The enzymes for genome size increase and maintenance of large (+)RNA viruses.

With sizes <50 kb, viral RNA genomes are at the crossroads of genetic, biophysical, and biochemical stability in their host cell. Here, we analyze the enzymatic assets accompanying large RNA genome viruses, mostly based on recent scientific advances in Coronaviridae. We argue that, in addition to the presence of an RNA exonuclease (ExoN), two markers for the large size of viral RNA genomes are (i) the presence of one or more RNA methyltransferases (MTases) and (ii) a specific architecture of the RNA-dependent RNA polymerase active site. We propose that RNA genome expansion and maintenance are driven by an evolutionary ménage-à-trois made of fast and processive RNA polymerases, RNA repair ExoNs, and RNA MTases that relates to the transition between RNA- to DNA-based life.

Global RNA modifications to the MALAT1 triple helix differentially affect thermostability and weaken binding to METTL16.

Therapeutic mRNAs are generated using modified nucleotides, namely N1-methylpseudouridine (m1Ψ) triphosphate, so that the mRNA evades detection by the immune system. RNA modifications, even at a single-nucleotide position, perturb RNA structure, although it is not well understood how structure and function is impacted by globally modified RNAs. Therefore, we examined the metastasis-associated lung adenocarcinoma transcript 1 triple helix, a highly structured stability element that includes single-, double-, and triple-stranded RNA, globally modified with N6-methyladenosine (m6A), pseudouridine (Ψ), or m1Ψ. UV thermal denaturation assays showed that m6A destabilizes both the Hoogsteen and Watson-Crick faces of the RNA by ∼20 °C, Ψ stabilizes the Hoogsteen and Watson-Crick faces of the RNA by ∼12 °C, and m1Ψ has minimal effect on the stability of the Hoogsteen face of the RNA but increases the stability of the Watson-Crick face by ∼9 °C. Native gel-shift assays revealed that binding of the methyltransferase-like protein 16 to the metastasis-associated lung adenocarcinoma transcript 1 triple helix was weakened by at least 8-, 99-, and 23-fold, respectively, when RNA is globally modified with m6A, Ψ, or m1Ψ. These results demonstrate that a more thermostable RNA structure does not lead to tighter RNA-protein interactions, thereby highlighting the regulatory power of RNA modifications by multiple means.

5-methylcytosine modification by Plasmodium NSUN2 stabilizes mRNA and mediates the development of gametocytes.

5-methylcytosine (m5C) is an important epitranscriptomic modification involved in messenger RNA (mRNA) stability and translation efficiency in various biological processes. However, it remains unclear if m5C modification contributes to the dynamic regulation of the transcriptome during the developmental cycles of Plasmodium parasites. Here, we characterize the landscape of m5C mRNA modifications at single nucleotide resolution in the asexual replication stages and gametocyte sexual stages of rodent (Plasmodium yoelii) and human (Plasmodium falciparum) malaria parasites. While different representations of m5C-modified mRNAs are associated with the different stages, the abundance of the m5C marker is strikingly enhanced in the transcriptomes of gametocytes. Our results show that m5C modifications confer stability to the Plasmodium transcripts and that a Plasmodium ortholog of NSUN2 is a major mRNA m5C methyltransferase in malaria parasites. Upon knockout of P. yoelii nsun2 (pynsun2), marked reductions of m5C modification were observed in a panel of gametocytogenesis-associated transcripts. These reductions correlated with impaired gametocyte production in the knockout rodent malaria parasites. Restoration of the nsun2 gene in the knockout parasites rescued the gametocyte production phenotype as well as m5C modification of the gametocytogenesis-associated transcripts. Together with the mRNA m5C profiles for two species of Plasmodium, our findings demonstrate a major role for NSUN2-mediated m5C modifications in mRNA transcript stability and sexual differentiation in malaria parasites.

Application of mutational profiling: New functional analyses reveal the tRNA recognition mechanism of tRNA m1A22 methyltransferase.

