key: cord-0258246-qpt5sotl
authors: Qi, Shan; Mota, Javier; Chan, Siu-Hong; Villarreal, Johanna; Dai, Nan; Arya, Shailee; Hromas, Robert A.; Rao, Manjeet K.; Corrêa, Ivan R.; Gupta, Yogesh K.
title: RNA binding to human METTL3-METTL14 restricts N6-deoxyadenosine methylation of DNA in vitro
date: 2022-01-09
journal: bioRxiv
DOI: 10.1101/2022.01.08.475504
sha: 373d80831f2b2ad8d1c1bf2eab03262dad891fd8
doc_id: 258246
cord_uid: qpt5sotl

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely regulated by structured RNA elements prevalent in long noncoding and other cellular RNAs.

Introduction 42 43 N 6 -methyladenosine (m 6 A) is considered a major covalent modification of the adenosine (A) base 44 in coding and noncoding (nc) RNAs 1-5 . It is linked to diverse physiologic processes, including -45 but not limited to -RNA turnover 6-8 , stem cell differentiation 9, 10 , oncogenic translation 11- 14 preferentially methylates N 6 dA on ssDNAs to m 6 dA, and this activity is largely regulated by 84 structured RNA elements prevalent in long noncoding (lnc) RNAs, that are also found in other 85 cellular RNAs. Thus, our work provides a framework to explore a new regulatory axis of RNA-86 mediated restriction of N 6 -deoxyadenosine (m 6 dA) methylation in mammalian genomes. 87

We compared the MTase cores of (ModB) of EcoP15I (aa 90 -132, 169 -261, 385 -511, PDB:  90 4ZCF 20 ) and METTL14 (aa 116 -402, PDB: 5IL0 21 ). A secondary structure-based superposition 36 91 of these two structures revealed common features and similar arrangement of canonical MTase 92 motifs (motifs I, and IV-X), including the motif IV (D/EPPY/W/L) that surrounds the Watson-93 crick edge of the flipped target adenine base ( fig. 1 a-e and supplementary fig. 1 propensity to form a perfect stem-loop (23-mer rTCE23 or also known as SRE 39 ) without a 118 DRACH motif, and a 30-mer rNEAT2 (or MALAT1) encompassing one DRACH motif and 119 predicted to form a stem-loop with a bulged stem ( fig. 1 h-i) . . 2g ). We confirmed that deoxyadenosine on d6T* and adenosine on 214 rNEAT2 were the only modified bases. In the absence of rNEAT2 or rTCE23, 86.3% of dAs on 215 d6T* were modified. When rNEAT2 or rTCE23 were present, only 14.4 and 7.4%, respectively, 216 of dAs on d6T* were modified. Additionally, 5.8% of the rAs on rNEAT2 were modified. As 217 expected, no methylation on rTCE23 was detected (no RRACH motif). These results are consistent 218 with those obtained by oligonucleotide intact mass analysis. Altogether, the methyltransferase and 219 binding assays confirm that the binding to structured RNAs, especially those lacking the GGACU 220 sequence, almost completely abolishes the methyltransferase activity METTL3-METTL14. 221

The c-terminal RGG repeat motif of METTL14 contributes to RNA binding and activity of 223 . 3a) . Thus, we hypothesized that the absence of RGG motif in 224 METTL3-METTL14 should diminish its ability to bind RNA and consequently attenuate the effect 225 of RNA on DNA methylation. In fact, we observed a 2-to 10-fold decrease in binding affinity by 226 METTL3-METTL14-RGG ( fig. 3b, and table 1 ). This suggests that the RGG motifs play a major 227 role in RNA and DNA substrates, while other parts of the enzyme (e.g., CCCH-type zinc finger 228 domains in METTL3 44 and MTase core of METTL14 35 ) may also contribute to the overall binding 229 ( fig. 3b ). As expected, the deletion of RGG caused 60% reduction in DNA methylation activity. 230

In the presence of rNEAT2, the activity of METTL3-METTL14-RGG on d6T* DNA was reduced 231 to about 75% of its activity in absence of the RNA; comparatively, a >90% activity reduction was 232 fig. 1a-c, fig. 3a ). Of note, there 

Promoter-bound 447 METTL3 maintains myeloid leukaemia by m(6)A-dependent translation control

The N6-methyladenosine (m6A)-forming enzyme METTL3 453 controls myeloid differentiation of normal hematopoietic and leukemia cells

mRNA circularization by METTL3-eIF3h 457 enhances translation and promotes oncogenesis

460 (2017) RNA m6A methylation regulates the ultraviolet-induced DNA damage response, 461

