A Folded Viral Noncoding RNA Blocks Host Cell Exoribonucleases Through a Conformationally Dynamic RNA Structure

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 flavivirus 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 degradation. 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 potential xrRNA sequence from the 3′ UTR of plant-infecting dianthoviruses (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 annotation (41). 3′ exoribonucleolytic degradation through dynamic changes in The Xrn1 stop site is denoted by the labeled arrow. (E) Ribbon representa- RNA structure that we term “codegradational” RNA remodeling. tion of the SCNMV xrRNA structure. Colors match those in D. The “single- This work reveals RNA-dependent exoribonuclease resistance be- stranded” RNA at the 3′ end was ordered due to intermolecular interactions yond the realm of flaviviruses, conferred by diverse structured in the crystal, described in Fig. 2 D–F and SI Appendix, Fig. S4. RNA elements that nonetheless share a common underlying topology. Thus, convergent evolution has given rise to this mechanism at least twice, indicating it to be a generally useful pathway withtheenzyme(SI Appendix,Fig.S1G–I). That this matches the that may be widespread across biology. behavior of flavivirus xrRNAs suggests potential similarity in the molecular mechanisms of these xrRNAs. This result also justifies Results the use of recombinant yeast Xrn1 in our experiments as a proxy The Dianthovirus 3′ UTR Contains a Bona Fide xrRNA. We verified that for the larger family of eukaryotic XRN proteins, which is impor- dianthovirus infection results in accumulation of a subgenomic tant because dianthoviruses infect plant cells whose major cyto- RNA by infecting Nicotiana benthamiana with red clover necrotic plasmic 5′ → 3′ exoribonuclease is the evolutionarily related XRN mosaic virus (RCNMV) (28). Northern blot analysis of RNA from protein Xrn4 (2). infected leaves with probes targeting the viral 3′ UTR revealed the presence of a discrete RNA species consistent with the previously Dianthovirus xrRNAs Adopt an Unexpected Structure. Dianthovirus reported SR1f RNA (Fig. 1A),whichhasbeenshowntobethe xrRNAs are significantly shorter than flaviviral xrRNAs and there result of 5′ → 3′ exoribonuclease resistance (27). To determine if is no readily identified sequence similarity between the two, sug- RNAs with sequences from the dianthovirus 3′ UTR are sufficient gesting that a different structure confers Xrn1 resistance. To un- to block 5′ → 3′ exoribonucleolytic decay without trans-acting derstand the structural basis of Xrn1 resistance by the dianthovirus BIOCHEMISTRY proteins, we challenged in vitro-transcribed RNA from RCNMV xrRNAs, we aimed to solve the structure of a dianthovirus xrRNA. with recombinant Xrn1 (SI Appendix,Fig.S1B). A 130-nt-long Only the SCNMV xrRNA yielded diffracting crystals, and we RNA from the RCNMV 3′ UTR blocked exoribonucleolytic de- solved the structure of the complete 43-nt SCNMV xrRNA to cay with no protein cofactors, revealing that RCNMV contains an 2.9-Å resolution using X-ray crystallography (SI Appendix,Fig.S2and authentic xrRNA (Fig. 1B). We mapped the Xrn1 halt site using Table S1). The SCNMV xrRNA adopts a global conformation with reverse transcription and capillary electrophoresis (SI Appendix, no immediate similarity to the mosquito-borne flaviviral xrRNA Fig. S1 D–F), observing that the enzyme halts at a specific point. structures (7, 18). Overall, the SCNMV xrRNA comprises a stem- We then determined the 3′ terminus of the resistant sequence loop (SL) structure with a single-stranded 3′ tail (Fig. 1 D and E). using test RNAs truncated at their 3′ ends, identifying a 43-nt RNA The stem contains coaxially positioned helices P1 and P2, which element as the minimal RCNMV xrRNA (Fig. 1B). Thus, the are linked by stacking of bases and a noncanonical base pair in the RCNMV 3′ UTR has an authentic xrRNA contained in a discrete L1 internal loop. Helix P2 is capped by L2, an atypical bipartite RNA sequence that can operate outside of its natural context. loop. The 5′ portion of the loop contains a run of single-stranded The xrRNA sequence identified in RCNMV is highly conserved stacked bases (L2A), followed by a sharp turn in the backbone and in all three known members of the dianthovirus family —RCNMV, a compact hairpin structure (L2B) embedded within the larger sweet clover necrotic mosaic virus (SCNMV), and carnation ring- loop structure. This L2B element forms long-distance tertiary spot virus (CRSV) (SI Appendix,Fig.S1C)—and, accordingly, all stacking interactions with the L1 internal loop, bringing L2B into viral 3′ UTR sequences confer Xrn1 resistance in vitro (Fig. 1C). close proximity to the stem, thus causing L2 to adopt an overall As the mosquito-borne flavivirus xrRNAs are able to block diverse “tilted” conformation. Sequence conservation between the dia- exoribonucleases and thus act as general mechanical blocks to nthoviral xrRNAs suggests that this structure is representative of these enzymes (20), we tested the three dianthovirus xrRNAs for all three known members (SI Appendix,Fig.S1C). this ability. All three blocked two 5′ → 3′ exoribonucleases unrelated to Xrn1—yeast decapping and exoribonuclease protein 1 The Dianthoviral xrRNA Crystal Structure Is an RNA Folding Intermediate. (Dxo1) and bacterial 5′ → 3′ exoribonuclease RNase J1—in- The SCNMV xrRNA structure did not immediately suggest a dicating that they function as general roadblocks against exoribo- mechanism for Xrn1 resistance, but functional data provided in- nucleolytic degradation rather than through specific interactions sight. The Xrn1 stop site is at the base of the P1 stem (Fig. 1D).

