Precise Antibody-Independent m6A Identification via 4SedTTP

Feb 28, 2018 - Innovative detection techniques to achieve precise m6A distribution within mammalian transcriptome can advance our understanding of its...
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Communication Cite This: J. Am. Chem. Soc. 2018, 140, 5886−5889

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Precise Antibody-Independent m6A Identification via 4SedTTPInvolved and FTO-Assisted Strategy at Single-Nucleotide Resolution Tingting Hong,‡,† Yushu Yuan,‡,† Zonggui Chen,§ Kun Xi,# Tianlu Wang,† Yalun Xie,† Zhiyong He,† Haomiao Su,† Yu Zhou,§ Zhi-Jie Tan,# Xiaocheng Weng,*,† and Xiang Zhou*,† †

College of Chemistry and Molecular Sciences, Wuhan University, Wuhan, Hubei 430072, China College of Life Science, Wuhan University, Wuhan, Hubei 430072, China # Center for Theoretical Physics and Key Laboratory of Artificial Micro- & Nano-structures of Ministry of Education, School of Physics and Technology, Wuhan University, Wuhan, Hubei 430072, China §

S Supporting Information *

However, the low yield and false-positive results associated with immunoprecipitation19 made us consider whether an antibodyindependent method can add a new dimension for m6A mapping. Because m6A is chemically stable and unable to introduce mutational or truncated signatures during reverse transcription, dTTP modifications would be a potent way to alter the ability of T to base pair with A and m6A during cDNA synthesis. Atoms specifically adopted in substituted nucleobases, such as sulfur and selenium, which belong to the same elemental family but possess much larger radii than oxygen (O: 0.73 Å, S: 1.02 Å, Se: 1.16 Å), can afford exclusive hybridization properties when incorporated into nucleobases due to their unique steric and electronic efforts.20−22 Accordingly, we introduced the atom-specific replacement of oxygen with sulfur and selenium at 4-position of deoxythymidine triphosphate expecting that there might exist a valid candidate that allowed for efficient A-T* pairing, while discouraging m6A-T* pairing during cDNA synthesis. So herein, 4SedTTP was demonstrated for its unique ability to confer specific RT signature of truncation on m6A during reverse transcription. The unfavorable 4SeT-m6A base pairing compared with 4SeT-A resulted in aborted cDNA synthesis opposite m6A sites, thereby allowing for the precise identification of m6A in an antibody-independent way. We first prepared these S- and Se-modified deoxythymidine triphosphates (4SdTTP and 4SedTTP, 4ST and 4SeT for short, Figure 1a) and carried out single-nucleotide incorporation reactions using A/m6A-containing oligo RNAs as templates to validate their abilities to base pair with A and m6A. The 3′ end of FAM-labeled primers were designed to enable the direct incorporation of T/4ST/4SeT opposite A or m6A residue of interest by reverse transcriptase. Because the AT* base pairing was stable enough to accommodate the larger sulfur and selenium atoms,21 M-Mulv RT was capable of affording extended primers with T/4ST/4SeT incorporated opposite A residue (Figure 1b). Similarly, for m6A site, normal T and 4ST also could be efficiently incorporated into primer by M-Mulv RT to base pair with m6A without any visible differences. However, different from A site, the incorporation

ABSTRACT: Innovative detection techniques to achieve precise m6A distribution within mammalian transcriptome can advance our understanding of its biological functions. We specifically introduced the atom-specific replacement of oxygen with progressively larger atoms (sulfur and selenium) at 4-position of deoxythymidine triphosphate to weaken its ability to base pair with m6A, while maintaining A-T* base pair virtually the same as the natural one. 4SedTTP turned out to be an outstanding candidate that endowed m6A with a specific signature of RT truncation, thereby making this “RT-silent” modification detectable with the assistance of m6A demethylase FTO through next-generation sequencing. This antibody-independent, 4SedTTP-involved and FTO-assisted strategy is applicable in m6A identification, even for two closely gathered m6A sites, within an unknown region at single-nucleotide resolution.

