Effect of Ribose Conformation on RNA Cleavage via Internal

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Effect of Ribose Conformation on RNA Cleavage via Internal Transesterification Fengmin Guo, Zekun Yue, Marko Trajkovski, Xiaoping Zhou, Dong Cao, Qiang Li, Baifan Wang, Xin Wen, Janez Plavec, Qian Peng, Zhen Xi, and Chuanzheng Zhou J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b06313 • Publication Date (Web): 12 Sep 2018 Downloaded from http://pubs.acs.org on September 12, 2018

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Journal of the American Chemical Society

Effect of Ribose Conformation on RNA Cleavage via Internal Transesterification Fengmin Guo,† Zekun Yue,† Marko Trajkovski,§ Xiaoping Zhou,† Dong Cao,† Qiang Li,† Baifan Wang,† Xin Wen,† Janez Plavec,*,§ Qian Peng,*,† Zhen Xi† and Chuanzheng Zhou*,†,‡ † State Key Laboratory of Elemento-Organic Chemistry and Department of Chemical Biology, College of Chemistry, Nankai University, Tianjin 300071, China ‡ Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, China §Slovenian NMR Centre, National Institute of Chemistry, Hajdrihova 19, Ljubljana, University of Ljubljana, Faculty of Chemistry and Chemical Technology, Ljubljana, EN-FIST Centre of Excellence, Ljubljana, Slovenia.

Supporting Information Placeholder ABSTRACT: RNA cleavage via internal transesterification is a fundamental reaction involved in RNA processing and metabolism, and the regulation thereof. Herein, the influence of ribose conformation on this reaction was investigated with conformationally constrained ribonucleotides. RNA cleavage rates were found to decrease in the order South-constrained ribonucleotide > native ribonucleotide >> North-constrained counterpart, indicating that the ribose conformation plays an important role in modulating RNA cleavage via internal transesterification.

RNA cleavage via internal transesterification is a fundamental reaction that is involved in RNA processing and me1 tabolism, and the regulation thereof, but the mechanistic details have not been fully elucidated. Mechanistic studies will deepen our understanding not only of spontaneous RNA degradation under physiological conditions but also of ribozyme- and RNase-catalyzed RNA cleavage and splicing, and may facilitate the rational design of artificial ribonucleases for therapeutic RNA targeting. RNA cleavage via internal transesterification is thought to be initiated by attack of the deprotonated 2′-hydroxyl group on the vicinal 3′-phosphate to form a pentacoordinate transition state (TS), followed by the spontaneous departure of the 5′-oxyanion and generation 2,3 of the 2′,3′-cyclic phosphate (Figure 1). Effectors such as metal ions and specific and general acids and bases promote the reaction by deprotonating the 2′-OH, stabilizing the TS, 4-6 or protonating the 5′-oxyanion leaving group. More importantly, the ability of the ribonucleotide to adopt a “near 7 attack conformation” in which the attacking 2′-oxyanion is positioned in-line with the developing 5′-oxyanion group 8-10 strongly influences the internal transesterification rate. 4 The orientation is thought to enhance the rate >100 fold and has been observed in the active center of nucleolytic ribo5,11-15 zymes.

Figure 1. Proposed mechanism of RNA cleavage via internal transesterification. The ribose moieties in RNA mainly exist in two puckered conformations—designated North (N) and South (S)—that are in equilibrium; the former is thermally more stable and 16-18 thus predominates. During internal transesterification, the in-line TS can theoretically be reached via either conformation (Figure 1). Chattopadhyaya et al. reported that the cleavage site of lariat-RNA preferentially adopts the S con19-21 formation. Herein, by using conformationally constrained ribonucleotides as models, we demonstrate that RNA cleavage via the S-type TS (Pathway II) is kinetically more favorable than via the N-type TS (Pathway I), indicating that the ribose conformation plays an important role in modulating RNA cleavage via internal transesterification. Substituents on the ribose moiety can drive the ribonucleotide to adopt predominantly one conformation or the oth22,23 er. For example, in 6,3′-methanouridine (U6,3′-Methano) the covalent 6,3′-methylene bridge locks the ribose in the N con3 24 formation ( JH1′-H2′ = 0 Hz, Figures 2A and S9), whereas 4′methyluridine (U4′-Me) reportedly exists in the S confor25 mation. Therefore, we synthesized the phosphoramidites of U6,3′-Methano and U4′-Me and introduced them into oligonucleo-