Steckelberg et al. PNAS | June 19, 2018 | vol. 115 | no. 25 | 6405 Downloaded by guest on September 29, 2021 This is significant because the entry channel into Xrn1’s active site caused a complete loss of SR1f RNA formation, whereas com- accommodates only single-stranded RNA, mandating that the pensatory mutations partially rescued production (Fig. 2F). We helicase activity of the enzyme unwinds RNA so it can enter the also determined that the PK interaction does not function in enzyme’s interior. The distance from the surface of the enzyme to trans as a dimer (SI Appendix, Fig. S5 A–C). These results show the buried active site requires that at least five or six nucleotides of that the dianthovirus xrRNAs require the L2A-S2 PK interaction RNA downstream of the stop site be single-stranded (Fig. 2A)(3, to block Xrn1. 4). Thus, the location of the Xrn1 stop site on the dianthovirus xrRNA mandates that P1 be unwound at the moment when xrRNA PK Formation Creates a Protective Loop Around the 5′ End. To Xrn1 halts (Fig. 2B). understand how L2A-S2 PK formation could lead to exoribonu- This finding raised the question of whether formation of the clease resistance, we constructed a model with this interaction P1 stem is needed to block the enzyme. We tested this by measuring formed in cis and the P1 helix unwound, mimicking the structure Xrn1 resistance of an RNA mutated to disrupt P1 base pairing atthemomentwhenXrn1ishalted(Fig.3A and B). Even though (P1MUT)(allmutants;SI Appendix,Fig.S3). This mutation signifi- it is theoretically possible that the PK forms without unwinding P1, cantly reduced, but did not abolish, Xrn1 resistance in vitro, while this would require a much more dramatic distortion of the overall compensatory mutations (P1COMP) that restore Watson–Crick base structure. In addition, the experimentally determined Xrn1 stop pairing completely rescued Xrn1 resistance (Fig. 2C). Thus, for- site at the base of P1 indicates that the stem must unwind to allow mation of the P1 stem is not essential for Xrn1 resistance but does RNA entry into the active site, and thus P1 is not part of the Xrn1- contribute to full activity, yet P1 must be unwound during the resistant state. In the modeled conformation, the 3′ end of the exoribonuclease halting event. Therefore, we hypothesized that dianthovirus xrRNA encircles the 5′ end, thereby creating a to- the dianthovirus xrRNA structure observed in the crystal (with the pology similar to the protective ring-like fold of mosquito-borne P1 helix formed) is an important folding intermediate, but the RNA flaviviral xrRNAs (Fig. 3 B and C). Thus, these xrRNAs appear to must change conformation in a programmed way to block Xrn1. rely on a similar overarching strategy to block Xrn1, which is re- markable and unexpected given their very different sequences. An RNA PK Interaction Is Necessary for Xrn1 Resistance. The crystal In both cases, an apical loop forms long-range base pairs with a structure suggests what the aforementioned putative conforma- downstream element, causing intervening sequences to loop pro- tional change might entail. Within the crystal lattice, nucleotides tectively around the 5′ end (SI Appendix,Fig.S5D). In the flavivirus C43, U44, and C45 at the single-stranded 3′ end of one RNA xrRNAs, the loop is formed by two intact helical elements, while in molecule form Watson–Crick base pairs with G20, A19, and the dianthovirus xrRNAs, the P1 helix must be unwound. A com- G18 in the loop region (L2A) of an adjacent RNA molecule (SI parison of these two xrRNAs shows the ring-like structure to be an Appendix, Figs. S2C and S4A). The formation of these pairs in emerging theme of RNA structure-based 5′ → 3′ exoribonuclease trans in the crystal suggests that they could form in cis,creatinga resistance, and convergent evolution can select different RNA se- PK between the L2A and S2 regions (Fig. 2D). The potential to quences and structures to achieve this topology. form this PK is present in all three dianthovirus xrRNA sequences The SL conformation with P1 paired likely represents a fold- (SI Appendix,Fig.S4B), and in fact this putative PK can be ex- ing intermediate to position S2 in such a way that when the PK tended by one additional Watson–Crick base pair (C17-G46, not forms it will encircle the 5′ end of the RNA rather than forming a present in the crystallized RNA). Indeed, addition of G46 to the 3′ ring with the 5′ end outside the loop. This interpretation suggests end of the SCNMV xrRNA increased Xrn1 resistance in vitro and that the SL-to-PK switch itself is promoted by the tilted SL prompted us to perform all subsequent experiments using this 44- conformation created by tertiary interactions formed by the nt-long xrRNA sequence (SI Appendix,Fig.S4C). highly conserved sequence (SI Appendix, Fig. S6A). One such To determine the importance of the L2A-S2 PK, we mutated interaction is enabled by the conformation of L2B, which is es- either L2A or S2 in SCNMV, RCNMV, and CRSV xrRNA and sentially a smaller loop embedded in the larger L2 loop. The assessed Xrn1 resistance in vitro (Fig. 2E and SI Appendix,Fig. backbone of L2B is similar to the U-turn found in some RNA S4D). In all xrRNAs, disruption of the putative PK led to loss of tetraloops, but the loop is closed by a reverse Watson–Crick base Xrn1 resistance, whereas compensatory mutations (PKCOMP)par- pair between U21 and A28, with the latter base extruded from the tially rescued Xrn1 resistance. We tested the importance of the PK P2 helix (SI Appendix,Fig.S6B). The L2B structure creates a for the accumulation of dianthovirus SR1f RNA by infecting Nico- “docking point” forA7,whichisextrudedfrom the L1 internal loop tiana benthamiana with RNA transcripts generated from an to stack between A25 and A26, thereby inducing the overall tilted RCNMV RNA-1 infectious clone with the WT, PKS2,orPKCOMP conformation (SI Appendix,Fig.S6C). A similar long-range hair- xrRNA sequence. Matching the in vitro data, disruption of the PK pin–adenosine interaction is found in 16S and 18S rRNA (SI

