RNA G-quadruplex (rG4) structures are of fundamental importance to biology. A novel approach is introduced to detect and structurally map rG4s at single-nucleotide resolution in RNAs. The approach, denoted SHALiPE, couples selective 2'-hydroxyl acylation with lithium ion-based primer extension, and identifies characteristic structural fingerprints for rG4 mapping. We apply SHALiPE to interrogate the human precursor microRNA 149, and reveal the formation of an rG4 structure in this non-coding RNA. Additional analyses support the SHALiPE results and uncover that this rG4 has a parallel topology, is thermally stable, and is conserved in mammals. An in vitro Dicer assay shows that this rG4 inhibits Dicer processing, supporting the potential role of rG4 structures in microRNA maturation and post-transcriptional regulation of mRNAs.This is the accepted manuscript. The final version is available at http://dx.doi.org/10.1002/anie.201603562

Balasubramanian, Shankar

Kwok, Chun Kit

Sahakyan, Aleksandr B

Angewandte Chemie International Edition

English

Apollo (Cambridge)

German Edition: DOI: 10.1002/ange.201603562G-QuadruplexesInternational Edition: DOI: 10.1002/anie.201603562Structural Analysis using SHALiPE to Reveal RNAG-QuadruplexFormation in Human Precursor MicroRNAChun Kit Kwok, Aleksandr B. Sahakyan, and Shankar Balasubramanian*Abstract: RNA G-quadruplex (rG4) structures are of funda-mental importance to biology. A novel approach is introducedto detect and structurally map rG4s at single-nucleotideresolution in RNAs. The approach, denoted SHALiPE,couples selective 2’-hydroxyl acylation with lithium ion-basedprimer extension, and identifies characteristic structural finger-prints for rG4 mapping. We apply SHALiPE to interrogate thehuman precursor microRNA 149, and reveal the formation ofan rG4 structure in this non-coding RNA. Additional analysessupport the SHALiPE results and uncover that this rG4 hasa parallel topology, is thermally stable, and is conserved inmammals. An in vitro Dicer assay shows that this rG4 inhibitsDicer processing, supporting the potential role of rG4 struc-tures in microRNA maturation and post-transcriptional regu-lation of mRNAs.RNAG-quadruplexes (rG4s) appear to be important in generegulation and disease.[1] This structural motif comprises G-quartets (Figure 1A) with connecting loops, and can bestabilized by cations, especially potassium (K+). Recently,rG4s have been visualized in human cells,[2] and ligands, suchas pyridostatin (PDS)[3] (Figure 1B), have been shown tofurther stabilize rG4 structures.[2a]Our understanding of rG4 structure and function is still inits infancy. Biophysical characterization of rG4s has typicallyinvolved circular dichroism (CD) spectroscopy,[4] UV-thermalmelting,[5] and NMR spectroscopy,[6] which report rG4features, but largely without considering the effects offlanking sequences and an extended sequence context onrG4 formation. In-line probing can address these shortcom-ings and identifies rG4s;[7] however, the long experimentaltime (two-day reaction) poses some practical limitations.Reverse transcriptase stalling (RTS)[8] can map the 3’-end ofthe rG4 in a short time (15-minute reaction), but does notprovide nucleotide resolution details of the G-quartets andloops.SHAPE[9] (selective 2’-hydroxyl acylation analyzed byprimer extension) exploits differential, structure-dependentkinetics of acylation of 2’-OH of RNA, which is measured byreverse transcriptase stop at the 2’-O adduct. SHAPE hasmapped various RNA structures,[10] including motifs such asRNA hairpins, but not rG4s. Herein, we show a new methodto map rG4s that overcomes limitations of the classicalSHAPE method. Specifically, we introduce lithium ion (Li+)-based primer extension (LiPE) with 2-methylnicotinic acidimidazolide (NAI) to map rG4s at single nucleotide resolu-tion (Figure 1C). Importantly, we apply it to reveal rG4formation in biologically important RNAs, and report the roleof rG4s in Dicer processing.We first evaluated the current SHAPE approach on anrG4 structure, using a construct comprising 5’ and 3’ stemloops (SL)[11] and the telomeric TERRA[12] rG4, UUAGG-GUUAGGGUUAGGGUUAGGGUUA (Supporting Infor-mation, Table S1 and Figure S1). Using NAI-acylation (5-minute reaction),[13] we observed that patterns of NAI-induced modifications are obscured by strong RTS byTERRA rG4 formation, in the standard K+-containing PEbuffer (Figure 2, lanes 1–6, green). This suggests that K+should be avoided in the PE buffer[14] to enable SHAPEanalysis of rG4s.As rG4 structures are less stable in Li+,[15] we preparedand evaluated a Li+-based PE buffer to reduce rG4 stallingand unmask patterns of rG4-dependent 2’-OH acylation.When the reaction of RNA with NAI was performed in thepresence of Li+, the 2’-OH of most nucleotides in the TERRArG4 region were strongly modified (Figure 2, lane 10),indicating that the rG4 region is unstructured. In contrast,Figure 1. Structure probing of rG4s using SHALiPE. Chemical structureof A) a G-quartet and B) pyridostatin (PDS). C) Overview of SHALiPE.RNA is folded under Li+, K+, or K++PDS conditions, followed byselective 2’-hydroxyl acylation (SHA) using NAI. Li+-based primerextension (LiPE) is conducted to analyze the NAI-modified RNA.Controls are performed with no NAI.