A

s the most prevalent internal modification of mRNA and lncRNA,1 m6A has emerged as a new frontier in RNA epigenetic studies.2,3 The intrinsically reversible feature of m6A achieved by METTL3-METTL14-WATP4−6 methyltransferase complex and two vital m6A demethylases (FTO7 and ALKBH58) endows m6A with significant regulatory roles in fine-tuning gene expression and various biological processes.9,10 Thus far, for m6A identification, a variety of strategies have been developed with their own merits but also with plenty of room for improvement. Among these, selective RNA reverse transcriptase based strategies can be implemented for m6A identification at definite sites but is not feasible for sequencing.11,12 The SCARLET method13 is effective to validate the precise location and fraction of m6A, while its laborious and time-consuming nature impede its wider application. m6A-seq (or MeRIP-Seq)14−16 facilitates the profiling of m6A distribution in mammalian transcriptomes but falls short in pinpointing the precise location of m6A. PAm6A-Seq17 takes advantage of prior 4SU incorporation and RNase digestion, which allows for the identification of m6A within ∼23 nt. miCLIP18 achieves single-nucleotide resolution owing to the specific pattern of mutations or truncations in cDNA during reverse transcription upon UV 245 nm exposure. © 2018 American Chemical Society

Received: January 4, 2018 Published: February 28, 2018 5886

DOI: 10.1021/jacs.7b13633 J. Am. Chem. Soc. 2018, 140, 5886−5889

Communication

Journal of the American Chemical Society

able to provide visible truncated cDNA products for methylated RNA, albeit to a different extent (Figures S2 and S3), which demonstrated that the RT truncation caused by m6A was independent of specific reverse transcriptase. Note that the stalling efficiency would be affected by the incubation temperature (Figure S4) and the concentrations of Mg2+, 4SedTTP and reverse transcriptase. Too low concentrations of these components would cause undesirable RT truncation for normal A sites, on the contrary, too high concentrations would result in depressed stalling efficiency for m6A site (Figures S5− S8). Under the optimal condition, the percentages of truncated products caused by m6A showed a linear relation with the methylation level with the aid of 4SeT (Figure S9), demonstrating its successful implementation for the quantitative evaluation of methylation degree. We postulated that this unfavorable incorporation of 4SeT opposite m6A by different reverse transcriptase might be caused by the destabilization of 4SeT-m6A base pair. Thus, we performed all-atom MD simulations28,29 to gain insight into the distinct base-pairing pattern of 4SeT-m6A at atomic level. The replacement of O with Se would reduce the possibility of hydrogen bonds formation to a certain degree due to the weaker electronegativity of Se. Conversely, the base stacking interaction would be slightly disturbed by the methyl group at the N6-position of m6A because of the excluded volume effect (Table S2, Figures S10 and S11). Considering these two effects together, once the Se replacement and methyl group coexisted simultaneously, the stability of base pair would be dramatically decreased as a result of the combined effect of reduced stacking stabilization and the loss of hydrogen bond energy30−32 (Table S3), which was consistent with the phenomenon that only m6A-4SeT was unfavorably recognized by reverse transcriptase during cDNA synthesis while other base pairs (A-T, m6A-T, A4ST, m6A-4ST and A-4SeT) were not. Encouraged by these observations, we proceeded to implement this 4SeT-based strategy in more complicated RNA models that carried multiple potential m6A motifs, including GGACU and GAACU. A-RNA, m6A-RNA and 2m6A-RNA represented three RNA templates with identical sequences but diverse methylation statuses. A-RNA was designed as an unmethylated control to allow the comparison of cDNA products with those from the methylated ones. With the addition of 4SeT combined with the three other natural nucleobases, cDNA synthesis smoothly proceeded until it encountered m6A sites, leaving intense truncated cDNA bands that allowed for the precise identification of m6A at singlenucleotide resolution. Although relatively slight cDNA truncation would be also generated by normal A sites due to the minor destabilization caused by Se substitution, they can be regarded as background truncation signals because the same extent of truncations also existed for the unmethylated templates (Figure 2). Through comparison, even two closely gathered m6A sites (for 2-m6A-RNA) whose signals might be easily overlapped in the antibody-associated strategies can be accurately pinpointed in a one-tube reaction (Figure 2). It is noteworthy that no observable m6A information can be provided when natural T is adopted, furtherly emphasizing the unique function of 4SeT for the identification of m6A. Another series of RNA templates carrying various methylation statuses were also chosen to verify the feasibility of this strategy for the precise identification of gathered m6A sites (Figure S12).