Fig. 2. Partial unfolding of SCNMV xrRNA contrib- A B C utes to Xrn1 resistance. (A, Left) Structure of Dro- C G A G active C U P1 P1 U U WT MUT COMP sophila melanogaster Xrn1 [Protein Data Bank (PDB) A A G site C-G A C _ _ _ A Xrn1: + + + ID code 2Y35] (3). Red spheres denote active site C G U- A C-G input residues; black, 8-nt RNA substrate. Five single- C-G A G A RNA A A stranded nucleotides span the distance from the U- A G-C P1 resistant ’ 5 nt distance C-G Xrn1 halt active site to the enzyme s surface. (A, Right) Sec- G-C P1 RNA A AG UU CC unwound ondary structure of SCNMV xrRNA, with the 5 nt 90±10 54±13 99±1 Xrn1 stop Xrn1 stop site indicated. Yellow denotes the portion % resistance of the P1 stem that must unwind. (B) Scheme of Xrn1 unwinding the P1 stem (red). (C) In vitro D EFWT PKS2 PKL2A PK COMP PK S2 PK COMP 32 _ _ _ _ Xrn1 degradation assay on P-3′ end-labeled WT Xrn1: + + + + mock WT 1212 input and mutant SCNMV xrRNAs. Data are average (± SD) ? RNA1 RNA 1000 percent resistance from three individual experi- resistant 600 SR1f ments. (D) Scheme of putative PK interaction (SI RNA 400 RNA Appendix, Fig. S4). (E) In vitro Xrn1 degradation as- 90±5 2±1 3±2 33±6 5.8S say on 32P-3′ end-labeled WT and mutant SCNMV % resistance rRNA 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.