[*] Dr. C. K. Kwok, Dr. A. B. Sahakyan, Prof. S. BalasubramanianThe University of Cambridge, Department of ChemistryLensfield Road, Cambridge, CB2 1EW (UK)E-mail: sb10031@cam.ac.ukSupporting information for this article can be found under:http://dx.doi.org/10.1002/anie.201603562.Ó 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co.KGaA. This is an open access article under the terms of the CreativeCommons Attribution Non-Commercial NoDerivs License, whichpermits use and distribution in any medium, provided the originalwork is properly cited, the use is non-commercial, and nomodifications or adaptations are made.AngewandteChemieCommunications8958 Ó 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2016, 55, 8958 –8961NAI reaction in the presence of K+, led to protection of someGs in the rG4 region (Figure 2, lanes 10 and 12, purpleasterisks), suggestive of rG4 formation. Interestingly, themodifications at the remaining Gs in the rG4 motif were moreintense in K+ compared to Li+ (Figure 2, lanes 10 and 12, blueasterisks), but more protected in the presence of both K+ andthe rG4 stabilizing ligand, namely PDS (K++PDS) (Figure 2,lanes 12 and 14, blue asterisks). These Gs were mostly locatedat the 3’-end of each G-tract. Similar findings were observedfor TERC rG4 and NRAS rG4 (Supporting Information,Figures S2,S3), suggesting that the 2’-OHs of the 3’-G-quartetare more flexible than those of the 5’-G-quartet. These datashow that SHALiPE provides a distinctive pattern for rG4structure that can be used to fine map rG4s, along with otherRNA structures (Supporting Information, Figure S4) at singlenucleotide resolution.RNA structures are dynamic and hairpin to G-quadruplexconformational transition[16] may have regulatory roles inbiological processes. We surveyed the miRBase[17] to identifyprecursor microRNA (pre-miRNA) hairpins that containputative rG4s (Supporting Information, Table S2), andselected a conserved candidate (Supporting Information,Table S3), pre-miRNA 149, for further investigation. Theprocessing of pre-miRNA 149 by Dicer produces miR149and miR149*, both of which have been shown to function asoncogenic regulators in cancers.[18]Using comparative sequence analysis, we identified a con-served rG4 sequence that partially overlaps withmiRNA 149*(Figure 3A). We confirmed rG4 formation in the putativerG4 sequence by CD and UV-melting spectroscopic analyses(Figure 3B; Supporting Information, Figure S5). Under150 mmK+ conditions, the CD spectrum of the wildtype rG4sequence, but not the mutated sequence, showed a distinctCD profile consistent with a parallel rG4 topology[4] (Fig-ure 3B; Supporting Information, Table S1). UV-melting anal-ysis of the wildtype rG4 sequence displayed a hypochromicFigure 2. SHALiPE development and analysis. TERRA RNA was probedwith NAI under Li+, K+, and K++PDS conditions, followed by primerextension (PE) using either the SSIII commercial (com.) K+-containingPE buffer[14] (left gel) or home-made Li+-containing PE buffer (rightgel). On the left, intense stalling was observed (lanes 1–6), caused byformation of TERRA rG4 (stabilized by K+ in the commercial PE buffer)in the PE step. On the right, stalling was reduced using the Li+-basedPE buffer. Lanes 9–14 show (¢) and (+) NAI signals under Li+, K+,and K++PDS conditions. TERRA rG4 was unstructured in Li+ con-ditions. Under physiological K+ conditions, TERRA rG4 displayeddistinct NAI profiles (low NAI signals in several Gs involved in rG4;purple asterisks, see lanes 10 and 12). Blue asterisks indicate Gs thatwere protected upon PDS addition (lanes 12 and 14). The loops (L1,L2, and L3) and loop nucleotides (UUA) are shown in red. Lanes 7–8show sequencing of G. 5’ SL, 5’ stem loop (Supporting Information,Figure S1).Figure 3. Computational analysis and biophysical characterization of a putative rG4 in human pre-miRNA 149. A) Comparative sequence analysisof pre-miRNA 149. The miR149, miR149*, and putative rG4 are highlighted. Conserved nucleotides are marked by asterisks and the length ofRNAs are shown. The name of the species, presented here with three-letter codes, can be found in the Supporting Information, Table S3. B) CDspectrum of wildtype rG4 sequence under K+ condition shows a characteristic signature that indicates formation of a parallel topology rG4structure. Mutant shows no such signature. The Y axis is molar ellipticity per nucleotide (De). C) The secondary structure model for the humanpre-miRNA 149 is derived using the sequences in (A) and TurboFold.[19]AngewandteChemieCommunications8959Angew. Chem. Int. Ed. 2016, 55, 8958 –8961 Ó 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.orgshift at 295 nm (Supporting Information, Figure S5), a hall-mark for rG4 formation.