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tides by means of solid-supported DNA and RNA synthesis. Because of the possible conformational flexibility of U4′-Me, the solution structures of U4′-Me-modified short singlestranded RNA (oligo 1: 5′-r[CCAUU4′-MeAUAGC]) and DNA (oligo 2: 5′-d[CCATU4′-MeATAGC]) were characterized by 1 NMR spectroscopy. The lack of imino signals in the H NMR spectra of the two oligos indicates the absence of welldefined structures. Unfortunately, extensive signal overlap in the DQF COSY NMR spectrum of oligo 1 prevented unambiguous assignment of the U4′-Me conformation (Figure S1). 3 However, the large coupling constant ( JH1′-H2′ = 8 Hz) for U4′1 Me in the H and DQF COSY NMR spectra of oligo 2 clearly indicates that U4′-Me existed in the S conformation in the context of a single-stranded oligonucleotide (Figures 2B and 26 S2). In addition, replica exchange molecular dynamics simulations of 5′-r[UUA] (Figure 2c) showed that the native uridine residue in the middle preferred the N conformation (accounting for 70% of all sampled conformations), whereas U4′-Me in 5′-r[UU4′-MeA] existed predominantly in the S conformation (~85% of all sampled conformations).

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and MgCl2 (5 mM) at moderate temperature (23ºC). Kinetics studies showed that the ribonucleotide cleavage rates under these conditions decreased in the order U4′-Me > uridine >> U6,3′-Methano. U4′-Me cleavage was 2 times as fast as uridine cleavage, which was in turn ~20 times as fast as U6,3′-Methano cleavage.

Table 1. Half-lives for cleavage of single-stranded DNA with a single embedded ribonucleotide.a 5′′-FAM-d(CTTCTTTNTTACTTC) Oligo 3

Oligo 4

Oligo 5

N = U4′-Me

N=U

N = U6,3′-Methano

(S-type)

(S

N)

(N-type)

pKa of 2′′-OH

12.9

12.6

12.5

t1/2 at pH 12.4

1.4 ± 0.1 h

3.3 ± 0.3 h

62 ± 5 h

t1/2 at pH 14.0

8 ± 1 min

33 ± 2 min

39 ± 2 h

t1/2 at pH 4.0

206± 19 h

379 ± 11 h

1579 ± 39 h

a

Reaction conditions: Each oligo (0.5 µM) was incubated in an aqueous solution containing KCl (1 M) and MgCl2 (5 mM) at 23ºC (for pH 12.4 and 14.0) or at 55ºC (for pH 4.0). The pH was adjusted with NaOH or HCl.

Figure 2. Ribose conformation of 6,3′-methanouridine (U6,3′Methano) and 4′-methyluridine (U4′-Me). a) Structures of U6,3′Methano and U4′-Me. b) DQF COSY NMR spectrum of oligo 2 (5′d[CCATU4′-MeATAGC]) showing the resolved H1′-H2′ cross1 peak of the U4′-Me residue. The portion of the H NMR spectrum corresponding to the H1′ region is shown above the DQF COSY plot. c) Probability distributions of the phase angles for the middle ribonucleotides in 5′-r[UUA] and 5′r[UU4′-MeA]. Data were obtained by means of replica exchange molecular dynamics simulations at 300 K. Under physiological conditions, RNA cleavage via internal transesterification is rather slow. In the laboratory, acid- and alkali-promoted RNA cleavage conditions have been extensively utilized to interrogate factors that affect the kinetics 2,27 and thermodynamics of this reaction. Therefore, we began our study of the impact of ribose conformation on internal transesterification by carrying out alkali-catalyzed cleavage of single-stranded DNA molecules containing a single embedded ribonucleotide. We found that incubation of oligos 3–5 (Table 1) in aqueous NaOH solution (pH 12.4) resulted in 27 strand cleavage at the ribonucleotide site (Figure S3). To minimize the influence of other effectors, we incubated the oligos in the presence of high concentrations of KCl (1 M)

At pH 12.4, U4′-Me cleavage appeared to be nearly 45 times as fast as U6,3′-Methano cleavage. However, this rate difference cannot be attributed simply to the difference in ribose conformations. Under alkaline conditions, the internal transesterification rate also depends on the extent to which the attacking 2′-OH is deprotonated, which is in turn deter27 mined by its pKa. To explore this effect, we measured the pKa values of the 2′-OH groups of U4′-Me, uridine, and U6,3′28 and found them to be Methano by means of NMR titration 12.9, 12.6, and 12.5, respectively (Table 1 and Figure S8). The 0.4 difference in pKa between U6,3′-Methano and U4′-Me can be expected to decrease the cleavage rate of U4′-Me by a factor 1/2. Thus, after correction for the impact of the 2′-OH pKa, we concluded that the rate of cleavage of the S-constrained ribonucleotide was roughly 100 times that of the Nconstrained ribonucleotide. Strand cleavage reactions of oligos 3–5 were also carried out at pH 14 (1 M KCl, 5 mM MgCl2, 23ºC). At this pH, the 2′OH groups of U4′-Me, uridine, and U6,3′-Methano were predominantly deprotonated, and thus the impact of their pKa values 27 was marginal. Under these conditions, half-lives for strand cleavage at U4′-Me, uridine, and U6,3′-Methano were 8 min, 33 min, and 39 h, respectively (Table 1 and Figure S4), corroborating our conclusion that RNA transesterification via the S conformation is hundreds of times as fast as transesterification via the N conformation. To assess the influence of ribose conformation on acidcatalyzed RNA cleavage via internal transesterification, we incubated oligos 3–5 in aqueous HCl (pH 4.0, 1 M KCl, 5 mM MgCl2). Because acid-promoted RNA cleavage is rather slow at 23ºC, the temperature was increased to 55ºC. Under these conditions, the half-lives for RNA cleavage of oligos 3–5 were 206, 379, and 1579 h, respectively (Table 1 and Figure S5). Thus, it is likely that the ribose conformation had similar effects on both acid-catalyzed and base-catalyzed cleavage.