6406 | www.pnas.org/cgi/doi/10.1073/pnas.1802429115 Steckelberg et al. Downloaded by guest on September 29, 2021 decreased from 10 mM to near-physiological 1 mM (SI Appendix, Fig. S9). smFRET traces following individual molecules over time demonstrate two types of WT SCNMV xrRNAs: RNAs “locked in” a high-FRET state and RNAs that undergo rapid conformational dynamics between the two states (Fig. 4C and SI Appendix,Fig.S8C). This suggests two configurations in the PK state, indistinguishable by FRET: an unstable PK, which rapidly transitions back into the SL state, and a stable PK that remains folded for a longer period (perhaps stabilized by additional interactions). A subset of PKS2 molecules display a very rapid exchange between mid- and high- FRET states (Fig. 4E and SI Appendix,Fig.S8D), but none remain in the high-FRET state; these molecules may sample the PK confor- Fig. 3. PK formation creates a protective ring around the 5′ end. (A) Crys- mation but revert back to the SL state. tallized SCNMV xrRNA. The arrow indicates the conformational change Of note, P1MUT xrRNA has the same fraction of RNA molecules needed to form the PK interaction. (B) Modeled xrRNA PK conformation (all in the high-FRET state as WT xrRNA, despite being less efficient 3 nt of P1 unwound, PK formed). PK is in orange. The 3′ end (blue) encircles in blocking Xrn1 (SI Appendix,Fig.S10C and D). This suggests the 5′ end (red). (C) Structure of ZIKV xrRNA1 (PDB ID code 5TPY) with 5′ end that in the absence of P1, the xrRNA can still adopt a PK state, but (red), ring-like fold (blue), and PK (orange) matching the colors in A and B. either one that is Xrn1-resistant or one that is misfolded and is not resistant (SI Appendix,Fig.S10E). The misfolded RNA would be indistinguishable from properly folded xrRNA by smFRET (SI Appendix,Fig.S7). The importance of this interaction and thus the Appendix,Fig.S10A–D). A 50:50 distribution of properly folded overall tilted SL conformation is revealed by an A7U mutant, which and misfolded RNA would produce the 50% resistance efficiency abolishes Xrn1 resistance in vitro and prevents SR1f RNA pro- observed for P1MUT. Overall, these data are consistent with the idea duction during RCNMV infection (SI Appendix,Fig.S6D and E). that the formation of P1 acts as a folding intermediate to ensure that the 5′ end is encircled when the PK forms. Conformational Dynamics of Dianthovirus xrRNA. Our model suggests that formation of the essential S2-L2A PK involves a Codegradational Remodeling of Dianthovirus xrRNA Contributes to conformational switch between the SL and PK states. To test this Xrn1 Resistance. The Xrn1 resistance of dianthoviruses relies on idea, we used single-molecule Förster resonance energy transfer the SL-to-PK conformational change, yet only the PK state can (smFRET). Using the crystal structure and the PK model as a block Xrn1. These observations raise several questions: What guide, we designed an RNA with dye modifications at sites that happens if Xrn1 encounters an xrRNA in the SL conformation? Is would result in high FRET if the PK was formed and a mid- it able to quickly degrade the RNA, or does the xrRNA refold in FRET state if not (Fig. 4A and SI Appendix, Fig. S8A). We the presence of the exoribonuclease? Degradation-coupled un- verified that SCNMV xrRNA labeled with acceptor dye (Cy5) at winding of the P1 stem could potentially liberate the 3′ end of the position U15 was Xrn1-resistant (SI Appendix, Fig. S8B), then RNA structure, favoring the formation of the PK and inducing annealed this RNA to a surface-immobilized DNA oligo labeled Xrn1 resistance through codegradational RNA remodeling. If this with a donor dye (Cy3) and monitored FRET efficiency by total were so, then this model would require that RNA remodeling be internal reflection fluorescence (TIRF) microscopy. faster than Xrn1-induced RNA hydrolysis. WT SCNMV xrRNA exists predominantly in two states with To test how an RNA in the SL conformation responds to Xrn1 roughly 0.8 and 0.4 FRET efficiency (Fig. 4 B and C). To assign these degradation, we constructed an xrRNA with a 7-nt 5′ extension high- and mid-FRET states to specific conformations, in smFRET that captures the 3′ endoftheRNAinanextendedP1stem experiments we used the PKS2 mutant, which predominantly adopts (P1EXT), preventing PK formation (Fig. 5A and SI Appendix,Fig. – D E the mid-FRET associated conformation (Fig. 4 and ). Surpris- S10F). As expected, the P1EXT xrRNA almost exclusively adopted ingly, >80% of the WT RNA molecules adopted the high-FRET a mid-FRET state corresponding to the SL conformation, in-

state, indicating that the PK state is more prevalent in solution than dicating that the PK is not formed in this mutant (Fig. 5B). BIOCHEMISTRY 2+ the SL state. This was true even when the Mg concentration was However, P1EXT is Xrn1-resistant (Fig. 5C), suggesting that on this

A B WT C Pseudoknot Stem loop CGAG 0.20 n=3238 1.2 1.2 (high-FRET) (mid-FRET) 0.15 0.78 1.0 1.0 0.8 0.8 0.10 0.6 0.6 Cy5 RET F

FRET 0.4 0.4

Frequency 0.05 0.40 0.2 0.2 0.0 0.0 SCNMV 0.00 RNA 5’ 5’ -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 Cy5 N(10) CUCG ~60Å FRET Time (s) Time (s) ~30Å Cy3 Cy3 D PKS2 E

CGAG 0.30 0.25 n=3868 1.2 1.2 DNA 1.0 1.0 0.20 0.39 0.8 0.8 handle Biotin- 0.15 0.6 0.6 streptavidin RET RET F linkage X 0.10 F 0.4 0.4 0.2 0.2 Frequency PEG 0.05 0.0 0.0 Coverslip 0.00 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 35 40 N(10) UCUA FRET Time (s) Time (s)

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 PKS2 xrRNA. (Left) Scheme of the RNA construct. (E) Representative smFRET traces of two individual PKS2 xrRNA molecules.