Transfer RNAs undergo diverse posttranscriptional modifications to regulate a myriad of cellular events including translation, stress response, and viral replication. These posttranscriptional modifications are synthesized by site-specific modification enzymes. Recent RNA-seq techniques have revealed multiple features of tRNA such as tRNA abundance, tRNA modification, and tRNA structure. Here, we adapt a tRNA-sequencing technique and design a new functional analysis where we perform mutational profiling of tRNA modifications to gain mechanistic insights into how tRNA modification enzymes recognize substrate tRNA. Profiling of Geobacillus stearothermophilus tRNAs and protein orthology analysis predict the existence of natural modifications in 44 tRNA molecular species of G. stearothermophilus. We selected the 1-methyladenosine modification at position 22 (m1A22) and tRNA (m1A22) methyltransferase (TrmK) for further analysis. Relative quantification of m1A22 levels in 59 tRNA transcripts by mutational profiling reveals that TrmK selectively methylates a subset of tRNAs. Using 240 variants of tRNALeu transcripts, we demonstrate the conserved nucleosides including U8, A14, G15, G18, G19, U55, Purine57, and A58 are important for the methyl transfer reaction of TrmK. Additional biochemical experiments reveal that TrmK strictly recognizes U8, A14, G18, and U55 in tRNA. Furthermore, these findings from tRNALeu variants were crossvalidated using variants of three different tRNA species. Finally, a model of the TrmK-tRNA complex structure was constructed based on our findings and previous biochemical and structural studies by others. Collectively, our study expands functional analyses of tRNA modification enzyme in a high-throughput manner where our assay rapidly identifies substrates from a large pool of tRNAs.

Posttranscriptional Regulation by Proteins and Noncoding RNAs.

Posttranscriptional regulation comprises those mechanisms occurring after the initial copy of the DNA sequence is transcribed into an intermediate RNA molecule (i.e., messenger RNA) until such a molecule is used as a template to generate a protein. A subset of these posttranscriptional regulatory mechanisms essentially are destined to process the immature mRNA toward its mature form, conferring the adequate mRNA stability, providing the means for pertinent introns excision, and controlling mRNA turnover rate and quality control check. An additional layer of complexity is added in certain cases, since discrete nucleotide modifications in the mature RNA molecule are added by RNA editing, a process that provides large mature mRNA diversity. Moreover, a number of posttranscriptional regulatory mechanisms occur in a cell- and tissue-specific manner, such as alternative splicing and noncoding RNA-mediated regulation. In this chapter, we will briefly summarize current state-of-the-art knowledge of general posttranscriptional mechanisms, while major emphases will be devoted to those tissue-specific posttranscriptional modifications that impact on cardiac development and congenital heart disease.

Structure, dynamics, and molecular inhibition of the Staphylococcus aureus m1A22-tRNA methyltransferase TrmK.

The enzyme m1A22-tRNA methyltransferase (TrmK) catalyzes the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection but has no homolog in mammals, making it a promising target for antibiotic development. Here, we characterize the structure and function of S. aureus TrmK (SaTrmK) using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the SaTrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favorable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favorable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for SaTrmK-catalyzed methylation of A22 mutants of tRNALeu demonstrate that the adenine at position 22 is absolutely essential. In silico screening of compounds suggested the multifunctional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of SaTrmK, laying a foundation for future structure-based drug discovery.

Crystal structure and functional analysis of mycobacterial erythromycin resistance methyltransferase Erm38 reveals its RNA-binding site.

Erythromycin resistance methyltransferases (Erms) confer resistance to macrolide, lincosamide, and streptogramin antibiotics in Gram-positive bacteria and mycobacteria. Although structural information for ErmAM, ErmC, and ErmE exists from Gram-positive bacteria, little is known about the Erms in mycobacteria, as there are limited biochemical data and no structures available. Here, we present crystal structures of Erm38 from Mycobacterium smegmatis in apoprotein and cofactor-bound forms. Based on structural analysis and mutagenesis, we identified several catalytically critical, positively charged residues at a putative RNA-binding site. We found that mutation of any of these sites is sufficient to abolish methylation activity, whereas the corresponding RNA-binding affinity of Erm38 remains unchanged. The methylation reaction thus appears to require a precise ensemble of amino acids to accurately position the RNA substrate, such that the target nucleotide can be methylated. In addition, we computationally constructed a model of Erm38 in complex with a 32-mer RNA substrate. This model shows the RNA substrate stably bound to Erm38 by a patch of positively charged residues. Furthermore, a π-π stacking interaction between a key aromatic residue of Erm38 and a target adenine of the RNA substrate forms a critical interaction needed for methylation. Taken together, these data provide valuable insights into Erm-RNA interactions, which will aid subsequent structure-based drug design efforts.