Figure 1. Sulfur and selenium substituted thymidine and their abilities to base pair with A/m6A. (a) The replacement of oxygen with sulfur and selenium at 4-position of thymidine. (b) PAGE analysis of the single-nucleotide extension with T/4ST/4SeT to be incorporated opposite the definite A or m6A site. (c) Reverse transcriptions employing A, C, G and T/4ST/4SeT with primer distant from A/m6A site.

opposite m6A site would be significantly stalled and resulted in distinct extension products with the addition of 4SeT (Figure 1b). This distinct receptivity of A/m6A-4SeT base pairs by reverse transcriptase made 4SeT a potent candidate for the discrimination of m6A from A in RNA. Meanwhile, 4SeT also showed high potency for the identification of diverse adenine methylation statuses in dsDNA (6mA, a counterpart of m6A),23−27 with pronounced differences in the extended primer products that were observed for unmethylated, hemimethylated and methylated dsDNA (Figure S1). Notably, single-nucleotide incorporation could identify m6A at definite sites but fell short in profiling the m6A distribution within an unknown region. In a feasibility test, a FAM-labeled primer that was distant from A/m6A site of interest was designed, and reverse transcriptions were achieved by comparison of these substituent nucleotides (T, 4ST or 4SeT) together with three other natural nucleotides (A, C and G). With the accommodation of sulfur and selenium atoms in the A-T* base pair, both 4ST and 4SeT could afford fulllength cDNA products for unmethylated templates just as natural T did (Figure 1c). As expected, even with the presence of m6A in the detected RNA region, 4ST could still be efficiently incorporated into the cDNA by reverse transcriptase like normal T when encountered a m6A site without leaving any visible trace in the cDNA products. Intriguingly, 4SeT could cause RT truncation opposite the m6A site under identical treatments because of the unfavorable formation of m6A-4SeT, thus resulting in an apparently aborted cDNA product besides the full-length cDNA and making the “RTsilent” modification detectable during reverse transcription process (Figure 1c). To validate this phenomenon, three other different commercially available reverse transcriptase were chosen to monitor the cDNA synthesis in the presence of T/4ST/4SeT. Similarly, only when 4SeT was adopted were these enzymes 5887

DOI: 10.1021/jacs.7b13633 J. Am. Chem. Soc. 2018, 140, 5886−5889

Communication

Journal of the American Chemical Society

comparing the cDNA products generated by (−) and (+) FTO samples, we were able to detect a significant truncated cDNA band for (−) FTO sample, which, however, was disappeared for (+) FTO sample, verifying that the RT truncation was indeed caused by m6A modification (Figure 3c). As comparison, no significant differences were observed between FTO treated or untreated samples if the input RNA was unmethylated (Figure 3c). This 4SedTTP-involved and FTO-assisted RT strategy enabled the exact identification of m6A within totally unknown positions. Furthermore, to acquire precise single-base information at the 3′ end of the truncated cDNA caused by m6A, we combined the 4SedTTP-involved and FTO-assisted RT strategy with next-generation sequencing (NGS). RT products generated from (−) and (+) FTO samples were circularized, followed by PCR amplification, including barcoding, sequencing and data analysis with mapping of identified sequences to the original one (Figure 4a). The significant RT stop signal

Figure 2. Gathered m6A identification through employing 4SeT instead of normal T for cDNA synthesis. RNA sequence was displayed on the right with potential m6A sites labeled as A*. Marker on the left indicated the exact positions of the initial primer, A or m6A sites and full-length cDNA from the bottom up.