Steckelberg et al. PNAS | June 19, 2018 | vol. 115 | no. 25 | 6407 Downloaded by guest on September 29, 2021 AFP1EXT BCRNA only unlabeled RNA 0.30 xrRNA CGAG P1 0.25 n = 3410 WT EXT _ _ 5' 3' 0.20 0.29 Xrn1: + + Fig. 5. Codegradational remodeling of SCNMV 0.15 input 0.10 xrRNA structure. (A) Schematic of smFRET construct

Frequency RNA 0.05 with an extended P1 stem (P1EXT). (B) smFRET histo- 0.00 resistant Exonuclease -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 SL RNA halting grams of P1 xrRNA. Average FRET values are FRET state EXT +Xrn1 +Xrn1 D208A shown. n, number of molecules in the histogram. (C) D0.12 E0.30 Degradation n = 2335 0.25 n=4001 0.09 In vitro Xrn1 resistance assay of WT and P1EXT xrRNA. 0.20 0.27 0.06 0.15 (D and E) smFRET histograms of P1EXT xrRNA pre- Codegradational

Frequency 0.10 Frequency 0.03 C 0.05 PK remodeling treated with WT Xrn1 (D) or mutant Xrn1(D208A) U 0.00 0.00 C state (E). (F) Scheme of the conformational changes and N(10) G -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 FRET FRET codegradational remodeling of SCNMV xrRNA.

RNA, the SL-to-PK switch required for exoribonuclease resistance been found in other RNAs, and thus they might be a defining is triggered during degradation, likely through unwinding of P1. and necessary feature of xrRNAs. Consistent with this, treating P1EXT xrRNA with Xrn1 and exam- Although flaviviral and dianthoviral xrRNAs share several sim- ining the resultant RNA product by smFRET showed a redistri- ilarities, there are important differences. In the flavivirus version, bution of molecules from a mid-FRET to a high-FRET state (Fig. the fold requires a three-way junction, which interacts with the 5′ 5D), demonstrating that the enzyme has shifted the xrRNA con- end directly to form a second PK interwoven with the first PK (7, formation from the SL state to the PK state. Treatment with a 18). The stretch of RNA that forms the ring is almost entirely base- catalytically inactive Xrn1 did not result in this redistribution (Fig. paired, and a number of other tertiary interactions, such as base 5E), indicating that the enzyme’s degradation-linked helicase ac- triples and long-range noncanonical base pairs, stabilize the final tivity is necessary for the observed conformational change of fold (SI Appendix,Fig.S5C). The dianthoviral xrRNAs are sub- P1EXT. Specifically, this result strongly suggests that unwinding of stantially shorter, there is no three-way junction, and most of the the P1 stem liberates the 3′ end of the structure to encircle the 5′ RNA that forms the ring appears to be single-stranded as a result end and form the PK. These data show that Xrn1 can be co-opted of the unwinding of the P1 stem (SI Appendix,Fig.S5C). These into a “pseudochaperone” function to favor the structure that ul- differences reveal how very divergent sequences and secondary timately prevents enzyme progression (Fig. 5F). structures can give rise to the ring-like topology. Because the crystal does not contain the dianthoviral xrRNA in the PK form, Discussion we do not yet know the full repertoire of tertiary interactions that Exoribonuclease resistance as a means of generating noncoding stabilize this conformation; local remodeling of the structure could RNA from larger precursors is an emerging theme in virology give rise to additional interactions. A specifically configured PK (21–25, 27). We have found that exoribonuclease-blocking se- may be a common feature of all xrRNAs, but more structures are quences from plant-infecting dianthoviruses are authentic xrRNAs, needed to test this and to identify other shared features. generating viral noncoding RNAs in a process dependent on a spe- The unusual nature of the xrRNA fold invites speculation as to cific but dynamic 3D RNA fold. Furthermore, these xrRNAs can how it forms correctly; RNA PKs are ubiquitous, but only xrRNAs fold spontaneously in vitro using a defined conformational in- have been observed to form a protective ring. This implies that the termediate, and also are capable of using the RNA unwinding high levels of Xrn1 resistance observed in vitro require a mechanism to ensure that the ring always wraps around the 5′ end of the activity of the exoribonuclease to promote or stabilize their active structure. In flaviviral xrRNAs, the structure shows that the 5′ end structure in a form of codegradational remodeling. These findings must interact directly with the three-way junction for the junction establish xrRNAs as a distinct functional class of RNAs and sug- to fold and form the ring structure (19). In dianthoviral xrRNAs, gest that RNA structure-dependent exoribonuclease resistance is a the data suggest that the P1 helix initially forms to position the 5′ general mechanism of RNA maturation. and 3′ ends correctly (SL state), and then a programmed con- Flaviviruses and dianthoviruses belong to unrelated virus formational change leads to P1 helix unwinding and PK formation families and do not infect the same host species, and the xrRNA (PK state). This pathway could help ensure that PK formation sequences have no similarity. Nonetheless, in both a folded ′ ′ establishes the ring around the 5 end rather than forming a ring xrRNA in the 3 UTR of a longer viral RNA is used to produce with the 5′ end outside (SI Appendix,Fig.S10E). Accordingly, shorter noncoding RNAs that then play multiple (but different) – mutations affecting the putative folding intermediate (i.e., muta- important roles during viral infection (8 17, 27). Thus, a similar tions to the P1 stem) lead to an approximate 50% reduction in RNA maturation strategy has emerged in very divergent viruses, Xrn1 resistance; the most straightforward interpretation of this attesting to its utility and potential pervasiveness. Xrn1 can de- finding is that failure to form the P1 stem decouples PK and ring grade most cellular RNAs, even highly structured ones (2); the formation from 5′ end placement, and approximately 50% of the fact that discrete xrRNA elements can stop the enzyme suggests molecules are misfolded. that they have characteristics distinct from most folded RNAs. Additional evidence for the folding model described above is Indeed, while the 3D structures of class 1 flaviviral xrRNAs and the fact that dianthoviral xrRNAs retain their activity even when dianthoviral xrRNAs are different, they have similarities that the PK state is prevented from forming through an extended suggest a shared overarching strategy. Both flaviviral and dia- P1 stem. This observation and the shift toward the PK state when nthoviral xrRNAs rely on a PK, a common RNA fold with many Xrn1 degrades the RNA reveals that RNA remodeling can occur different configurations (29–31). However, in these xrRNAs, the codegradationally, and that the conformational switch between PK creates a ring-like fold around the 5′ end of the structure, a the SL and PK states is faster than the progression of the enzyme topology not observed in other PK structures. The ring comprises through the structure. Thus, the helicase activity of Xrn1 can be sequences within the PK and downstream of the protected 5′ co-opted into a pseudochaperone function; by unfolding P1, end; thus, exoribonuclease hydrolysis-coupled helicase activity is Xrn1 liberates the nucleotides required for PK formation and unable to access or unfold the structure when the ring braces helps shift the conformation to the resistant state. against the enzyme’s surface (7). In addition, this shared topol- Although smFRET shows that PK formation can occur spon- ogy suggests a way to confer directionality; the protective ring taneously in vitro, within the cell and in the context of the full- poses an obstacle to 5′ → 3′ decay, yet can be easily unwound by length viral RNA, codegradational remodeling may be important. enzymes approaching from the 3′ side, such as the viral RNA- It could allow the xrRNA to respond to the RNA decay machinery dependent RNA polymerase. These ring-like folds have not yet while remaining sufficiently flexible for 3′ → 5′ unfolding by the