Epigenetic inactivation of the 5-methylcytosine RNA methyltransferase NSUN7 is associated with clinical outcome and therapeutic vulnerability in liver cancer.

BACKGROUND: RNA modifications are important regulators of transcript activity and an increasingly emerging body of data suggests that the epitranscriptome and its associated enzymes are altered in human tumors. METHODS: Combining data mining and conventional experimental procedures, NSUN7 methylation and expression status was assessed in liver cancer cell lines and primary tumors. Loss-of-function and transfection-mediated recovery experiments coupled with RNA bisulfite sequencing and proteomics determined the activity of NSUN7 in downstream targets and drug sensitivity. RESULTS: In this study, the initial screening for genetic and epigenetic defects of 5-methylcytosine RNA methyltransferases in transformed cell lines, identified that the NOL1/NOP2/Sun domain family member 7 (NSUN7) undergoes promoter CpG island hypermethylation-associated with transcriptional silencing in a cancer-specific manner. NSUN7 epigenetic inactivation was common in liver malignant cells and we coupled bisulfite conversion of cellular RNA with next-generation sequencing (bsRNA-seq) to find the RNA targets of this poorly characterized putative RNA methyltransferase. Using knock-out and restoration-of-function models, we observed that the mRNA of the coiled-coil domain containing 9B (CCDC9B) gene required NSUN7-mediated methylation for transcript stability. Most importantly, proteomic analyses determined that CCDC9B loss impaired protein levels of its partner, the MYC-regulator Influenza Virus NS1A Binding Protein (IVNS1ABP), creating sensitivity to bromodomain inhibitors in liver cancer cells exhibiting NSUN7 epigenetic silencing. The DNA methylation-associated loss of NSUN7 was also observed in primary liver tumors where it was associated with poor overall survival. Interestingly, NSUN7 unmethylated status was enriched in the immune active subclass of liver tumors. CONCLUSION: The 5-methylcytosine RNA methyltransferase NSUN7 undergoes epigenetic inactivation in liver cancer that prevents correct mRNA methylation. Furthermore, NSUN7 DNA methylation-associated silencing is associated with clinical outcome and distinct therapeutic vulnerability.

Biallelic variants in NSUN6 cause an autosomal recessive neurodevelopmental disorder.

PURPOSE: 5-methylcytosine RNA modifications are driven by NSUN methyltransferases. Although variants in NSUN2 and NSUN3 were associated with neurodevelopmental diseases, the physiological role of NSUN6 modifications on transfer RNAs and messenger RNAs remained elusive. METHODS: We combined exome sequencing of consanguineous families with functional characterization to identify a new neurodevelopmental disorder gene. RESULTS: We identified 3 unrelated consanguineous families with deleterious homozygous variants in NSUN6. Two of these variants are predicted to be loss-of-function. One maps to the first exon and is predicted to lead to the absence of NSUN6 via nonsense-mediated decay, whereas we showed that the other maps to the last exon and encodes a protein that does not fold correctly. Likewise, we demonstrated that the missense variant identified in the third family has lost its enzymatic activity and is unable to bind the methyl donor S-adenosyl-L-methionine. The affected individuals present with developmental delay, intellectual disability, motor delay, and behavioral anomalies. Homozygous ablation of the NSUN6 ortholog in Drosophila led to locomotion and learning impairment. CONCLUSION: Our data provide evidence that biallelic pathogenic variants in NSUN6 cause one form of autosomal recessive intellectual disability, establishing another link between RNA modification and cognition.

TRIMETHYLGUANOSINE SYNTHASE1 mutations decanalize female germline development in Arabidopsis.

Here, we report the characterization of a plant RNA methyltransferase, orthologous to yeast trimethylguanosine synthase1 (Tgs1p) and whose downregulation was associated with apomixis in Paspalum grasses. Using phylogenetic analyses and yeast complementation, we determined that land plant genomes all encode a conserved, specific TGS1 protein. Next, we studied the role of TGS1 in female reproduction using reporter lines and loss-of-function mutants in Arabidopsis thaliana. pAtTGS1:AtTGS1 reporters showed a dynamic expression pattern. They were highly active in the placenta and ovule primordia at emergence but, subsequently, showed weak signals in the nucellus. Although expressed throughout gametophyte development, activity became restricted to the female gamete and was also detected after fertilization during embryogenesis. TGS1 depletion altered the specification of the precursor cells that give rise to the female gametophytic generation and to the sporophyte, resulting in the formation of a functional aposporous-like lineage. Our results indicate that TGS1 participates in the mechanisms restricting cell fate acquisition to a single cell at critical transitions throughout the female reproductive lineage and, thus, expand our current knowledge of the mechanisms governing female reproductive fate in plants.