[5] Furthermore, the melting temper-ature for the rG4 sequence under the 150 mmK+ conditionwas found to be > 90 8C, suggestive of a highly thermostablerG4 under physiological K+ conditions.Next, we predicted the secondary structure of the humanpre-miRNA 149 by TurboFold,[19] which takes into consider-ation the sequences of orthologues and generates a consensusRNA structural model (Figure 3C). Since the rG4 partiallyoverlaps with the canonical hairpin structure in pre-miRNA149 (Figure 3C), we hypothesize that the formation of an rG4structure may affect the folding of the hairpin structure in thispre-miRNA.We applied SHALiPE to probe the structure of pre-miRNA 149 under Li+, K+, and K++PDS conditions (Fig-ure 4A). The results revealed a number of important findings.First, NAI modification (reactivity) profiles for Li+ versus K+and Li+ versus K++PDS were weakly correlated, withPearsonÏs correlation coefficients (PCC) of 0.10 and 0.19respectively (Figure 4B, left and middle plots), suggestingthat the major RNA conformations under Li+ and K+ (orK++PDS) conditions are different.Second, we found that in Li+ (Figure 4A, lanes 1–2), NAImodifications mapped consistently to the predicted hairpinstructure (Figure 4A,C, green asterisks). In contrast, underK+ and K++PDS conditions (Figure 4A, lanes 3–6), NAImodifications did not agree with the hairpin structure. Forexample, A56, A60, and A64 were strongly modified by NAI(Figure 4A, lanes 3–4), despite being assigned as base-pairednucleotides in the hairpin structure (Figure 4C). To verify theSHALiPE results, a nucleobase-specific reagent dimethylsulfate (DMS) was used, which modifies the Watson–Crickface of As (N1A) and Cs (N3C)[20] in a manner that stallsreverse transcriptase. As such, DMSLiPE provides a measureof nucleobase-specific DMS reactivity with RNA. Similar toNAI, we observed that A56, A60, and A64 were stronglymodified by DMS under K+ and K++PDS conditions but notin Li+ conditions (Supporting Information, Figure S6), sug-gesting that A56, A60, and A64 are not base-paired and arethe loop nucleotides of the rG4 (Figure 4A,D). Our resultssupport that the major RNA conformation under K+ andK++PDS conditions contains an rG4 (Figure 4D).Furthermore, we performed SHALiPE on a mutantconstruct that cannot form an rG4, and found that the NAImodification profiles were almost identical under Li+, K+, andK++PDS conditions (Supporting Information, Figure S7).Also, the NAI modifications mapped well to the predictedstructure of this construct (Supporting Information, Fig-ure S7). This verified that the change in NAI profilesobserved in the natural sequence (Figure 4A) was indeeddue to rG4 formation.Third, we observed that the PCC for NAI reactivity valuesunder K+ and K++PDS conditions was 0.65 (Figure 4B, rightplot). Visually, three nucleotides (G63, G55, and G59) stoodout, and by removing them from the correlation analysisyielded a PCC of 0.95 (Figure 4B, right plot), similar to theFigure 4. SHALiPE and Dicer cleavage assays on human pre-miRNA 149 reveal an rG4 that inhibits in vitro Dicer processing. A) SHALiPE on pre-miRNA 149 under Li+, K+, and K++PDS conditions (lanes 1–6). Lanes 7–10 show sequencing of U, C, G, and A respectively. The rG4 loops are inred. G that are protected with the addition of PDS (G55, G59, G63) are marked with blue asterisks. B) The PCC of the normalized NAI reactivityfor K+ versus Li+ (left), K++PDS versus Li+ (middle), and K++PDS versus K+ (right). The PCC increases from 0.65 to 0.95 by removing G55, G59and G63 (right). C) The hairpin conformation of pre-miRNA 149. Some nucleotides are marked with green asterisks. D) The rG4-containingconformation of pre-miRNA 149. G55, G59, and G63 are marked with blue asterisks. E) Results of in vitro Dicer assay on pre-miRNA 149 wildtype(WT) and mutant (MUT). Relative miRNA ratio (K+/Li+ or K++PDS/Li+) are reported. Values are from 3 biological replicates and error barsdepict standard deviations. The constructs for the Dicer assay do not contain the 5’ and 3’SL (Supporting Information, Table S1).AngewandteChemieCommunications8960 www.angewandte.org Ó 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2016, 55, 8958 –8961PCC of 0.93 for DMS reactivity (only considers As and Cs)[20](Supporting Information, Figure S6). Interestingly, thesethree nucleotides were the 3’-G of each G-tract in the rG4(Figure 4D, blue asterisks). They were highly modified byNAI under K+, and protected with the addition of PDS(Figure 4A, lanes 4 and 6), as for the other rG4 examples wetested (Figure 2; Supporting Information, Figures S2,S3).Different acylation reagents and rG4 ligands yielded thesame results (Supporting Information, Figure S8), suggestingthat this feature is not reagent- or rG4 ligand-dependent, butrather due to the flexible nature of the 3’-G-quartet and/or theunique conformation of the 3’-Gs of the rG4, making themsusceptible for 2’-OH acylation attack.Taken together, these results provide strong evidence forthe formation of an rG4 under K+ and K++PDS conditions(Figure 4D), whereas a hairpin conformation predominatesunder Li+ conditions (Figure 4C) in the pre-miRNA 149wildtype. It is possible that two (or more) RNA conforma-tions exist under K+ and K++PDS conditions.To assess the role of the rG4 structure in this pre-miRNA 149, we performed an in vitro Dicer cleavage assayon