6408 | www.pnas.org/cgi/doi/10.1073/pnas.1802429115 Steckelberg et al. Downloaded by guest on September 29, 2021 viral RNA-dependent RNA polymerase. At the very least, the pentatricopeptide repeat proteins function as protein barriers for presence of the enzyme favors formation of the PK by unwinding a exoribonucleolytic decay and thus define mRNA termini in chlo- potentially competing structure. To our knowledge, there are no roplasts and mitochondria (34–36). Likewise, rRNA processing other known examples of codegradational RNA structure forma- involves precise trimming by exoribonucleases (37, 38). Cellular tion or remodeling, but our discovery suggests that a similar xrRNAs might exist to generate biologically active 5′-truncated pseudochaperone function dependent on exoribonucleases or decay intermediates; indeed, many RNA-centered processes used helicases may exist elsewhere. by cells were first discovered in viruses, including RNA structures Exoribonuclease resistance conferred by RNA structure may be that block 3′ → 5′ decay (39, 40). Thus, RNA elements identified a more common mechanism than hitherto anticipated, and recent studies have identified putative xrRNAs in several virus families. in viruses can inform the search for related molecular structures A conserved sequence that blocks 5′ → 3′ exoribonucleolytic decay elsewhere in biology and motivate ongoing research to understand was recently found in the 3′ UTR of plant-infecting benyviruses the enormous complexity and diversity of structured RNAs. and cucumoviruses, both of which have multipartite positive-sense RNA genomes (23–25, 32). Moreover, Xrn1 is confounded by Materials and Methods RNA elements in the 5′ UTRs of hepaciviruses and pestiviruses Detailed information on RNA and protein production, viral infections, (21), as well as by a G-rich sequence structure in the N mRNA of Northern blot analysis, halt site mapping, smFRET, and RNA structure de- Rift Valley fever virus and multiple RNA structures in ambisense- termination is provided in SI Appendix, Materials and Methods. derived transcripts of arenaviruses (22). There are no obvious common sequence patterns, and most await detailed functional ACKNOWLEDGMENTS. We thank C. Musselman and O. Rissland for their and structural characterization. critical reading of the manuscript. Support was provided by the National The range of biological functions of ncRNAs produced by Institutes of Health (Grants R35GM118070 and R01 AI133348 to J.S.K. and xrRNAs (8–17, 23, 27), as well as their presence in diverse virus F32GM117730 to B.M.A.) and the European Molecular Biology Laboratory families, suggest that regulated exoribonuclease resistance could (Fellowship ALTF 611-2015, to A.-L.S.). The University of Colorado Denver X-Ray Facility is supported by National Institutes of Health Grants P30CA046934 and be a pathway for RNA maturation in cells. G-rich RNA sequences S10OD012033. The Advanced Light Source is supported by the Office of Science, installed artificially into mRNAs have been shown to confound Office of Basic Energy Sciences of the US Department of Energy under Contract Xrn1 activity in yeast (presumably through G-quadruplex struc- DE-AC02-05CH11231. T.L.S. acknowledges support from the North Carolina tures), but the same G-rich sequences appear unable to stall Agricultural Research Service in the College of Agriculture and Life Sciences at exoribonucleolytic decay in mammalian cells (33). Moreover, North Carolina State University.

