ 10 ]. Unlike the classical RIG-I ligand, the eRL is not phosphorylated at the 5′ end, but its immunostimulatory activity depends on a 2′3′ cyclic phosphate and GTP binding by internal G-quadruplex structures [ 10 ].…”