the wildtype and mutant constructs (without 5’ and 3’ SL)under Li+, K+, and K++PDS conditions (Figure 4E; Sup-porting Information, Figure S9, Table S1). Our results showedthat the rG4 inhibits Dicer processing activity. The yield ofmiRNAunder K+ and K++PDS conditions were 2.5–2.7-foldless than under the Li+ condition for the wildtype (Figure 4E,blue bars, 0.37 0.05 for K+/Li+ and 0.39 0.04 for K++PDS/Li+), in contrast to 1.1–1.2-fold for the mutant (Fig-ure 4E, red bars, 0.91 0.12 for K+/Li+ and 0.82 0.08 04 forK++PDS/Li+). The repressive role of the rG4 in pre-miRNA 149 is consistent with recently reported cases ofpre-miRNA 92b and pre-miRNA let-7e,[21] suggestinga broader role for rG4s in miRNA maturation. Our bioinfor-matics analysis shows 85 putative rG4s in human pre-miRNAs(using G3+L1-12-based motif, see the Supporting Information)and many others across organisms (Supporting Information,Table S2), suggested the phenomenon may be widespread.In summary, SHALiPE (and DMSLiPE) has enabled finemapping of rG4 structures in RNAs. The approach hasnumerous benefits over prior methods and we have used it toreveal an rG4 structure in the biologically important pre-miRNA 149, and showed that the rG4 inhibits Dicer process-ing in vitro. Our findings provide insights into the biologicalroles of rG4 structures and the approach may ultimately haveutility in transcriptome-wide probing of rG4s.AcknowledgementsThis work is supported by a European Research CouncilAdvanced grant (No. 339778). C.K.K. received some supportfrom the Croucher Foundation. A. Sahakyan was supportedby a Herchel Smith Fellowship. We thank V. Chambers, Dr. B.Herdy, Dr. L. K. M. Chan, and Dr. M. Di Antonio fordiscussions.Keywords: Dicer processing · G-quadruplexes ·precursor miRNA · RNA structure · structure probingHow to cite: Angew. Chem. Int. Ed. 2016, 55, 8958–8961Angew. Chem. 2016, 128, 9104–9107[1] a) S. Millevoi, H. Moine, S. Vagner, WIREs RNA 2012, 3, 495 –507; b) R. Simone, P. Fratta, S. Neidle, G. N. Parkinson, A. M.Isaacs, FEBS Lett. 2015, 589, 1653 – 1668.[2] a) G. Biffi, M. Di Antonio, D. Tannahill, S. Balasubramanian,Nat. Chem. 2014, 6, 75 – 80; b) A. Laguerre, K. Hukezalie, P.Winckler, F. Katranji, G. Chanteloup, M. Pirrotta, J. M. Perrier-Cornet, J. M. Wong, D. Monchaud, J. Am. Chem. Soc. 2015, 137,8521 – 8525.[3] R. Rodriguez, S. Muller, J. A. Yeoman, C. Trentesaux, J. F. Riou,S. Balasubramanian, J. Am. Chem. Soc. 2008, 130, 15758 – 15759.[4] M. Vorlcˇkov, I. Kejnovsk, J. Sagi, D. Rencˇiuk, K. Bednrˇov,J. Motlov, J. Kypr, Methods 2012, 57, 64 – 75.[5] J.-L. Mergny, A.-T. Phan, L. Lacroix, FEBS Lett. 1998, 435, 74 –78.[6] M. Webba da Silva, Methods 2007, 43, 264 – 277.[7] a) J. D. Beaudoin, R. Jodoin, J. P. Perreault, Methods 2013, 64,79 – 87; b) C. K. Kwok, Y. Ding, S. Shahid, S. M. Assmann, P. C.Bevilacqua, Biochem. J. 2015, 467, 91 – 102.[8] C. K. Kwok, S. Balasubramanian, Angew. Chem. Int. Ed. 2015,54, 6751 – 6754; Angew. Chem. 2015, 127, 6855 – 6858.[9] K. A.Wilkinson, E. J. Merino, K. M.Weeks,Nat. Protoc. 2006, 1,1610 – 1616.[10] K. M. Weeks, D. M. Mauger, Acc. Chem. Res. 2011, 44, 1280 –1291.[11] This design allows information near the 5’- and 3’-end of thesequence of interest to be retrieved. For gel-based analysis, the5’-end of the RNA has band compression and intense full lengthband, whereas the 3’-end of the RNA requires a primer bindngsite.[12] B. Luke, J. Lingner, EMBO J. 2009, 28, 2503 – 2510.[13] R. C. Spitale, P. Crisalli, R. A. Flynn, E. A. Torre, E. T. Kool,H. Y. Chang, Nat. Chem. Biol. 2013, 9, 18 – 20.[14] Reverse transcriptases, for example, Superscript, AMV, Proto-script, Thermoscript, Maxima, and Tth Pol, contain 150–375 mmK+ in their 5X primer extension buffers.[15] S. Neidle, S. Balasubramanian, Quadruplex nucleic acids, Vol. 7,Royal Society of Chemistry, Cambridge, 2006.[16] A. Bugaut, P. Murat, S. Balasubramanian, J. Am. Chem. Soc.2012, 134, 19953 – 19956.[17] A. Kozomara, S. Griffiths-Jones, Nucleic Acids Res. 2014, 42,D68 – 73.[18] a) L. Jin, W. L. Hu, C. C. Jiang, J. X. Wang, C. C. Han, P. Chu,L. J. Zhang, R. F. Thorne, J. Wilmott, R. A. Scolyer, P. Hersey,X. D. Zhang, M. Wu, Proc. Natl. Acad. Sci. USA 2011, 108,15840 – 15845; b) A. Bischoff, B. Huck, B. Keller, M. Strotbek, S.Schmid, M. Boerries, H. Busch, D. Muller, M. A. Olayioye,Cancer Res. 2014, 74, 5256 – 5265; c) S. H. Chan, W. C. Huang,J. W. Chang, K. J. Chang, W. H. Kuo, M. Y. Wang, K. Y. Lin,Y. H. Uen, M. F. Hou, C. M. Lin, T. H. Jang, C. W. Tu, Y. R. Lee,Y. H. Lee, M. T. Tien, L. H. Wang, Oncogene 2014, 33, 4496 –4507.[19] A. Harmanci, G. Sharma, D. Mathews, BMC Bioinf. 2011, 12,108.[20] DMS also modifies the unprotected, Hoogsteen face of Gs(N7G); however, they are undetected in reverse transcription.[21] a) G. Mirihana Arachchilage, A. C. Dassanayake, S. Basu,Chem. Biol. 2015, 22, 262 – 272; b) S. Pandey, P. Agarwala,G. G. Jayaraj, R. Gargallo, S. Maiti,Biochemistry 2015, 54, 7067 –7078.Received: April 12, 2016Published online: June 29, 2016AngewandteChemieCommunications8961Angew. Chem. Int. Ed. 2016, 55, 8958 –8961 Ó 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org