1. Jones CI, Zabolotskaya MV, Newbury SF (2012) The 5′ → 3′ exoribonuclease XRN1/ 22. Charley PA, Wilusz CJ, Wilusz J (2018) Identification of phlebovirus and arenavirus RNA Pacman and its functions in cellular processes and development. Wiley Interdiscip Rev sequences that stall and repress the exoribonuclease XRN1. JBiolChem293:285–295. RNA 3:455–468. 23. Flobinus A, et al. (2016) A viral noncoding RNA complements a weakened viral RNA 2. Nagarajan VK, Jones CI, Newbury SF, Green PJ (2013) XRN 5′→3′ exoribonucleases: silencing suppressor and promotes efficient systemic host infection. Viruses 8:E272. Structure, mechanisms and functions. Biochim Biophys Acta 1829:590–603. 24. Peltier C, et al. (2012) Beet necrotic yellow vein virus subgenomic RNA3 is a cleavage 3. Jinek M, Coyle SM, Doudna JA (2011) Coupled 5′ nucleotide recognition and proc- product leading to stable non-coding RNA required for long-distance movement. essivity in Xrn1-mediated mRNA decay. Mol Cell 41:600–608. J Gen Virol 93:1093–1102. 4. Chang JH, Xiang S, Xiang K, Manley JL, Tong L (2011) Structural and biochemical 25. Flobinus A, et al. (2018) Beet necrotic yellow vein virus noncoding RNA production studies of the 5′→3′ exoribonuclease Xrn1. Nat Struct Mol Biol 18:270–276. depends on a 5′→3′ Xrn exoribonuclease activity. Viruses 10:E137. 5. Moon SL, Wilusz J (2013) Cytoplasmic viruses: Rage against the (cellular RNA decay) 26. Okuno T, Hiruki C (2013) Molecular biology and epidemiology of dianthoviruses. Adv machine. PLoS Pathog 9:e1003762. Virus Res 87:37–74. 6. Chapman EG, Moon SL, Wilusz J, Kieft JS (2014) RNA structures that resist degradation 27. Iwakawa HO, et al. (2008) A viral noncoding RNA generated by cis element-mediated by Xrn1 produce a pathogenic dengue virus RNA. eLife 3:e01892. protection against 5′→3′ RNA decay represses both cap-independent and cap-dependent 7. Chapman EG, et al. (2014) The structural basis of pathogenic subgenomic flavivirus translation. JVirol82:10162–10174. RNA (sfRNA) production. Science 344:307–310. 28. Xiong ZG, Lommel SA (1991) Red clover necrotic mosaic virus infectious transcripts 8. Pijlman GP, et al. (2008) A highly structured, nuclease-resistant, noncoding RNA synthesized in vitro. Virology 182:388–392. produced by flaviviruses is required for pathogenicity. Cell Host Microbe 4:579–591. 29. Brierley I, Pennell S, Gilbert RJ (2007) Viral RNA pseudoknots: Versatile motifs in gene 9. Moon SL, et al. (2012) A noncoding RNA produced by arthropod-borne flaviviruses inhibits expression and replication. Nat Rev Microbiol 5:598–610.

