A folded viral noncoding RNA blocks host cell exoribonucleases through a conformationally dynamic RNA structure Anna-Lena Steckelberga, Benjamin M. Akiyamaa, David A. Costantinoa, Tim L. Sitb, Jay C. Nixc, and Jeffrey S. Kiefta,d,1 aDepartment of Biochemistry and Molecular Genetics, School of Medicine, University of Colorado, Aurora, CO 80045; bDepartment of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC 27606; cMolecular Biology Consortium, Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; and dRNA BioScience Initiative, School of Medicine, University of Colorado, Aurora, CO 80045 Edited by Joan A. Steitz, HHMI and Yale University, New Haven, CT, and approved May 7, 2018 (received for review February 8, 2018) Folded RNA elements that block processive 5′ → 3′ cellular exor- maturation pathway that relies on a compact and unusual RNA ibonucleases (xrRNAs) to produce biologically active viral noncoding fold. Specifically, these xrRNAs form an interwoven pseudoknot RNAs have been discovered in flaviviruses, potentially revealing (PK) conformation centered on a conserved three-helix junction, a new mode of RNA maturation. However, whether this RNA creating a protective ring-like structure that wraps around the 5′ structure-dependent mechanism exists elsewhere and, if so, end of the xrRNA (7, 18). This unique topology acts as a me- whether a singular RNA fold is required, have been unclear. Here chanical block to Xrn1 when the ring-like structure braces against we demonstrate the existence of authentic RNA structure-dependent the surface of the enzyme (7, 19, 20). To date, this topology and xrRNAs in dianthoviruses, plant-infecting viruses unrelated to animal- resultant mechanism has been observed only in the flaviviruses. infecting flaviviruses. These xrRNAs have no sequence similarity to Recruiting Xrn1 to a larger precursor RNA and then blocking known xrRNAs; thus, we used a combination of biochemistry and the enzyme using a compact structured RNA element to gen- virology to characterize their sequence requirements and mechanism erate a biologically active smaller RNA could represent a useful of stopping exoribonucleases. By solving the structure of a diantho- general mechanism for RNA maturation. Consistent with this, virus xrRNA by X-ray crystallography, we reveal a complex fold that is xrRNAs have been identified in a broad range of flaviviruses, very different from that of the flavivirus xrRNAs. However, both including those that are tick-borne, those specific to insects, and versions of xrRNAs contain a unique topological feature, a pseudo- those with no known arthropod vector (20). Based on secondary knot that creates a protective ring around the 5′ end of the RNA structural patterns, flaviviral xrRNAs can be grouped into two structure; this may be a defining structural feature of xrRNAs. classes; all xrRNAs from mosquito-borne flaviviruses identified Single-molecule FRET experiments reveal that the dianthovirus to date belong to class 1 xrRNAs, while others compose class “ 2 xrRNAs (20). The 3D structure of a class 2 flavivirus RNA has xrRNAs undergo conformational changes and can use codegrada- “ tional remodeling,” exploiting the exoribonucleases’ degradation- not been solved; thus, for simplicity, here we use the term fla- vivirus xrRNA” to refer to the well-characterized class 1 flavivi- linked helicase activity to help form their resistant structure; such a rus xrRNAs unless specified otherwise. In addition, RNA mechanism has not previously been reported. Convergent evolution sequences that appear to resist 5′ → 3′ exoribonuclease degra- has created RNA structure-dependent exoribonuclease resistance in dation have been identified in a few other virus families (21–25). different contexts, which establishes it as a general RNA matura- However, nothing is known about the molecular processes or tion mechanism and defines xrRNAs as an authentic functional structures of putative xrRNAs outside the flavivirus family. We class of RNAs. do not know whether other candidate exoribonuclease–resistant noncoding RNA maturation | RNA structure | RNA dynamics | single-molecule FRET | exoribonuclease resistance Significance Folded RNA elements are essential for diverse biological pro- uring eukaryotic cellular RNA decay, 5′ → 3′ hydrolysis by cesses. Recently discovered examples include viral xrRNAs, which an XRN protein is important for degrading decapped mes- D co-opt the cellular RNA decay machinery within a novel non- senger RNA (mRNA) and other 5′-monophosphorylated RNAs coding RNA production pathway. Here we characterize an xrRNA including fragments of endonucleolytic cleavage, ribosomal RNA with no apparent evolutionary link or sequence homology to (rRNA), and