Structural Analysis using SHALiPE to Reveal RNA G-Quadruplex Formation in Human Precursor MicroRNA.

RNA G-quadruplex (rG4) structures are of fundamental importance to biology. A novel approach is introduced to detect and structurally map rG4s at single-nucleotide resolution in RNAs. The approach, denoted SHALiPE, couples selective 2'-hydroxyl acylation with lithium ion-based primer extension, and identifies characteristic structural fingerprints for rG4 mapping. We apply SHALiPE to interrogate the human precursor microRNA 149, and reveal the formation of an rG4 structure in this non-coding RNA. Additional analyses support the SHALiPE results and uncover that this rG4 has a parallel topology, is thermally stable, and is conserved in mammals. An in vitro Dicer assay shows that this rG4 inhibits Dicer processing, supporting the potential role of rG4 structures in microRNA maturation and post-transcriptional regulation of mRNAs

Kwok, CK

Sahakyan, Aleksandr B.

Balasubramanian, S

ORA - Oxford University Research Archive

Structural analysis using SHALiPE to reveal RNA G-quadruplex formation in human precursor MicroRNA.

AbstractRNA G‐quadruplex (rG4) structures are of fundamental importance to biology. A novel approach is introduced to detect and structurally map rG4s at single‐nucleotide resolution in RNAs. The approach, denoted SHALiPE, couples selective 2′‐hydroxyl acylation with lithium ion‐based primer extension, and identifies characteristic structural fingerprints for rG4 mapping. We apply SHALiPE to interrogate the human precursor microRNA 149, and reveal the formation of an rG4 structure in this non‐coding RNA. Additional analyses support the SHALiPE results and uncover that this rG4 has a parallel topology, is thermally stable, and is conserved in mammals. An in vitro Dicer assay shows that this rG4 inhibits Dicer processing, supporting the potential role of rG4 structures in microRNA maturation and post‐transcriptional regulation of mRNAs.</jats:p