the cellular exoribonuclease XRN1 and alters host mRNA stability. RNA 18:2029–2040. 30. Peselis A, Serganov A (2014) Structure and function of pseudoknots involved in gene BIOCHEMISTRY 10. Moon SL, et al. (2015) Flavivirus sfRNA suppresses antiviral RNA interference in cultured cells expression control. Wiley Interdiscip Rev RNA 5:803–822. and mosquitoes and directly interacts with the RNAi machinery. Virology 485:322–329. 31. Wyatt JR, Puglisi JD, Tinoco I, Jr (1989) RNA folding: Pseudoknots, loops and bulges. 11. Schnettler E, et al. (2012) Noncoding flavivirus RNA displays RNA interference sup- BioEssays 11:100–106. pressor activity in insect and mammalian cells. J Virol 86:13486–13500. 32. Ratti C, et al. (2009) Beet soil-borne mosaic virus RNA-3 is replicated and encapsidated 12. Schuessler A, et al. (2012) West Nile virus noncoding subgenomic RNA contributes to in the presence of BNYVV RNA-1 and -2 and allows long-distance movement in Beta viral evasion of the type I interferon-mediated antiviral response. JVirol86:5708–5718. macrocarpa. Virology 385:392–399. 13. Bidet K, Dadlani D, Garcia-Blanco MA (2014) G3BP1, G3BP2 and CAPRIN1 are required 33. Muhlrad D, Decker CJ, Parker R (1994) Deadenylation of the unstable mRNA encoded for translation of interferon-stimulated mRNAs and are targeted by a dengue virus by the yeast MFA2 gene leads to decapping followed by 5′→3′ digestion of the non-coding RNA. PLoS Pathog 10:e1004242. transcript. Genes Dev 8:855–866. 14. Liu Y, Liu H, Zou J, Zhang B, Yuan Z (2014) Dengue virus subgenomic RNA induces 34. Pfalz J, Bayraktar OA, Prikryl J, Barkan A (2009) Site-specific binding of a PPR protein apoptosis through the Bcl-2–mediated PI3k/Akt signaling pathway. Virology 448:15–25. defines and stabilizes 5′ and 3′ mRNA termini in chloroplasts. EMBO J 28:2042–2052. 15. Roby JA, Pijlman GP, Wilusz J, Khromykh AA (2014) Noncoding subgenomic flavivirus 35. Prikryl J, Rojas M, Schuster G, Barkan A (2011) Mechanism of RNA stabilization and trans- RNA: Multiple functions in West Nile virus pathogenesis and modulation of host re- lational activation by a pentatricopeptide repeat protein. Proc Natl Acad Sci USA 108:415–420. sponses. Viruses 6:404–427. 36. Zhelyazkova P, et al. (2012) Protein-mediated protection as the predominant mech- 16. Manokaran G, et al. (2015) Dengue subgenomic RNA binds TRIM25 to inhibit in- anism for defining processed mRNA termini in land plant chloroplasts. Nucleic Acids terferon expression for epidemiological fitness. Science 350:217–221. Res 40:3092–3105. 17. Göertz GP, et al. (2016) Noncoding subgenomic flavivirus RNA is processed by the 37. Tomecki R, Sikorski PJ, Zakrzewska-Placzek M (2017) Comparison of preribosomal mosquito RNA interference machinery and determines West Nile virus transmission by RNA processing pathways in yeast, plant and human cells: Focus on coordinated ac- Culex pipiens mosquitoes. J Virol 90:10145–10159. tion of endo- and exoribonucleases. FEBS Lett 591:1801–1850. 18. Akiyama BM, et al. (2016) Zika virus produces noncoding RNAs using a multi-pseudoknot 38. Geerlings TH, Vos JC, Raué HA (2000) The final step in the formation of 25S rRNA in structure that confounds a cellular exonuclease. Science 354:1148–1152. Saccharomyces cerevisiae is performed by 5′→ 3′ exonucleases. RNA 6:1698–1703. 19. Kieft JS, Rabe JL, Chapman EG (2015) New hypotheses derived from the structure of a flaviviral 39. Brown JA, et al. (2014) Structural insights into the stabilization of MALAT1 noncoding Xrn1-resistant RNA: Conservation, folding, and host adaptation. RNA Biol 12:1169–1177. RNA by a bipartite triple helix. Nat Struct Mol Biol 21:633–640. 20. MacFadden A, et al. (2018) Mechanism and structural diversity of exoribonuclease- 40. Mitton-Fry RM, DeGregorio SJ, Wang J, Steitz TA, Steitz JA (2010) Poly(A) tail recognition resistant RNA structures in flaviviral RNAs. Nat Commun 9:119. by a viral RNA element through assembly of a triple helix. Science 330:1244–1247. 21. Moon SL, et al. (2015) XRN1 stalling in the 5′ UTR of hepatitis C virus and bovine viral di- 41. Leontis NB, Westhof E (2001) Geometric nomenclature and classification of RNA arrhea virus is associated with dysregulated host mRNA stability. PLoS Pathog 11:e1004708. base pairs. RNA 7:499–512.

Steckelberg et al. PNAS | June 19, 2018 | vol. 115 | no. 25 | 6409 Downloaded by guest on September 29, 2021