transfer RNA (tRNA). Xrn1 is the dominant cyto- ′ → ′ those described previously. Our results show that xrRNAs are an plasmic 5 3 exoribonuclease in most eukaryotic cells (1, 2), authentic class of functional RNAs that have arisen independently where its processive translocation-coupled unwinding of RNA helices in different contexts, suggesting that they may be widespread. allows it to efficiently hydrolyze structured RNA substrates with- The detailed 3D structure of one of these xrRNAs reveals that an out releasing partially degraded intermediates (3, 4). Xrn1 plays underlying structural topology may be the key feature that a central role in constitutive mRNA turnover and RNA quality confers exoribonuclease resistance to diverse xrRNAs. control and has been implicated in degrading viral RNAs as part of the cell’s antiviral response (5). Despite the efficiency of Xrn1, Author contributions: A.-L.S., B.M.A., T.L.S., and J.S.K. designed research; A.-L.S., B.M.A., some viruses have evolved RNA sequences that robustly block the D.A.C., T.L.S., and J.C.N. performed research; A.-L.S., B.M.A., T.L.S., J.C.N., and J.S.K. ana- enzyme’s progression. This ability is conferred by their folded 3D lyzed data; and A.-L.S., B.M.A., D.A.C., and J.S.K. wrote the paper. structures without the help of accessory proteins; thus, we refer to The authors declare no conflict of interest. them as Xrn1-resistant RNAs (xrRNAs) (6, 7). This article is a PNAS Direct Submission. xrRNAs were originally identified in the positive-sense RNA Published under the PNAS license. genomes of mosquito-borne flaviviruses (e.g., Dengue virus, Zika Data deposition: The atomic coordinates and structure factors have been deposited in the virus, West Nile virus) where they protect the viral genome’s3′ Protein Data Bank, www.wwpdb.org (PDB ID code 6D3P). UTR from degradation, generating biologically active noncoding 1To whom correspondence should be addressed. Email: [email protected]. RNAs involved in cytopathic outcomes and pathogenicity during This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. infection (8–17). Thus, these mosquito-borne flaviviruses usurp 1073/pnas.1802429115/-/DCSupplemental. Xrn1’s powerful degradation activity as part of an elegant RNA Published online June 4, 2018. 6404–6409 | PNAS | June 19, 2018 | vol. 115 | no. 25 www.pnas.org/cgi/doi/10.1073/pnas.1802429115 Downloaded by guest on September 29, 2021 RNAs operate with the help of protein factors or if they are stably structured elements sufficient to block processive degra- dation. If RNA structure plays a role in diverse putative xrRNAs, we do not know whether there is a universal structural feature that confers Xrn1 resistance or many structures that can block Xrn1. The degree to which different xrRNAs operate by creating a mechanical block for Xrn1 versus using some other means to stop the enzyme remains unexplored. Indeed, if viruses outside the flavivirus family use diverse sequences and structures to block exoribonucleases in a programmed way, this would indicate that this mechanism is a general pathway, and that xrRNAs are a true functional class of RNAs. To address these questions, we investigated an unexplored po- tential xrRNA sequence from the 3′ UTR of plant-infecting dia- nthoviruses (26, 27) (SI Appendix,Fig.S1A). Dianthoviruses belong to the Tombusviridae family and have a bipartite positive-sense single-stranded RNA genome consisting of RNA1 and RNA2 (26). A recent study described the accumulation of a noncoding Fig. 1. Structure of an authentic xrRNA in dianthovirus 3′ UTRs. (A) viral RNA, SR1f RNA, during dianthovirus infection, generated Northern blot of total RNA from mock- or RCNMV-infected N. benthamiana. ′ from the 3′ UTR of viral RNA1 through an unexplored mechanism Probes are against viral genomic 3 UTR and 5.8S rRNA. Full-length RNA1 and SI Appendix A exoribonuclease-resistant degradation product (SR1f) are indicated. (B)In of incomplete degradation ( ,Fig.S1 )(27).Usinga 32 ′ ′ combination of virology and biochemistry, we show that the dia- vitro Xrn1 degradation assay of P-3 end-labeled RCNMV 3 UTR sequences. ′ (C) In vitro Xrn1 degradation assay on minimal xrRNAs from RCNMV, nthovirus 3 UTRs contain a bona fide xrRNA sequence that in- ± ′ → ′ cis SCNMV, and CRSV. Data are average ( SD) percent resistance from three hibits 5 3 exoribonucleolytic decay in without the help of individual experiments. (D) Secondary structure of the crystallized SCNMV protein factors. X-ray crystallography and single-molecule FRET ′ → RNA. Lowercase letters represent sequences altered to facilitate transcrip- experiments reveal a unique RNA fold that operates to block 5 tion. Non–Watson–Crick base pairs are in Leontis–Westhof