German Edition: DOI: 10.1002/ange.201603562G-QuadruplexesInternational Edition: DOI: 10.1002/anie.201603562Structural Analysis using SHALiPE to Reveal RNAG-QuadruplexFormation in Human Precursor MicroRNAChun Kit Kwok, Aleksandr B. Sahakyan, and Shankar Balasubramanian*Abstract: RNA G-quadruplex (rG4) structures are of funda-mental importance to biology. A novel approach is introducedto detect and structurally map rG4s at single-nucleotideresolution in RNAs. The approach, denoted SHALiPE,couples selective 2’-hydroxyl acylation with lithium ion-basedprimer extension, and identifies characteristic structural finger-prints for rG4 mapping. We apply SHALiPE to interrogate thehuman precursor microRNA 149, and reveal the formation ofan rG4 structure in this non-coding RNA. Additional analysessupport the SHALiPE results and uncover that this rG4 hasa parallel topology, is thermally stable, and is conserved inmammals. An in vitro Dicer assay shows that this rG4 inhibitsDicer processing, supporting the potential role of rG4 struc-tures in microRNA maturation and post-transcriptional regu-lation of mRNAs.RNAG-quadruplexes (rG4s) appear to be important in generegulation and disease.[1] This structural motif comprises G-quartets (Figure 1A) with connecting loops, and can bestabilized by cations, especially potassium (K+). Recently,rG4s have been visualized in human cells,[2] and ligands, suchas pyridostatin (PDS)[3] (Figure 1B), have been shown tofurther stabilize rG4 structures.[2a]Our understanding of rG4 structure and function is still inits infancy. Biophysical characterization of rG4s has typicallyinvolved circular dichroism (CD) spectroscopy,[4] UV-thermalmelting,[5] and NMR spectroscopy,[6] which report rG4features, but largely without considering the effects offlanking sequences and an extended sequence context onrG4 formation. In-line probing can address these shortcom-ings and identifies rG4s;[7] however, the long experimentaltime (two-day reaction) poses some practical limitations.Reverse transcriptase stalling (RTS)[8] can map the 3’-end ofthe rG4 in a short time (15-minute reaction), but does notprovide nucleotide resolution details of the G-quartets andloops.SHAPE[9] (selective 2’-hydroxyl acylation analyzed byprimer extension) exploits differential, structure-dependentkinetics of acylation of 2’-OH of RNA, which is measured byreverse transcriptase stop at the 2’-O adduct. SHAPE hasmapped various RNA structures,[10] including motifs such asRNA hairpins, but not rG4s. Herein, we show a new methodto map rG4s that overcomes limitations of the classicalSHAPE method. Specifically, we introduce lithium ion (Li+)-based primer extension (LiPE) with 2-methylnicotinic acidimidazolide (NAI) to map rG4s at single nucleotide resolu-tion (Figure 1C). Importantly, we apply it to reveal rG4formation in biologically important RNAs, and report the roleof rG4s in Dicer processing.We first evaluated the current SHAPE approach on anrG4 structure, using a construct comprising 5’ and 3’ stemloops (SL)[11] and the telomeric TERRA[12] rG4, UUAGG-GUUAGGGUUAGGGUUAGGGUUA (Supporting Infor-mation, Table S1 and Figure S1). Using NAI-acylation (5-minute reaction),[13] we observed that patterns of NAI-induced modifications are obscured by strong RTS byTERRA rG4 formation, in the standard K+-containing PEbuffer (Figure 2, lanes 1–6, green). This suggests that K+should be avoided in the PE buffer[14] to enable SHAPEanalysis of rG4s.As rG4 structures are less stable in Li+,[15] we preparedand evaluated a Li+-based PE buffer to reduce rG4 stallingand unmask patterns of rG4-dependent 2’-OH acylation.When the reaction of RNA with NAI was performed in thepresence of Li+, the 2’-OH of most nucleotides in the TERRArG4 region were strongly modified (Figure 2, lane 10),indicating that the rG4 region is unstructured. In contrast,Figure 1. Structure probing of rG4s using SHALiPE. Chemical structureof A) a G-quartet and B) pyridostatin (PDS). C) Overview of SHALiPE.RNA is folded under Li+, K+, or K++PDS conditions, followed byselective 2’-hydroxyl acylation (SHA) using NAI. Li+-based primerextension (LiPE) is conducted to analyze the NAI-modified RNA.Controls are performed with no NAI.[*] Dr. C. K. Kwok, Dr. A. B. Sahakyan, Prof. S. BalasubramanianThe University of Cambridge, Department of ChemistryLensfield Road, Cambridge, CB2 1EW (UK)E-mail: sb10031@cam.ac.ukSupporting information for this article can be found under:http://dx.doi.org/10.1002/anie.201603562.⌫ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co.KGaA. This is an open access article under the terms of the CreativeCommons Attribution Non-Commercial NoDerivs License, whichpermits use and distribution in any medium, provided the originalwork is properly cited, the use is non-commercial, and nomodifications or adaptations are made.AngewandteChemieCommunications8958 ⌫ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2016, 55, 8958 –8961NAI reaction in the presence of K+, led to protection of someGs in the rG4 region (Figure 2, lanes 10 and 12, purpleasterisks), suggestive of rG4 formation. Interestingly, themodifications at the remaining Gs in the rG4 motif were moreintense in K+ compared to Li+ (Figure 2, lanes 10 and 12, blueasterisks), but more protected in the presence of both K+ andthe rG4 stabilizing ligand, namely PDS (K++PDS) (Figure 2,lanes 12 and 14, blue asterisks). These Gs were mostly locatedat the 3’-end of each G-tract. Similar findings were observedfor TERC rG4 and NRAS rG4 (Supporting Information,Figures S2,S3), suggesting that the 2’-OHs of the 3’-G-quartetare more flexible than those of the 5’-G-quartet. These datashow that SHALiPE provides a distinctive pattern for rG4structure that can be used to fine map rG4s, along with otherRNA structures (Supporting Information, Figure S4) at singlenucleotide resolution.RNA structures are dynamic and hairpin to G-quadruplexconformational transition[16] may have regulatory roles inbiological processes. We surveyed the miRBase[17] to identifyprecursor microRNA (pre-miRNA) hairpins that containputative rG4s (Supporting Information, Table S2), andselected a conserved candidate (Supporting Information,Table S3), pre-miRNA 149, for further investigation. Theprocessing of pre-miRNA 149 by Dicer produces miR149and miR149*, both of which have been shown to function asoncogenic regulators in cancers.