Promote Homologous Recombination-Mediated Repair of DSBs by Modulating DNA-465 RNA Hybrid Accumulation

A METTL3-METTL14 complex mediates 468 mammalian nuclear RNA N6-adenosine methylation

Structure 470 prediction and phylogenetic analysis of a functionally diverse family of proteins 471 homologous to the MT-A70 subunit of the human mRNA:m(6)A methyltransferase

Beta 474 class amino methyltransferases from bacteria to humans: evolution and structural 475 consequences

Structural basis of 477 asymmetric DNA methylation and ATP-triggered long-range diffusion by EcoP15I

Structural basis of N(6)-adenosine methylation by 481 the METTL3-METTL14 complex

Structural insights into the molecular mechanism of the 483 m(6)A writer complex

Structure-guided analysis reveals nine 485 sequence motifs conserved among DNA amino-methyltransferases, and suggests a 486 catalytic mechanism for these enzymes

Single-nucleotide-resolution mapping of m6A and m6Am throughout the 489 transcriptome

The methylation state of poly A-containing 491 messenger RNA from cultured hamster cells

Transcription Impacts the Efficiency of mRNA Translation via Co-494 transcriptional N6-adenosine Methylation

Histone H3 trimethylation 500 at lysine 36 guides m(6)A RNA modification co-transcriptionally

) m(6)A RNA methylation promotes XIST-mediated transcriptional 503 repression

N(6)-methyladenosine-505 dependent RNA structural switches regulate RNA-protein interactions

RNA m(6)A methylation regulates the ultraviolet-induced DNA damage response, 510

Targeting the m(6)A RNA 514 modification pathway blocks SARS-CoV-2 and HCoV-OC43 replication

2021) METTL3 regulates viral m6A RNA 518 modification and host cell innate immune responses during SARS-CoV-2 infection

Human MettL3-MettL14 complex is a 522 sequence-specific DNA adenine methyltransferase active on single-strand and unpaired 523 DNA in vitro

The origin of genomic 525 N(6)-methyl-deoxyadenosine in mammalian cells

Structural Basis for Cooperative Function 527 of Mettl3 and Mettl14 Methyltransferases

Secondary-structure matching (SSM), a new tool for 529 fast protein structure alignment in three dimensions

Defining the 532 RGG/RG motif

Interactions, localization, and phosphorylation of 535 the m(6)A generating METTL3-METTL14-WTAP complex

Shape-specific recognition in the structure of the Vts1p SAM domain with RNA

N6-540 methyladenosine marks primary microRNAs for processing

) ncRNA-and Pc2 methylation-dependent gene relocation 543 between nuclear structures mediates gene activation programs

Deciphering the "m(6)A Code" via Antibody-Independent Quantitative Profiling

Maintains Tumorigenicity of Glioblastoma Stem-like Cells by Sustaining FOXM1 551 Expression and Cell Proliferation Program

Solution structure of the RNA recognition domain of 554 METTL3-METTL14 N(6)-methyladenosine methyltransferase

N(6)-methyladenosine regulates the stability of 559 RNA:DNA hybrids in human cells

Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA

Perturbation of m6A writers reveals two distinct 567 classes of mRNA methylation at internal and 5' sites

No 569 evidence for DNA N (6)-methyladenine in mammals

RNA promotes 572 the formation of spatial compartments in the nucleus, bioRxiv

Structural 575 basis of RNA cap modification by SARS-CoV-2

The MC-Fold and MC-Sym pipeline infers RNA 577 structure from sequence data

Figure 1. Structural similarity, purification of methyltransferases, and substrate designing

All three members belong to the β-class of SAM-dependent MTases 613 and exhibit a sequential arrangement of motifs (IV-X followed by motif I) 18

/L) and I are associated with the recognition of target adenine base

ModB MTase (blue), non-methylating DNA strand (grey), target 617 (methylating) DNA strand (orange)

of ModB and ModA are shown in dark blue and light pink, respectively. The Res subunit of EcoP15I 619 was omitted for clarity. d. MTase domains of two Mod subunits (ModA/B) of EcoP15I with target 620

CTD and TRD) are omitted for 621 clarity. Only the region encompassing the MTase core

EcoP15I Mod was selected for the alignment with the methyltransferase core of METTL3 (aa 358-623 580) and METTL14 (aa 165-378). e. An overlay of MTase domains of EcoP15I and METTL14 624 (PDB ID: 5IL0) shows structural similarity within MTase folds

RNA strand here was modeled based on the respective methylating strand in the EcoP15I structure

