Structural and functional insights into the molecular mechanism of rRNA m6A methyltransferase RlmJ

RlmJ catalyzes the m⁶A2030 methylation of 23S rRNA during ribosome biogenesis in Escherichia coli. Here, we present crystal structures of RlmJ in apo form, in complex with the cofactor S-adenosyl-methionine and in complex with S-adenosyl-homocysteine plus the substrate analogue adenosine monophosphate (AMP). RlmJ displays a variant of the Rossmann-like methyltransferase (MTase) fold with an inserted helical subdomain. Binding of cofactor and substrate induces a large shift of the N-terminal motif X tail to make it cover the cofactor binding site and trigger active-site changes in motifs IV and VIII. Adenosine monophosphate binds in a partly accommodated state with the target N6 atom 7 Å away from the sulphur of AdoHcy. The active site of RlmJ with motif IV sequence ₁₆₄DPPY₁₆₇ is more similar to DNA m⁶A MTases than to RNA m⁶₂A MTases, and structural comparison suggests that RlmJ binds its substrate base similarly to DNA MTases T4Dam and M.TaqI. RlmJ methylates in vitro transcribed 23S rRNA, as well as a minimal substrate corresponding to helix 72, demonstrating independence of previous modifications and tertiary interactions in the RNA substrate. RlmJ displays specificity for adenosine, and mutagenesis experiments demonstrate the critical roles of residues Y4, H6, K18 and D164 in methyl transfer.     

Shared requirements for key residues in the antibiotic resistance enzymes ErmC and ErmE suggest a common mode of RNA recognition

Erythromycin-resistance methyltransferases are SAM dependent Rossmann fold methyltransferases that convert A2058 of 23S rRNA to m⁶ ₂A2058. This modification sterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multidrug-resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, whereas ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24% sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity–binding assays. We demonstrate that residues in α4, α5, and the α5-α6 linker are essential for methyltransferase function, including an aromatic residue on α4 that likely forms stacking interactions with the substrate adenosine and basic residues in α5 and the α5-α6 linker that likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.     

YTH Domain: A Family of N6-methyladenosine (m6A) Readers

Like protein and DNA, different types of RNA molecules undergo various modifications. Accumulating evidence suggests that these RNA modifications serve as sophisticated codes to mediate RNA behaviors and many important biological functions. N⁶-methyladenosine (m⁶A) is the most abundant internal RNA modification found in a variety of eukaryotic RNAs, including but not limited to mRNAs, tRNAs, rRNAs, and long non-coding RNAs (lncRNAs). In mammalian cells, m⁶A can be incorporated by a methyltransferase complex and removed by demethylases, which ensures that the m⁶A modification is reversible and dynamic. Moreover, m⁶A is recognized by the YT521-B homology (YTH) domain-containing proteins, which subsequently direct different complexes to regulate RNA signaling pathways, such as RNA metabolism, RNA splicing, RNA folding, and protein translation. Herein, we summarize the recent progresses made in understanding the molecular mechanisms underlying the m⁶A recognition by YTH domain-containing proteins, which would shed new light on m⁶A-specific recognition and provide clues to the future identification of reader proteins of many other RNA modifications.     

West Nile virus methyltransferase catalyzes two methylations of the viral RNA cap through a substrate-repositioning mechanism

Flaviviruses encode a single methyltransferase domain that sequentially catalyzes two methylations of the viral RNA cap, GpppA-RNA-->m(7)GpppA-RNA-->m(7)GpppAm-RNA, by using S-adenosyl-l-methionine (SAM) as a methyl donor. Crystal structures of flavivirus methyltransferases exhibit distinct binding sites for SAM, GTP, and RNA molecules. Biochemical analysis of West Nile virus methyltransferase shows that the single SAM-binding site donates methyl groups to both N7 and 2'-O positions of the viral RNA cap, the GTP-binding pocket functions only during the 2'-O methylation, and two distinct sets of amino acids in the RNA-binding site are required for the N7 and 2'-O methylations. These results demonstrate that flavivirus methyltransferase catalyzes two cap methylations through a substrate-repositioning mechanism. In this mechanism, guanine N7 of substrate GpppA-RNA is first positioned to SAM to generate m(7)GpppA-RNA, after which the m(7)G moiety is repositioned to the GTP-binding pocket to register the 2'-OH of the adenosine with SAM, generating m(7)GpppAm-RNA. Because N7 cap methylation is essential for viral replication, inhibitors designed to block the pocket identified for the N7 cap methylation could be developed for flavivirus therapy.     