[18]Using comparative sequence analysis, we identified a con-served rG4 sequence that partially overlaps withmiRNA 149*(Figure 3A). We confirmed rG4 formation in the putativerG4 sequence by CD and UV-melting spectroscopic analyses(Figure 3B; Supporting Information, Figure S5). Under150 mmK+ conditions, the CD spectrum of the wildtype rG4sequence, but not the mutated sequence, showed a distinctCD profile consistent with a parallel rG4 topology[4] (Fig-ure 3B; Supporting Information, Table S1). UV-melting anal-ysis of the wildtype rG4 sequence displayed a hypochromicFigure 2. SHALiPE development and analysis. TERRA RNA was probedwith NAI under Li+, K+, and K++PDS conditions, followed by primerextension (PE) using either the SSIII commercial (com.) K+-containingPE buffer[14] (left gel) or home-made Li+-containing PE buffer (rightgel). On the left, intense stalling was observed (lanes 1–6), caused byformation of TERRA rG4 (stabilized by K+ in the commercial PE buffer)in the PE step. On the right, stalling was reduced using the Li+-basedPE buffer. Lanes 9–14 show (ˇ) and (+) NAI signals under Li+, K+,and K++PDS conditions. TERRA rG4 was unstructured in Li+ con-ditions. Under physiological K+ conditions, TERRA rG4 displayeddistinct NAI profiles (low NAI signals in several Gs involved in rG4;purple asterisks, see lanes 10 and 12). Blue asterisks indicate Gs thatwere protected upon PDS addition (lanes 12 and 14). The loops (L1,L2, and L3) and loop nucleotides (UUA) are shown in red. Lanes 7–8show sequencing of G. 5’ SL, 5’ stem loop (Supporting Information,Figure S1).Figure 3. Computational analysis and biophysical characterization of a putative rG4 in human pre-miRNA 149. A) Comparative sequence analysisof pre-miRNA 149. The miR149, miR149*, and putative rG4 are highlighted. Conserved nucleotides are marked by asterisks and the length ofRNAs are shown. The name of the species, presented here with three-letter codes, can be found in the Supporting Information, Table S3. B) CDspectrum of wildtype rG4 sequence under K+ condition shows a characteristic signature that indicates formation of a parallel topology rG4structure. Mutant shows no such signature. The Y axis is molar ellipticity per nucleotide (De). C) The secondary structure model for the humanpre-miRNA 149 is derived using the sequences in (A) and TurboFold.[19]AngewandteChemieCommunications8959Angew. Chem. Int. Ed. 2016, 55, 8958 –8961 ⌫ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.orgshift at 295 nm (Supporting Information, Figure S5), a hall-mark for rG4 formation.[5] Furthermore, the melting temper-ature for the rG4 sequence under the 150 mmK+ conditionwas found to be > 90 8C, suggestive of a highly thermostablerG4 under physiological K+ conditions.Next, we predicted the secondary structure of the humanpre-miRNA 149 by TurboFold,[19] which takes into consider-ation the sequences of orthologues and generates a consensusRNA structural model (Figure 3C). Since the rG4 partiallyoverlaps with the canonical hairpin structure in pre-miRNA149 (Figure 3C), we hypothesize that the formation of an rG4structure may affect the folding of the hairpin structure in thispre-miRNA.We applied SHALiPE to probe the structure of pre-miRNA 149 under Li+, K+, and K++PDS conditions (Fig-ure 4A). The results revealed a number of important findings.First, NAI modification (reactivity) profiles for Li+ versus K+and Li+ versus K++PDS were weakly correlated, withPearson s correlation coefficients (PCC) of 0.10 and 0.19respectively (Figure 4B, left and middle plots), suggestingthat the major RNA conformations under Li+ and K+ (orK++PDS) conditions are different.Second, we found that in Li+ (Figure 4A, lanes 1–2), NAImodifications mapped consistently to the predicted hairpinstructure (Figure 4A,C, green asterisks). In contrast, underK+ and K++PDS conditions (Figure 4A, lanes 3–6), NAImodifications did not agree with the hairpin structure. Forexample, A56, A60, and A64 were strongly modified by NAI(Figure 4A, lanes 3–4), despite being assigned as base-pairednucleotides in the hairpin structure (Figure 4C). To verify theSHALiPE results, a nucleobase-specific reagent dimethylsulfate (DMS) was used, which modifies the Watson–Crickface of As (N1A) and Cs (N3C)[20] in a manner that stallsreverse transcriptase. As such, DMSLiPE provides a measureof nucleobase-specific DMS reactivity with RNA. Similar toNAI, we observed that A56, A60, and A64 were stronglymodified by DMS under K+ and K++PDS conditions but notin Li+ conditions (Supporting Information, Figure S6), sug-gesting that A56, A60, and A64 are not base-paired and arethe loop nucleotides of the rG4 (Figure 4A,D). Our resultssupport that the major RNA conformation under K+ andK++PDS conditions contains an rG4 (Figure 4D).Furthermore, we performed SHALiPE on a mutantconstruct that cannot form an rG4, and found that the NAImodification profiles were almost identical under Li+, K+, andK++PDS conditions (Supporting Information, Figure S7).Also, the NAI modifications mapped well to the predictedstructure of this construct (Supporting Information, Fig-ure S7). This verified that the change in NAI profilesobserved in the