Enzymatic characterization of mRNA cap adenosine-N6 methyltransferase PCIF1 activity on uncapped RNAs

Yu, Dan; Dai, Nan; Wolf, Eric J; Corrêa, Ivan R.; Zhou, Jain; Wu, Tao; Blumenthal, Robert; Xing, Zhang; Cheng, Xiaodong

doi:10.1016/j.jbc.2022.101751

Cited by 10 publications

(17 citation statements)

References 58 publications

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“…First, we observed high activity of MettL3-MettL14 on the ssDNA (K app = 130 h −1 ; Figure 4A), which is the same as or even better than the corresponding RNA containing GGACU (K app = 120 h −1 ). 56 Second, as expected, we observed no activity of HemK2-Trm112 on DNA (Figure 4B). Observations on both the positive control (MettL3-MettL14) 25−27 and the negative control (HemK2-Trm1112) 17−19 agree with previous publications.…”

Section: Alkbh1supporting

confidence: 82%

“…Here, we analyzed existing structures for human FTO and PCIF1, which illustrated a general arrangement of a wellcharacterized catalytic domain (methylase or dioxygenase) in association with a less-characterized helical domain. In sum, FTO and PCIF1 have opposite enzymatic activities on capspecific m 6 A m, 39,45 on internal adenosines (m 6 A or m 6 A m ) in the context of RNA, 38,40,56 and on DNA adenine ( 35,38,41 and this study). We note that some of the activities are observed in vitro.…”

Section: ■ Discussionmentioning

confidence: 48%

“…The underlined As in R2 oligo indicates the RNA methylation sites confirmed by mass spectrometry. 56 The CATT site in D3 oligo is used in Figure 3 , and the GGACT site in D4 oligo is used in Figure 4 . (B) Comparison of PCIF1 activities on single-stranded oligos (D2 and D3) and double-stranded duplexes (R2/D3 and D2/D3) ( N = 4).…”

Section: Resultsmentioning

confidence: 99%

“…Next, we designed a short 14-mer single-strand (ss)DNA oligo (CATT) containing a single internal A, and compared the PCIF1 enzymatic activity on this potential DNA substrate to that on the RNA equivalent (CAUU). First, we showed that PCIF1 activity is linear under the conditions used: like the RNA substrates (whether capped or uncapped), 50,56 PCIF1 activity on DNA CATT is concentration-and temperature-dependent (Figure 3A,B). Second, we observed about half the PCIF1 activity on DNA that was observed on the corresponding RNA (up to 4 h), though eventually, the same level of methylation was reached in the saturated overnight reaction (Figure 3C).…”

Section: Alkbh1mentioning

confidence: 95%

“…56 For the RNA R2 oligo, PCIF1 showed high activity, resulting in fully N6-methylated 5′ A, with a second methylation site at the internal adenosine, three or four residues away from the 5′ end (AGCGAA). 56 In the case of the DNA D2 oligo, PCIF1 exhibited substantial activity, at about half the level, after a saturated overnight reaction, which was observed on the corresponding RNA (Figure 2A). The 5′ adenine is not required for PCIF1 activity on ssDNA, as the complimentary strand D3 yields similar activity to that of D2 (Figure 2B).…”

Section: Alkbh1mentioning

confidence: 99%

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Enzymatic Characterization of In Vitro Activity of RNA Methyltransferase PCIF1 on DNA

Zhou

Chen

et al. 2022

Biochemistry

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PCIF1 and FTO are a pair of human mRNA cap-specific modification enzymes that have opposing activities. PCIF1 adds a methyl group to the N6-position of 2′ O -methyladenosine (A m ), generating N6, 2′ O -dimethyladenosine (m 6 A m ), when A m is the cap-proximal nucleotide. FTO removes the N6-methyl group from m 6 A m . In addition, FTO has a demethylase activity on a broad spectrum of various RNA substrates, as well as on D NA N6-methyldeoxyadenosine (m 6 dA). While the existence of m 6 dA in mammalian D NA remains controversial, we show here that PCIF1 has significant methylation activity on single stranded DNA deoxyadenosine, double stranded RNA/DNA hybrids, and double stranded DNA, though with lower catalytic efficiency than that on its preferred RNA substrate. PCIF1 has activities in the order ssRNA > RNA/DNA hybrid > ssDNA > dsDNA. We discuss the implications of PCIF1 generation, and FTO removal, of DNA adenine methylation.

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Section: Alkbh1supporting

confidence: 82%

Section: ■ Discussionmentioning

confidence: 48%

Section: Resultsmentioning

confidence: 99%

Section: Alkbh1mentioning

confidence: 95%

Section: Alkbh1mentioning

confidence: 99%

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Enzymatic Characterization of In Vitro Activity of RNA Methyltransferase PCIF1 on DNA

Zhou

Chen

et al. 2022

Biochemistry

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RNA m6A methylation across the transcriptome

Sendinc¹,

Shi²

2023

Molecular Cell

136

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Systematic Design of Adenosine Analogs as Inhibitors of a Clostridioides difficile-Specific DNA Adenine Methyltransferase Required for Normal Sporulation and Persistence

et al. 2022

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Antivirulence agents targeting endospore-transmitted Clostridioides difficile infections are urgently needed. C. difficile-specific DNA adenine methyltransferase (CamA) is required for efficient sporulation and affects persistence in the colon. The active site of CamA is conserved and closely resembles those of hundreds of related S-adenosyl-l-methionine (SAM)-dependent methyltransferases, which makes the design of selective inhibitors more challenging. We explored the solvent-exposed edge of the SAM adenosine moiety and systematically designed 42 analogs of adenosine carrying substituents at the C6-amino group (N6) of adenosine. We compare the inhibitory properties and binding affinity of these diverse compounds and present the crystal structures of CamA in complex with 14 of them in the presence of substrate DNA. The most potent of these inhibitors, compound 39 (IC50 ∼ 0.4 μM and K D ∼ 0.2 μM), is selective for CamA against closely related bacterial and mammalian DNA and RNA adenine methyltransferases, protein lysine and arginine methyltransferases, and human adenosine receptors.

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Enzymatic characterization of mRNA cap adenosine-N6 methyltransferase PCIF1 activity on uncapped RNAs

Cited by 10 publications

References 58 publications

Enzymatic Characterization of In Vitro Activity of RNA Methyltransferase PCIF1 on DNA

Enzymatic Characterization of In Vitro Activity of RNA Methyltransferase PCIF1 on DNA

RNA m6A methylation across the transcriptome

Systematic Design of Adenosine Analogs as Inhibitors of a Clostridioides difficile-Specific DNA Adenine Methyltransferase Required for Normal Sporulation and Persistence

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