To further interrogate the role of ribose conformation in RNA cleavage via internal transesterification, we studied the degradation of single-stranded RNA molecules (oligos 6–9, Figure 3A) under both alkaline and acidic conditions. At pH 12.4, native oligo 7 showed no obvious propensity for degradation at any particular position (Figures 3B, C and S6). However, for oligo 6, the rate of cleavage at U4′-Me was remarkably enhanced relative to that at other sites in the same strand; and for oligo 8, the rate of cleavage at U6,3′-Methano was dramatically depressed relative to that at other sites, and the same was true of the U2′-F residue of oligo 9. Under acidic conditions (pH 4.0), similar results were obtained (Figure S7), once again confirming that cleavage of the S-constrained ribonucleotide was markedly faster than cleavage of the Nconstrained counterpart.

tion was not the rate-limiting step in either case. A more reasonable explanation is that the total activation energy ∆G ≠ ≠ (∆G = GTS3 − Gint1) for U6,3′-Methano(p)Et (21.6 kcal/mol) was considerably higher than that for U4′-Me(p)Et (8.8 kcal/mol). Furthermore, our calculations demonstrated that internal transesterification of the native substrate U[p]Et (Figure 4, lower panel, R = H) occurred spontaneously via S-type TSs with a total activation energy of 9.3 kcal/mol. The energy barrier for flipping between the N and S conformations (via an O4′-endo TS) was calculated to be only 2.5 kcal/mol (Figure S10). Therefore, a rapid N to S conformational flip followed by internal transesterification via S-type TSs seems kinetically more favorable for RNA cleavage. ∆G (kcal/mol)

16.4 .3 1 1.68 2

2.97

1.62

I-TS3 4 2.4

1 1.7

1.15 1.30

6.8 O

5.3

I-TS2

NH

I-int2 O

1.0

NH

HO

H 2C

O

HO

OH Mg 2+

M g 2+

OH

H

O

O O

H

H

H

H

-14.5 I-int3 North conformation

O O H P 2+ O Mg O- H H Mg 2+ O HO O H

O H P O H O O Mg 2+ OM g 2+ OH HO O H

OH

H

O O

O

O- O

O Mg 2+ O

H

N

O

O

H

O

H 2C

HO N

O

P

O Mg2+

NH

O

NH

I-int1 O

O

O-

O H2 O

N

O

P O

H 2C

HO

O O

O

-5.2

N O

U6,3'-Methano[p]Et (Pathway I)

0.0 I-TS1 I-int0 North O conformation

H 2C

HO

Rate limiting step 2'O-P formation

2'-OH deprotonation

To gain insight into the more-rapid cleavage of Sconstrained ribonucleotides, we studied alkali-promoted cleavage of two model compounds, 6,3′-methanouridine-3′ethylphosphate (U6,3′-Methano[p]Et) and 4′-methyluridine-3′ethylphosphate (U4′-Me[p]Et), by means of density functional theory (DFT) calculations at the M062x/6-311++G(d,p)-SMD 29 2+ 6 level with two Mg ions. The results indicated that U6,3’[p]Et was cleaved via N-type TSs owing to the ring Methano strain in this compound (Figure 4, upper panel), whereas U4′Me[p]Et cleavage proceeded via S-type TSs (Figure 4, lower panel, R = CH3). In both cases, 2′O-P formation (TS2) and P5′O scission (TS3) occurred in separate steps, with the latter being the rate-limiting step, which is consistent with previ3,30 ously reported calculation results. We could not rule out the possibility that pseudorotation was involved in these 31 steps. One difference between the two pathways is that a distinct TS for 2′-OH deprotonation (I-TS1) was identified for U6,3’-Methano[p]Et, whereas for U4′-Me[p]Et, a deprotonated intermediate (II-int1) was obtained directly, and II-TS1 was 32 not located. The failure to locate II-TS1 in the latter case 2+ could be attributed to the fact that chelation of the Mg ion by O-2 of the base lowered the barrier for deprotonation. However, the dramatic difference in cleavage rate between these two model compounds could not arise solely from the difference in the deprotonation steps, given that deprotona-

1.6 7

2. 42

2.2 6

R = CH3 (R = H) 0.0(0.0)

99 1.

