A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally dynamic RNA structure

Abstract

Folded RNA elements that block processive 5' → 3' cellular exoribonucleases (xrRNAs) to produce biologically active viral noncoding RNAs have been discovered in flaviviruses, potentially revealing a new mode of RNA maturation. However, whether this RNA structure-dependent mechanism exists elsewhere and, if so, whether a singular RNA fold is required, have been unclear. Here we demonstrate the existence of authentic RNA structure-dependent xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal-infecting flaviviruses. These xrRNAs have no sequence similarity to known xrRNAs; thus, we used a combination of biochemistry and virology to characterize their sequence requirements and mechanism of stopping exoribonucleases. By solving the structure of a dianthovirus xrRNA by X-ray crystallography, we reveal a complex fold that is very different from that of the flavivirus xrRNAs. However, both versions of xrRNAs contain a unique topological feature, a pseudoknot that creates a protective ring around the 5' end of the RNA structure; this may be a defining structural feature of xrRNAs. Single-molecule FRET experiments reveal that the dianthovirus xrRNAs undergo conformational changes and can use "codegradational remodeling," exploiting the exoribonucleases' degradation-linked helicase activity to help form their resistant structure; such a mechanism has not previously been reported. Convergent evolution has created RNA structure-dependent exoribonuclease resistance in different contexts, which establishes it as a general RNA maturation mechanism and defines xrRNAs as an authentic functional class of RNAs.

Keywords: RNA dynamics; RNA structure; exoribonuclease resistance; noncoding RNA maturation; single-molecule FRET.

Fig. 1.

Structure of an authentic xrRNA in dianthovirus 3′ UTRs. (A) Northern blot of total RNA from mock- or RCNMV-infected N. benthamiana. Probes are against viral genomic 3′ UTR and 5.8S rRNA. Full-length RNA1 and exoribonuclease-resistant degradation product (SR1f) are indicated. (B) In vitro Xrn1 degradation assay of ³²P-3′ end-labeled RCNMV 3′ UTR sequences. (C) In vitro Xrn1 degradation assay on minimal xrRNAs from RCNMV, SCNMV, and CRSV. Data are average (± SD) percent resistance from three individual experiments. (D) Secondary structure of the crystallized SCNMV RNA. Lowercase letters represent sequences altered to facilitate transcription. Non–Watson–Crick base pairs are in Leontis–Westhof annotation (41). The Xrn1 stop site is denoted by the labeled arrow. (E) Ribbon representation of the SCNMV xrRNA structure. Colors match those in D. The “single-stranded” RNA at the 3′ end was ordered due to intermolecular interactions in the crystal, described in Fig. 2 D–F and SI Appendix, Fig. S4.

Fig. 2.

Partial unfolding of SCNMV xrRNA contributes to Xrn1 resistance. (A, Left) Structure of Drosophila melanogaster Xrn1 [Protein Data Bank (PDB) ID code 2Y35] (3). Red spheres denote active site residues; black, 8-nt RNA substrate. Five single-stranded nucleotides span the distance from the active site to the enzyme’s surface. (A, Right) Secondary structure of SCNMV xrRNA, with the Xrn1 stop site indicated. Yellow denotes the portion of the P1 stem that must unwind. (B) Scheme of Xrn1 unwinding the P1 stem (red). (C) In vitro Xrn1 degradation assay on ³²P-3′ end-labeled WT and mutant SCNMV xrRNAs. Data are average (± SD) percent resistance from three individual experiments. (D) Scheme of putative PK interaction (SI Appendix, Fig. S4). (E) In vitro Xrn1 degradation assay on ³²P-3′ end-labeled WT and mutant SCNMV xrRNAs. Data are average (± SD) percent resistance from three individual experiments. (F) Northern blot of total RNA from two replicates of mock- or RCNMV-infected N. benthamiana. Probe: RCNMV 3′ UTR or 5.8S rRNA.

Fig. 3.

PK formation creates a protective ring around the 5′ end. (A) Crystallized SCNMV xrRNA. The arrow indicates the conformational change needed to form the PK interaction. (B) Modeled xrRNA PK conformation (all 3 nt of P1 unwound, PK formed). PK is in orange. The 3′ end (blue) encircles the 5′ end (red). (C) Structure of ZIKV xrRNA1 (PDB ID code 5TPY) with 5′ end (red), ring-like fold (blue), and PK (orange) matching the colors in A and B.

Fig. 4.

Conformational dynamics of SCNMV xrRNA. (A) smFRET labeling strategy. (B) smFRET histograms of WT xrRNA. (Left) RNA construct. Average FRET values are shown. n, number of molecules in the histogram. 18% and 82% of molecules populate the mid-FRET state and high-FRET state, respectively. (C) Representative smFRET traces of individual WT xrRNAs. (Left) Dynamic population. (Right) Stable high-FRET population. (D) smFRET histograms of PK_S2 xrRNA. (Left) Scheme of the RNA construct. (E) Representative smFRET traces of two individual PK_S2 xrRNA molecules.

Fig. 5.

Codegradational remodeling of SCNMV xrRNA structure. (A) Schematic of smFRET construct with an extended P1 stem (P1_EXT). (B) smFRET histograms of P1_EXT xrRNA. Average FRET values are shown. n, number of molecules in the histogram. (C) In vitro Xrn1 resistance assay of WT and P1_EXT xrRNA. (D and E) smFRET histograms of P1_EXT xrRNA pretreated with WT Xrn1 (D) or mutant Xrn1(D208A) (E). (F) Scheme of the conformational changes and codegradational remodeling of SCNMV xrRNA.