Synthesis of Bisubstrate Analogues for RNA Methylation Studies using two Transition-Metal-Catalyzed Reactions.

RNA methyltransferases (RNA MTases) are a family of enzymes that catalyze the methylation of RNA using the cofactor S-adenosyl-L-methionine. While RNA MTases are promising drug targets, new molecules are needed to fully understand their roles in disease and to develop effective drugs that can modulate their activity. Since RNA MTases are suitable for bisubstrate binding, we report an original strategy for the synthesis of a new family of m6A MTases bisubstrate analogues. Six compounds containing a S-adenosyl-L-methionine (SAM) analogue unit covalently tethered by a triazole ring to the N-6 position of an adenosine were synthesized. A procedure using two transition-metal-catalyzed reactions was used to introduce the α-amino acid motif mimicking the methionine chain of the cofactor SAM. First, a copper(I)-catalyzed alkyne-azide iodo-cycloaddition (iCuAAC) reaction afforded the 5-iodo-1,4-disubstituted-1,2,3-triazole which was functionalized by palladium-catalyzed cross-coupling to connect the α-amino acid substituent. Docking studies of our molecules in the active site of the m6A ribosomal MTase RlmJ show that the use of triazole as a linker provides additional interactions and the presence of the α-amino acid chain stabilizes the bisubstrate. The synthetic method developed here enhances the structural diversity of bisubstrate analogues to explore the active site of RNA modification enzymes and to develop new inhibitors.

Identification of an N6-methyladenosine-mediated positive feedback loop that promotes Epstein-Barr virus infection.

N6-methyladenosine (m6A) is among the most abundant mRNA modifications, particularly in eukaryotes, and is found in mammals, plants, and even some viruses. Although essential for the regulation of many biological processes, the exact role of m6A modification in virus-host interaction remains largely unknown. Here, using m6A -immunoprecipitation and sequencing, we find that Epstein-Barr virus (EBV) infection decreases the m6A modification of transcriptional factor KLF4 mRNA and subsequently increases its protein level. Mechanistically, EBV immediate-early protein BZLF1 interacts with the promoter of m6A methyltransferase METTL3, inhibiting its expression. Subsequently, the decrease of METTL3 reduces the level of KLF4 mRNA m6A modification, preventing its decay by the m6A reader protein YTHDF2. As a result, KLF4 protein level is upregulated and, in turn, promotes EBV infection of nasopharyngeal epithelial cells. Thus, our results suggest the existence of a positive feedback loop formed between EBV and host molecules via cellular mRNA m6A levels, and this feedback loop acts to facilitate viral infection. This mechanism contains multiple potential targets for controlling viral infectious diseases.

Structural basis for METTL6-mediated m3C RNA methylation.

RNA modifications play important roles in mediating the biological functions of RNAs. 3-methylcytidine (m3C), albeit less abundant, is found to exist extensively in tRNAs, rRNAs and mRNAs. Human METTL6 is a m3C methyltransferase for tRNAs, including tRNASER(UGA). We solved the structure of human METTL6 in the presence of S-adenosyl-L-methionine and found by enzyme assay that recombinant human METTL6 is active towards tRNASER(UGA). Structural analysis indicated the detailed interactions between S-adenosyl-L-methionine and METTL6, and suggested potential tRNA binding region on the surface of METTL6. The structural research, complemented by biochemistry enzyme assay, will definitely shed light on the design of potent inhibitors for METTL6 in near future.

The 18S ribosomal RNA m6 A methyltransferase Mettl5 is required for normal walking behavior in Drosophila.