NSUN6 is a human RNA methyltransferase that catalyzes formation of m5C72 in specific tRNAs

Many cellular RNAs require modification of specific residues for their biogenesis, structure, and function. 5-methylcytosine (m⁵C) is a common chemical modification in DNA and RNA but in contrast to the DNA modifying enzymes, only little is known about the methyltransferases that establish m⁵C modifications in RNA. The putative RNA methyltransferase NSUN6 belongs to the family of Nol1/Nop2/SUN domain (NSUN) proteins, but so far its cellular function has remained unknown. To reveal the target spectrum of human NSUN6, we applied UV crosslinking and analysis of cDNA (CRAC) as well as chemical crosslinking with 5-azacytidine. We found that human NSUN6 is associated with tRNAs and acts as a tRNA methyltransferase. Furthermore, we uncovered tRNA^Cys and tRNA^Thr as RNA substrates of NSUN6 and identified the cytosine C72 at the 3′ end of the tRNA acceptor stem as the target nucleoside. Interestingly, target recognition in vitro depends on the presence of the 3′-CCA tail. Together with the finding that NSUN6 localizes to the cytoplasm and largely colocalizes with marker proteins for the Golgi apparatus and pericentriolar matrix, our data suggest that NSUN6 modifies tRNAs in a late step in their biogenesis.     

The detection and functions of RNA modification m6A based on m6A writers and erasers

N⁶-methyladenosine (m⁶A) is the most frequent chemical modification in eukaryotic mRNA and is known to participate in a variety of physiological processes, including cancer progression and viral infection. The reversible and dynamic m⁶A modification is installed by m⁶A methyltransferase (writer) enzymes and erased by m⁶A demethylase (eraser) enzymes. m⁶A modification recognized by m⁶A binding proteins (readers) regulates RNA processing and metabolism, leading to downstream biological effects such as promotion of stability and translation or increased degradation. The m⁶A writers and erasers determine the abundance of m⁶A modifications and play decisive roles in its distribution and function. In this review, we focused on m⁶A writers and erasers and present an overview on their known functions and enzymatic molecular mechanisms, showing how they recognize substrates and install or remove m⁶A modifications. We also summarize the current applications of m⁶A writers and erasers for m⁶A detection and highlight the merits and drawbacks of these available methods. Lastly, we describe the biological functions of m⁶A in cancers and viral infection based on research of m⁶A writers and erasers and introduce new assays for m⁶A functionality via programmable m⁶A editing tools.     

Structures of Human ALKBH5 Demethylase Reveal a Unique Binding Mode for Specific Single-stranded N6-Methyladenosine RNA Demethylation

N⁶-Methyladenosine (m⁶A) is the most prevalent internal RNA modification in eukaryotes. ALKBH5 belongs to the AlkB family of dioxygenases and has been shown to specifically demethylate m⁶A in single-stranded RNA. Here we report crystal structures of ALKBH5 in the presence of either its cofactors or the ALKBH5 inhibitor citrate. Catalytic assays demonstrate that the ALKBH5 catalytic domain can demethylate both single-stranded RNA and single-stranded DNA. We identify the TCA cycle intermediate citrate as a modest inhibitor of ALKHB5 (IC₅₀, ∼488 μm). The structural analysis reveals that a loop region of ALKBH5 is immobilized by a disulfide bond that apparently excludes the binding of dsDNA to ALKBH5. We identify the m⁶A binding pocket of ALKBH5 and the key residues involved in m⁶A recognition using mutagenesis and ITC binding experiments.     