Chromatogram of final size exclusion chromatography (SEC) step of purification showing 627

full-length; right, METL3-METTL14[-RGG]) co-eluted as 628 single homogenous species. Blue, absorbance at 280 nm

Coomassie stained gels (lower panels) confirm high purity of METTL3-METTL14 proteins in the 630

All oligos 631 have a covalently attached 5'-fluorescein (not shown). h. Secondary structure of rNEAT2 and its 632 3-D model as predicted by MC-SYM 51

Secondary structure of rTCE23 RNA and its solution NMR structure (PDB ID: 2ES5)

Figure 2. RNA-mediated restriction of METTL3-METTL14 activity

Equilibrium 640 dissociation constants (Kd) for each oligo are shown on the right side of the isotherms. The data 641 were fit into one site specific binding model (Y=Bmax*X/(Kd + X). See methods section and 642 source data for details

RNA oligos (red), d6T* DNA alone (black) and equimolar mixture of the two (blue), measured by 644 radiometric assay. d. predicted secondary structures of each oligonucleotide. yellow

The values of the equilibrium dissociation constants (Kd) shown for each 646 oligonucleotide indicate an inverse relationship between binding affinity and methyltransferase 647 activity. e. dose-dependent inhibition of METTL3-METTL14 activity by RNA oligos rNEAT2 or 648 rTCE23, as measured by radiometric assay in a reaction buffer containing 5.0 mM NaCl (upper 649 panel) and 50.0 mM NaCl (lower panel). IC50, concentration of RNA required to achieve 50% 650 inhibition of the METTL3-METTL14 activity. f. Attenuation of the methyltransferase activity in 651 presence of rNEAT2 or rTCE23, as measured by oligonucleotide intact mass analysis. Quantitation 652 of modified dA (black circle) or rA

UHPLC chromatograms showing the reaction in absence (blue trace) or in the presence 655 of either rNEAT2 (red trace) or rTCE23 (green trace). The quantitation of the fraction of modified 656 bases in the nucleoside pool was consistent with the results from the oligonucleotide intact mass 657 analysis shown in f. The insert shows the full chromatographic trace with all detected nucleosides

with one standard deviation (s.d.) for each oligonucleotide (shown as error bars). Source data are 660 provided as a Source Data file

Role of RGG motifs and model of RNA-mediated regulation of methyltransferase 663

Domain architecture of METTL3 and METTL14. LH, leader helix; NLS, nuclear localization 665 signal

ZnF1/2, zinc-finger domain 1/2; RGG, arginine-glycine rich repeats motif. b. FP-based 666 binding assay for DNA and RNA oligos showing the highest affinity of

Equilibrium dissociation constants 668 (Kd) for each oligo are shown. The data were fit into one site specific binding model 669 (Y=Bmax*X/(Kd + X). See methods section and source data for details. c. Relative 670 methyltransferase activity of full-length METTL3-METTL14 and the truncated enzyme devoid of 671 the RGG motif in METTL14 ([METTL3-METTL14(-RGG)]) in the presence of d6T*, rNEAT2, or 672 an equimolar mixture of these two oligos, as measured by radiometric assay. Results presented are 673 the average of three independent experiments (n = 3) with one standard deviation (s.d.) for each 674 oligonucleotide (shown as error bars). The results of two groups were analyzed and compared 675 using two-tailed Student's unpaired T-test (P value <0.0001). Details about Student's T-test are 676 provided in the source data file

Structured motifs present in ncRNA/mRNAs (orange) can block the methyltransferase activity by 679 a shape-dependent binding of these RNAs to

RNA binding to human METTL3-METTL14 restricts N 6 -deoxyadenosine methylation of

Greehey Children's Cancer Research Institute, University of Texas Health at San Antonio 703

240 County Road

The conserved motifs in class β MTases are shown in magenta. The motif IV 726 (D/EPPY/W/L) that surrounds the watson-edge edge of the flipped target adenine base (red) is 727 shown in stick mode. b. Cyan ribbons, MTase core of human METTL14 (PDB: 5IL0) with a 728 modeled ssDNA as in ModB. The conserved motifs are colored in yellow. c. An overlay of MTases 729 of ModB and METTL14 was performed using the SSM method (secondary structure-based) that 730 yielded an rmsd of 2.55 over 282aa of METTL14 aligned with 278aa of ModB. The canonical 731 MTase motifs, including motif IV, in two MTases overlay well. Due to lack of structures of 732 METTL3 or ModA (in complex with a flipped adenine base), we chose ModB and METTL14 for 733 this superposition