RNA modifications have recently emerged as an important layer of gene regulation. N6-methyladenosine (m6 A) is the most prominent modification on eukaryotic messenger RNA and has also been found on noncoding RNA, including ribosomal and small nuclear RNA. Recently, several m6 A methyltransferases were identified, uncovering the specificity of m6 A deposition by structurally distinct enzymes. In order to discover additional m6 A enzymes, we performed an RNAi screen to deplete annotated orthologs of human methyltransferase-like proteins (METTLs) in Drosophila cells and identified CG9666, the ortholog of human METTL5. We show that CG9666 is required for specific deposition of m6 A on 18S ribosomal RNA via direct interaction with the Drosophila ortholog of human TRMT112, CG12975. Depletion of CG9666 yields a subsequent loss of the 18S rRNA m6 A modification, which lies in the vicinity of the ribosome decoding center; however, this does not compromise rRNA maturation. Instead, a loss of CG9666-mediated m6 A impacts fly behavior, providing an underlying molecular mechanism for the reported human phenotype in intellectual disability. Thus, our work expands the repertoire of m6 A methyltransferases, demonstrates the specialization of these enzymes, and further addresses the significance of ribosomal RNA modifications in gene expression and animal behavior.

RNA methyltransferase NSUN2 promotes hypopharyngeal squamous cell carcinoma proliferation and migration by enhancing TEAD1 expression in an m5C-dependent manner.

RNA methyltransferase NSUN2 is involved in cell proliferation and invasion in a variety of tumors. However, the expression, function, and mechanism of NSUN2 in hypopharyngeal squamous cell carcinoma (HPSCC) remains unknown. We used a bioinformatics database, polymerase chain reaction, cell culture and transfection, immunohistochemistry, cell proliferation assay, wound healing experiments, transwell assays, western blotting, RNA-seq detection, dual-luciferase reporter assay, in vivo experiments, and a dot blot assay to evaluate the role of NSUN2 in HPSCC. NSUN2 mRNA and protein were highly expressed in HPSCC; NSUN2 knockdown in vitro and in vivo decreased cell proliferation and invasion. Studies have shown that TEAD1, a transcription factor, may act downstream of NSUN2 in HPSCC. NSUN2 was found to promote the proliferation and invasion of HPSCC by upregulating TEAD1 in an 5-methylcytosine-dependent manner, thereby representing an oncogene and potential new target for treating HPSCC.

METTL16 promotes hepatocellular carcinoma progression through downregulating RAB11B-AS1 in an m6A-dependent manner.

BACKGROUND: The molecular mechanisms driving hepatocellular carcinoma (HCC) remain largely unclear. As one of the major epitranscriptomic modifications, N6-methyladenosine (m6A) plays key roles in HCC. The aim of this study was to investigate the expression, roles, and mechanisms of action of the RNA methyltransferase methyltransferase-like protein 16 (METTL16) in HCC. METHODS: The expression of METTL16 and RAB11B-AS1 was determined by RT-qPCR. The regulation of RAB11B-AS1 by METTL16 was investigated by RNA immunoprecipitation (RIP), methylated RIP (MeRIP), and RNA stability assays. In vitro and in vivo gain- and loss-of-function assays were performed to investigate the roles of METTL16 and RAB11B-AS1. RESULTS: METTL16 was upregulated in HCC, and its increased expression was correlated with poor prognosis of HCC patients. METTL16 promoted HCC cellular proliferation, migration, and invasion, repressed HCC cellular apoptosis, and promoted HCC tumoral growth in vivo. METTL16 directly bound long noncoding RNA (lncRNA) RAB11B-AS1, induced m6A modification of RAB11B-AS1, and decreased the stability of RAB11B-AS1 transcript, leading to the downregulation of RAB11B-AS1. Conversely to METTL16, RAB11B-AS1 is downregulated in HCC, and its decreased expression was correlated with poor prognosis of patients with HCC. Furthermore, the expression of RAB11B-AS1 was negatively correlated with METTL16 in HCC tissues. RAB11B-AS1 repressed HCC cellular proliferation, migration, and invasion, promoted HCC cellular apoptosis, and inhibited HCC tumoral growth in vivo. Functional rescue assays revealed that overexpression of RAB11B-AS1 reversed the oncogenic roles of METTL16 in HCC. CONCLUSIONS: This study identified the METTL16/RAB11B-AS1 regulatory axis in HCC, which represented novel targets for HCC prognosis and treatment.

Functional Characterization of Two RNA Methyltransferase Genes METTL3 and METTL14 Uncovers the Roles of m6A in Mediating Adaptation of Plutella xylostella to Host Plants.