Flavivirus RNA cap methyltransferase: structure,function, and inhibition

Many flaviviruses are significant human pathogens. The plus-strand RNA genome of a flavivirus contains a 5′ terminal cap 1 structure (m⁷GpppAmG). The flavivirus encodes one methyltransferase (MTase), located at the N-terminal portion of the NS5 RNA-dependent RNA polymerase (RdRp). Here we review recent advances in our understanding of flaviviral capping machinery and the implications for drug development. The NS5 MTase catalyzes both guanine N7 and ribose 2′-OH methylations during viral cap formation. Representative flavivirus MTases, from dengue, yellow fever, and West Nile virus (WNV), sequentially generate GpppA → m⁷GpppA → m⁷GpppAm. Despite the existence of two distinct methylation activities, the crystal structures of flavivirus MTases showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. This finding indicates that the substrate GpppA-RNA must be repositioned to accept the N7 and 2′-O methyl groups from SAM during the sequential reactions. Further studies demonstrated that distinct RNA elements are required for the methylations of guanine N7 on the cap and of ribose 2′-OH on the first transcribed nucleotide. Mutant enzymes with different methylation defects can trans complement one another in vitro, demonstrating that separate molecules of the enzyme can independently catalyze the two cap methylations in vitro. In the context of the infectious virus, defects in both methylations, or a defect in the N7 methylation alone, are lethal to WNV. However, viruses defective solely in 2′-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel and promising target for flavivirus therapy.     

m6A methyltransferase KIAA1429 accelerates oral squamous cell carcinoma via regulating glycolysis and ferroptosis

N⁶-methyladenosine (m⁶A) modification acts as the most prevalent modification on eukaryotic RNA, and its function on oral squamous cell carcinoma (OSCC) is still unclear. Here, the present research aimed to explore the novel function of m⁶A methyltransferase KIAA1429 in OSCC. Results illustrated that KIAA1429 up-regulated in the OSCC samples and cells. Gain/loss functional assays demonstrated that KIAA1429 repressed the ferroptosis of OSCC. Moreover, KIAA1429 positively accelerated the aerobic glycolysis of OSCC, including glucose uptake, lactate production, ATP level and ECAR. Mechanistically, KIAA1429 could install the m⁶A modification on the PGK1 mRNA, thereby up-regulating the methylated m⁶A level. Moreover, m⁶A reader YTHDF1 recognized the m⁶A modification site of PGK1 mRNA and enhanced its mRNA stability. Thus, KIAA1429 promoted the OSCC aerobic glycolysis and inhibited the ferroptosis of OSCC through YTHDF1-mediated PGK1 mRNA stability. Taken together, these findings reveal a novel insight for KIAA1429 on OSCC via m⁶A-dependent manner.     

Solution structure of the YTH domain in complex with N6-methyladenosine RNA: a reader of methylated RNA

N⁶A methylation is the most abundant RNA modification occurring within messenger RNA. Impairment of methylase or demethylase functions are associated with severe phenotypes and diseases in several organisms. Beside writer and eraser enzymes of this dynamic RNA epigenetic modification, reader proteins that recognize this modification are involved in numerous cellular processes. Although the precise characterization of these reader proteins remains unknown, preliminary data showed that most potential reader proteins contained a conserved YT521-B homology (YTH) domain. Here we define the YTH domain of rat YT521-B as a N⁶-methylated adenosine reader domain and report its solution structure in complex with a N⁶-methylated RNA. The structure reveals a binding preference for NGANNN RNA hexamer and a deep hydrophobic cleft for m⁶A recognition. These findings establish a molecular function for YTH domains as m⁶A reader domains and should guide further studies into the biological functions of YTH-containing proteins in m⁶A recognition.     

N~6 -methyl-adenosine (m ~6A) in RNA: An Old Modification with A Novel Epigenetic Function

N6 -methyl-adenosine (m6A) is one of the most common and abundant modifications on RNA molecules present in eukaryotes. However, the biological significance of m6A methylation remains largely unknown. Several independent lines of evidence suggest that the dynamic regulation of m6A may have a profound impact on gene expression regulation. The m6A modification is catalyzed by an unidentified methyltransferase complex containing at least one subunit methyltransferase like 3 (METTL3). m6A modification on messenger RNAs (mRNAs) mainly occurs in the exonic regions and 3’-untranslated region (3’-UTR) as revealed by high-throughput m6A-seq. One significant advance in m6A research is the recent discovery of the first two m6A RNA demethylases fat mass and obesity-associated (FTO) gene and ALKBH5, which catalyze m6A demethylation in an a-ketoglutarate (a-KG)-and Fe2+-dependent manner. Recent studies in model organisms demonstrate that METTL3, FTO and ALKBH5 play important roles in many biological processes, ranging from development and metabolism to fertility. Moreover, perturbation of activities of these enzymes leads to the disturbed expression of thousands of genes at the cellular level, implicating a regulatory role of m6A in RNA metabolism. Given the vital roles of DNA and histone methylations in epigenetic regulation of basic life processes in mammals, the dynamic and reversible chemical m6A modification on RNA may also serve as a novel epigenetic marker of profound biological significances.     