Figure 3. Degradation of single-stranded RNA oligos 6–9 at pH 12.4. a) Sequences of oligos 6–9. b) Denaturing PAGE analysis of the cleavage patterns for oligos 6–9 upon incubation at pH 12.4 for 1 h. c) Calculated percentages of cleavage at each position.

P-5'O cleavage

2.0 1

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Journal of the American Chemical Society

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6.1(5.3) II-TS3 O

0.9(0.2) II-TS2

II-int0 South conformation

-0.2(-1.3)

N

II-int2

O

O

-2.7(-4.0) II-int1

O

O

O O O

NH

NH

OH

N

N O

H O O H O

R O

O

Mg2+

O OMg

OH

O H

2+

H O H

OH O

R

Mg2+ O O O H O O H H H Mg2+ O OH H P

NH

O

N O

O O

P O

O

R

U4'-Me[p]Et and U[p]Et (Pathway II) O

OH

NH

OH

R

H O H O

O O P

-

O

O

Mg 2+

OMg2+ OH

O H H O H

O

P -

O H O

Mg 2+ H O H

H O H Mg 2+ O H

-15.7(-14.6) II-int3 South conformation

Figure 4. Calculated free energy profiles for RNA transesterification reactions of U6,3′-Methano[p]Et (upper panel), U4′Me[p]Et (lower panel), and U[p]Et (lower panel, in parentheses) at 298 K. The above-described results suggest that cleavage of the Sconstrained ribonucleotide was faster than cleavage of the native ribonucleotide, which was in turn markedly faster than cleavage of the N-constrained counterpart, suggesting that ribose conformation plays an important role in modulating RNA cleavage via internal transesterification. This has two important implications for our understanding of this fundamental biological reaction. First of all, it provides a plausible explanation for sequence-dependent degradation of 33,34 folded RNA. In folded RNA, ribonucleotides constrained



in the S conformation and those with the flexibility to undergo a conformational flip could be more susceptible to cleavage, whereas cleavage of N-constrained ribonucleotides may be retarded. Secondly, RNases and nucleolytic ribozymes may have evolved the ability to make use of the relationship between ribose conformation and the RNA internal transesterification rate to accomplish site-specific RNA degradation. This possibility is supported by at least two reported experimental observations. First, a recent statistical analysis of RNA–protein complexes in the Protein Data Bank (PDB) indicated that interaction of the 2′-OH with proteins leads to ribose conformational changes and a parallel enrichment in 35 S-type puckering. Second, by perusing the available crystal structures of nucleolytic ribozymes—glmS ribozyme (PDB: 11,36 12 2NZ4), pistol ribozyme (PDB: 5K7C), twister ribozyme 13,14 (PDB: 4OJI, 5DUN), hammerhead ribozyme (PDB: 37 38 2GOZ), and HDV ribozyme (PDB: 1DRZ) —we found that in these ribozymes, the ribonucleotides in the active center exhibit S-type puckering accompanied by in-line arrangement of the 2′-OH and the departing 5′-oxyanion. In summary, our results shed light on the effect of ribose conformation on RNA cleavage via internal transesterification and can be expected to improve our understanding of the mechanism of RNA cleavage catalyzed by RNases and ribozymes as well. In addition, the principle revealed herein may facilitate the rational design of catalysts for site-specific RNA cleavage and functionalized RNA molecules for biomedical applications.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. General experimental methods, gel images, NMR spectra, calculation details, supplementary figures and tables (PDF).

AUTHOR INFORMATION Corresponding Author *Janez Plavec. Email: [email protected] *Qian Peng. Email: [email protected] *Chuanzheng Zhou. Email: [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by NSFC (21572109, 21332004, 21702109, 21740002), the Natural Science Foundation of Tianjin City (15JCYBJC53300, 18JCYBJC21400), and the Slovenian Research Agency (ARRS, grant nos. P1–0242 and J1–6733). C. Z. and Q. P. are grateful for sponsorship from the National Thousand Young Talents Program of China and Tianjin. Q. P. gratefully acknowledges support from the Fundamental Research Funds for the Central Universities. The authors acknowledge the CERIC-ERIC Consortium for access to experimental facilities and for financial support.

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