N6-methyladenosine (m6A) is one of the major epigenetic modifications in eukaryotes. Although increasing functions of m6A have been identified in insects, its role in Plutella xylostella L. for host plant adaptation remains unclear. In the current study, we show that the m6A content of P. xylostella was relatively low in different developmental stages and tissues, with no significant differences. Two RNA methyltransferase genes, PxMETTL3 (methyltransferase-like 3) and PxMETTL14 (methyltransferase-like 14), were identified and characterized. PxMETTL3 could be transcribed into two transcripts, and PxMETTL14 had only one transcript; both of these genes were highly expressed in egg and adult stages and reproductive tissues. The CRISPR/Cas9-mediated knockout of PxMETTL3 (ΔPxMETTL3-2) or PxMETTL14 (ΔPxMETTL14-14) confirmed their function in m6A installation into RNA. Furthermore, upon transfer from an artificial diet to the host plant, the mutant strains were affected in terms of larval and pupal weight or adult emergence rate, while the wildtype (WT) strain did not exhibit any difference. In addition, the fecundity and egg hatching rate of the WT strain decreased significantly, whereas only the ΔPxMETTL14-14 mutant strain displayed significantly decreased fecundity. There seemed to be a tradeoff between the stress adaptation and reproduction in P. xylostella mediated by m6A modification. During host transfer, the expression of PxMETTL14 was consistent with the change in m6A content, which implied that PxMETTL14 could respond to host plant defense effectively, and may regulate m6A content. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the differentially expressed transcripts with changes in m6A levels revealed that the potential functions of m6A-related genes may be involved in steroid biosynthesis for larval performance and metabolic pathways for adult reproduction. Overall, our work reveals an epigenetic regulation mechanism for the rapid adaptation of P. xylostella to variations in the host environment.

RNA methyltransferase METTL3 induces intrinsic resistance to gefitinib by combining with MET to regulate PI3K/AKT pathway in lung adenocarcinoma.

Clinical research data show that gefitinib greatly improves the progression-free survival of patients, so it is used in advanced non-small cell lung cancer patients with EGFR mutation. However, some patients with EGFR sensitive mutations do not have good effects on initial gefitinib treatment, and this mechanism is rarely studied. METTL3, a part of N6-adenosine-methyltransferase, has been reported to play an important role in a variety of tumours. In this study, we found that METTL3 is up-regulated in gefitinib-resistant tissues compared to gefitinib-sensitive tissues. Cell function experiments have proved that under the treatment of gefitinib, METTL3 knockdown promotes apoptosis and inhibits proliferation of lung cancer cells. Mechanistic studies have shown that METTL3 combines with MET and causes the PI3K/AKT signalling pathway to be manipulated, which affects the sensitivity of lung cancer cells to gefitinib. Therefore, our research shows that METTL3 can be used as a molecular marker to predict the efficacy of EGFR-TKI therapy in patients, and METTL3 may be a potential therapeutic target.

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 m62A2058. 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.

NSun2 promotes cell migration through methylating autotaxin mRNA.

NSun2 is an RNA methyltransferase introducing 5-methylcytosine into tRNAs, mRNAs, and noncoding RNAs, thereby influencing the levels or function of these RNAs. Autotaxin (ATX) is a secreted glycoprotein and is recognized as a key factor in converting lysophosphatidylcholine into lysophosphatidic acid (LPA). The ATX-LPA axis exerts multiple biological effects in cell survival, migration, proliferation, and differentiation. Here, we show that NSun2 is involved in the regulation of cell migration through methylating ATX mRNA. In the human glioma cell line U87, knockdown of NSun2 decreased ATX protein levels, whereas overexpression of NSun2 elevated ATX protein levels. However, neither overexpression nor knockdown of NSun2 altered ATX mRNA levels. Further studies revealed that NSun2 methylated the 3'-UTR of ATX mRNA at cytosine 2756 in vitro and in vivo Methylation by NSun2 enhanced ATX mRNA translation. In addition, NSun2-mediated 5-methylcytosine methylation promoted the export of ATX mRNA from nucleus to cytoplasm in an ALYREF-dependent manner. Knockdown of NSun2 suppressed the migration of U87 cells, which was rescued by the addition of LPA. In summary, we identify NSun2-mediated methylation of ATX mRNA as a novel mechanism in the regulation of ATX.