Functional dichotomy in the 16S rRNA (m1A1408) methyltransferase family and control of catalytic activity via a novel tryptophan mediated loop reorganization

Methylation of the bacterial small ribosomal subunit (16S) rRNA on the N1 position of A1408 confers exceptionally high-level resistance to a broad spectrum of aminoglycoside antibiotics. Here, we present a detailed structural and functional analysis of the Catenulisporales acidiphilia 16S rRNA (m¹A1408) methyltransferase (‘CacKam’). The apo CacKam structure closely resembles other m¹A1408 methyltransferases within its conserved SAM-binding fold but the region linking core β strands 6 and 7 (the ‘β6/7 linker’) has a unique, extended structure that partially occludes the putative 16S rRNA binding surface, and sequesters the conserved and functionally critical W203 outside of the CacKam active site. Substitution of conserved residues in the SAM binding pocket reveals a functional dichotomy in the 16S rRNA (m¹A1408) methyltransferase family, with two apparently distinct molecular mechanisms coupling cosubstrate/ substrate binding to catalytic activity. Our results additionally suggest that CacKam exploits the W203-mediated remodeling of the β6/7 linker as a novel mechanism to control 30S substrate recognition and enzymatic turnover.     

A m6A Sensing Method by Its Impact on the Stability of RNA Double Helix

N⁶‐Methyladenosine (m⁶A) is one of the most important RNA modifications in epigenetics. The development of detection method for m⁶A is limited by its abundance and structure. Although it has been previously reported that its presence has an impact on the complementary pairing of RNA, few assays have been developed using this finding. We used this discovery and designed a detection method based on Cas13a system, which has different fluorescence signals for target RNAs containing m⁶A modification and target RNAs without m⁶A modification. We verified the fact that the presence of m⁶A could cause the instability of dsRNA using the Cas13a system and provided a new direction and strategy for the development of m⁶A detection methods in the future.     

m6A甲基转移酶家族Fiona/METT-10/METTL16的功能

RNA碱基上的化学修饰在其功能的精准调节中发挥关键作用,其中m⁶A是自然界中最普遍的RNA修饰之一,且该修饰在调控RNA稳定性、pre-mRNA剪接、翻译等方面具有重要功能。在真核生物中,m⁶A修饰主要由两种甲基转移酶完成,其在哺乳动物中分别命名为METTL3和METTL16。与METTL3相似,METTL16的底物多种多样,包括pre-mRNA、rRNA、snRNA和lncRNA等,因此似乎难以用一种分子机理解释METTL16对不同RNA底物进行m⁶A修饰的功能。此外,METTL16还在翻译调控中发挥重要作用,但此过程不依赖其甲基转移酶活性,这进一步增加了高度保守的METTL16的功能复杂性。本综述总结了METTL16及其同源蛋白质的结构域、甲基化底物以及它们的潜在功能,着重阐述了在不同物种中关于METTL16研究结果的矛盾之处,并推测METTL16调控S-腺苷基甲硫氨酸(SAM)代谢的功能是趋同进化的一个潜在案例。     

Human METTL16 is a N6‐methyladenosine (m6A) methyltransferase that targets pre‐mRNAs and various non‐coding RNAs

N⁶‐methyladenosine (m⁶A) is a highly dynamic RNA modification that has recently emerged as a key regulator of gene expression. While many m⁶A modifications are installed by the METTL3–METTL14 complex, others appear to be introduced independently, implying that additional human m⁶A methyltransferases remain to be identified. Using crosslinking and analysis of cDNA (CRAC), we reveal that the putative human m⁶A “writer” protein METTL16 binds to the U6 snRNA and other ncRNAs as well as numerous lncRNAs and pre‐mRNAs. We demonstrate that METTL16 is responsible for N⁶‐methylation of A43 of the U6 snRNA and identify the early U6 biogenesis factors La, LARP7 and the methylphosphate capping enzyme MEPCE as METTL16 interaction partners. Interestingly, A43 lies within an essential ACAGAGA box of U6 that base pairs with 5′ splice sites of pre‐mRNAs during splicing, suggesting that METTL16‐mediated modification of this site plays an important role in splicing regulation. The identification of METTL16 as an active m⁶A methyltransferase in human cells expands our understanding of the mechanisms by which the m⁶A landscape is installed on cellular RNAs.