In a bid to locate m6A position for unknown samples that have no parallel unmethylated control to be used as a comparison, we introduced FTO demethylase, a m6A “eraser” that performs excellent efficiency for m6A removal,7 to assist us to pick out the exact signals generated from m6A. The input RNA samples were divided into two identical parts, and one of them was subjected to FTO treatment to remove all the traces of m6A, whereas the other one remained to be unchanged. The (−) and (+) FTO samples were then synthesized into cDNA through the 4SedTTP-involved reverse transcription. The successful removal of m6A mediated by FTO was first verified by HPLC-MS/MS quantification, which appeared as a dramatic drop in m6A/A level after FTO treatment (Figure 3b). By

Figure 4. 4SedTTP-involved and FTO-assisted sequencing strategy for the identification of m6A at the single-nucleotide resolution. (a) The procedure of the proposed sequencing strategy. (b) RT stop signal of (−) and (+) FTO samples were compared to determine the exact signal generated from m6A. The precise base information on the 3′ end of the cDNA indicated that cDNA synthesis was actually aborted opposite the m6A site.

generated before m6A site verified that the cDNA synthesis indeed aborted opposite m6A site after comparing the strength of RT stop for m6A removed and unremoved samples (Figure 4b). In addition, negligible RT truncations caused by A, C, T and G were observed, verifying the unique RT signature of m6A in 4SedTTP-involved reverse transcription. These inspiring NGS results with single-nucleotide resolution encouraged us to implement this 4SedTTP-involved and FTO-assisted strategy for precise m6A mapping in the transcriptome in the near future. In summary, we first disclosed that the “RT-silent” modification, m6A, can be endowed with specific RT signature to make it distinguished from other bases through the introduction of 4SedTTP (4SeT for short), an artificially substituent nucleotide with selenium replacement of oxygen at 4-position of deoxythymidine triphosphate. 4SeT could smoothly pass the normal A during the process of reverse transcription while discouraging the formation of m6A-4SeT due to the perturbation of both hydrogen bonding and base stacking. The unfavorable m6A-4SeT base pair would arouse RT truncation and result in aborted cDNA, where cDNA synthesis was stalled across from the m6A site. With the assistance of m6A demethylase (FTO) and in combination with NGS, precise m6A identification at single-nucleotide resolution

Figure 3. m6A identification within an unknown region assisted with the biological demethylation process mediated by FTO. (a) Illustration of the 4SedTTP-involved and FTO-assisted strategy. (b) HPLC-MS/MS quantification of the m6A/A ratio of RNA with and without FTO treatment. (c) PAGE analysis of the cDNA products generated from the RNA samples with or without FTO treatment. Synthetic unmethylated sample RNA-A versus methylated sample RNA-m6A were used for comparison. 5888

DOI: 10.1021/jacs.7b13633 J. Am. Chem. Soc. 2018, 140, 5886−5889

Communication

Journal of the American Chemical Society

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could be achieved. We deem that this high resolution and specificity would potentially encourage the further implementation of this technique in developing sequencing technology to achieve transcriptome-wide mammalian m6A mapping. The atom-specific replacement strategy also adds a new perspective for the identification of epigenetic modifications that are not susceptive to chemical conversions in an antibody-independent manner.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b13633. Experiment methods, 4SedTTP synthesis, MD stimulation and NGS data analysis (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] ORCID

Zhiyong He: 0000-0001-7976-9928 Xiang Zhou: 0000-0002-1829-9368 Author Contributions ‡

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Natural Science Foundation of China (21432008, 91753201 and 21721005 to X.Z.; 21778040, 21572172 to X.W. The MD simulations were performed on the super computing system in the Super Computing Center of Wuhan University. The MD simulation is supported by the National Science Foundation of China grants (11575128 and 11774272 to Z.-J.T.).

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DOI: 10.1021/jacs.7b13633 J. Am. Chem. Soc. 2018, 140, 5886−5889