natural sequence (Figure 4A) was indeeddue to rG4 formation.Third, we observed that the PCC for NAI reactivity valuesunder K+ and K++PDS conditions was 0.65 (Figure 4B, rightplot). Visually, three nucleotides (G63, G55, and G59) stoodout, and by removing them from the correlation analysisyielded a PCC of 0.95 (Figure 4B, right plot), similar to theFigure 4. SHALiPE and Dicer cleavage assays on human pre-miRNA 149 reveal an rG4 that inhibits in vitro Dicer processing. A) SHALiPE on pre-miRNA 149 under Li+, K+, and K++PDS conditions (lanes 1–6). Lanes 7–10 show sequencing of U, C, G, and A respectively. The rG4 loops are inred. G that are protected with the addition of PDS (G55, G59, G63) are marked with blue asterisks. B) The PCC of the normalized NAI reactivityfor K+ versus Li+ (left), K++PDS versus Li+ (middle), and K++PDS versus K+ (right). The PCC increases from 0.65 to 0.95 by removing G55, G59and G63 (right). C) The hairpin conformation of pre-miRNA 149. Some nucleotides are marked with green asterisks. D) The rG4-containingconformation of pre-miRNA 149. G55, G59, and G63 are marked with blue asterisks. E) Results of in vitro Dicer assay on pre-miRNA 149 wildtype(WT) and mutant (MUT). Relative miRNA ratio (K+/Li+ or K++PDS/Li+) are reported. Values are from 3 biological replicates and error barsdepict standard deviations. The constructs for the Dicer assay do not contain the 5’ and 3’SL (Supporting Information, Table S1).AngewandteChemieCommunications8960 www.angewandte.org ⌫ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2016, 55, 8958 –8961PCC of 0.93 for DMS reactivity (only considers As and Cs)[20](Supporting Information, Figure S6). Interestingly, thesethree nucleotides were the 3’-G of each G-tract in the rG4(Figure 4D, blue asterisks). They were highly modified byNAI under K+, and protected with the addition of PDS(Figure 4A, lanes 4 and 6), as for the other rG4 examples wetested (Figure 2; Supporting Information, Figures S2,S3).Different acylation reagents and rG4 ligands yielded thesame results (Supporting Information, Figure S8), suggestingthat this feature is not reagent- or rG4 ligand-dependent, butrather due to the flexible nature of the 3’-G-quartet and/or theunique conformation of the 3’-Gs of the rG4, making themsusceptible for 2’-OH acylation attack.Taken together, these results provide strong evidence forthe formation of an rG4 under K+ and K++PDS conditions(Figure 4D), whereas a hairpin conformation predominatesunder Li+ conditions (Figure 4C) in the pre-miRNA 149wildtype. It is possible that two (or more) RNA conforma-tions exist under K+ and K++PDS conditions.To assess the role of the rG4 structure in this pre-miRNA 149, we performed an in vitro Dicer cleavage assayon the wildtype and mutant constructs (without 5’ and 3’ SL)under Li+, K+, and K++PDS conditions (Figure 4E; Sup-porting Information, Figure S9, Table S1). Our results showedthat the rG4 inhibits Dicer processing activity. The yield ofmiRNAunder K+ and K++PDS conditions were 2.5–2.7-foldless than under the Li+ condition for the wildtype (Figure 4E,blue bars, 0.37⌃ 0.05 for K+/Li+ and 0.39⌃ 0.04 for K++PDS/Li+), in contrast to 1.1–1.2-fold for the mutant (Fig-ure 4E, red bars, 0.91⌃ 0.12 for K+/Li+ and 0.82⌃ 0.08 04 forK++PDS/Li+). The repressive role of the rG4 in pre-miRNA 149 is consistent with recently reported cases ofpre-miRNA 92b and pre-miRNA let-7e,[21] suggestinga broader role for rG4s in miRNA maturation. Our bioinfor-matics analysis shows 85 putative rG4s in human pre-miRNAs(using G3+L1-12-based motif, see the Supporting Information)and many others across organisms (Supporting Information,Table S2), suggested the phenomenon may be widespread.In summary, SHALiPE (and DMSLiPE) has enabled finemapping of rG4 structures in RNAs. The approach hasnumerous benefits over prior methods and we have used it toreveal an rG4 structure in the biologically important pre-miRNA 149, and showed that the rG4 inhibits Dicer process-ing in vitro. Our findings provide insights into the biologicalroles of rG4 structures and the approach may ultimately haveutility in transcriptome-wide probing of rG4s.AcknowledgementsThis work is supported by a European Research CouncilAdvanced grant (No. 339778). C.K.K. received some supportfrom the Croucher Foundation. A. Sahakyan was supportedby a Herchel Smith Fellowship. We thank V. Chambers, Dr. B.Herdy, Dr. L. K. M. Chan, and Dr. M. 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Wang, Oncogene 2014, 33, 4496 –4507.[19] A. Harmanci, G. Sharma, D. Mathews, BMC Bioinf. 2011, 12,108.[20] DMS also modifies the unprotected, Hoogsteen face of Gs(N7G); however, they are undetected in reverse transcription.[21] a) G. Mirihana Arachchilage, A. C. Dassanayake, S. Basu,Chem. Biol. 2015, 22, 262 – 272; b) S. Pandey, P. Agarwala,G. G. Jayaraj, R. Gargallo, S. Maiti,Biochemistry 2015, 54, 7067 –7078.Received: April 12, 2016Published online: June 29, 2016AngewandteChemieCommunications8961Angew. Chem. Int. Ed. 2016, 55, 8958 –8961 ⌫ 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim www.angewandte.org

Structural Analysis using SHALiPE to Reveal RNA G‐Quadruplex Formation in Human Precursor MicroRNA

Chun Kit Kwok

Aleksandr B. Sahakyan

Shankar Balasubramanian

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Angewandte Chemie

Structural Analysis using SHALiPE to Reveal RNA G-Quadruplex Formation in Human Precursor MicroRNA

Oxford University Research Archive

https://www.repository.cam.ac.uk/bitstream/1810/256565/4/Kwok_et_al-2016-Angewandte_Chemie_International_Edition.pdf

Structural Analysis using SHALiPE to Reveal RNA G-Quadruplex Formation in Human Precursor MicroRNA

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Oxford University Research Archive