Methyltransferase-Glo: A Universal, Bioluminescent and Homogenous Assay for Monitoring all Classes of Methyltransferases

Hsiao, Kevin; Zegzouti, Hicham; Goueli, Said A.

doi:10.2217/epi.15.113

Cited by 83 publications

(95 citation statements)

References 41 publications

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“…The reactions were allowed to proceed at room temperature for indicated durations (5, 10, 15 and 20 min), and were terminated by the addition of TFA (Trifluoroacetic acid) to a final concentration of 0.1% (v/v). Then, 5 μl of reaction mixture was transferred to a low-volume 384-well plate, and the methylation activity was measured using the Promega bioluminescence assay (MTase-Glo TM ) (47). The bioluminescence assay was performed according to the manufacturer's protocol, in which the by-product SAH of the methylation reaction is converted into ATP in a two-step reaction and ATP is detected through a luciferase reaction.…”

Section: Steady-state Kinetic Measurementmentioning

confidence: 99%

METTL5, an 18S rRNA-specific m⁶A methyltransferase, modulates expression of stress response genes

Chen

Liu

et al. 2020

Preprint

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Tel. (617)919-3100.Running title: m 6 A on 18S rRNA is important for selective mRNA translation Abstract RNA N 6 -methyladenosine (m 6 A) modification is present in different RNA molecules, including protein-coding mRNAs and non-coding RNAs such as ribosomal RNAs (rRNAs). Previous studies identified m 6 A in both the 18S and 28S rRNAs, but the roles of these methylation events are poorly understood due to the lack of knowledge of the responsible methyltransferases. Here, we report that mammalian METTL5, a member of a highly conserved methyltransferase family, specifically methylates adenosine 1832 (A1832) in the 18S rRNA in vivo and in vitro. We identify TRMT112 as a near stoichiometric partner of METTL5 important for the enzymatic activity of METTL5. By mapping the positions of translating ribosomes (Ribo-seq), we found translation of multiple stress response-related mRNAs, including Atf4 mRNA, is selectively reduced in the Mettl5 knockout (KO) mouse B16 melanoma cells. Atf4 is a key transcription factor that mediates the Integrated Stress Response (ISR), as exemplified by the Endoplasmic Reticulum (ER) stress. Consistently, transcription of ISR effector genes is reduced in Mettl5 KO cells during ER stress, suggesting a compromised ISR. Our findings reveal a new mechanism that regulates expression of stress response genes and suggest that chemical modifications of ribosomal RNAs may play a key role in selectively impacting translation and possibly ISR.

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Section: Steady-state Kinetic Measurementmentioning

confidence: 99%

METTL5, an 18S rRNA-specific m⁶A methyltransferase, modulates expression of stress response genes

Chen

Liu

et al. 2020

Preprint

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“…1B). The MTase-Glo assay reagent measures methyltransferase activity through the coupling of the NSD2 reaction product, S-adenosyl-Lhomocysteine (SAH) to a bioluminescent signal (44). The assay was further optimized for the NSD2 mutants E1099K (Fig.…”

Section: Primary Assay Development and Optimizationmentioning

confidence: 99%

“…Of the 289 cherry picks, the activities of 48 were confirmed against NSD2 with the primary and orthogonal assays with no activity observed by the counter assays. The majority of these compounds were also active against the NSD2 mutants E1099K (45) and T1150A (44).…”

Section: Quantitative High-throughput Screen (Qhts) For Nsd2 Inhibitorsmentioning

confidence: 99%

“…4A). The compound shares structural commonality with the firefly luciferase inhibitor PTC-124 (44,50,51). As such it was likely a false-positive hit that inhibits the UltraGlo luciferase enzyme utilized in the MTase-Glo assay.…”

Section: Hit Confirmation and Secondary Screeningmentioning

confidence: 99%

“…Here we report biochemical assay development and pilot-scale screening using full-length recombinant wildtype, E1099K, and T1150A NSD2 enzymes with purified HeLa nucleosomes as a substrate. Chemical libraries were screened in 3-dose point quantitative high-throughput screening (qHTS) format (43) with the Methyltransferase-Glo (MTase-Glo™) methyltransferase bioluminescence assay (44). A number of counter and orthogonal assays were also developed to characterize the hits from the primary screen.…”

Section: Introductionmentioning

confidence: 99%

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Small Molecule Inhibitors of the Human Histone Lysine Methyltransferase NSD2 / WHSC1 / MMSET Identified from a Quantitative High-Throughput Screen with Nucleosome Substrate

Henderson

et al. 2017

Preprint

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The activity of the histone lysine methyltransferase NSD2 is thought to play a driving role in oncogenesis. Both overexpression of NSD2 and point mutations that increase its catalytic activity are associated with a variety of human cancers. While NSD2 is an attractive therapeutic target, no potent, selective and cell-active inhibitors have been reported to date, possibly due to the challenging nature of developing high-throughput assays for NSD2. To establish a platform for the discovery and development of selective NSD2 inhibitors, multiple assays were optimized and implemented. Quantitative high-throughput screening was performed with full-length wildtype NSD2 and a nucleosome substrate against a diverse collection of known bioactives comprising 16,251 compounds. Actives from the primary screen were further interrogated with orthogonal and counter assays, as well as activity assays with the clinically relevant NSD2 mutants E1099K and T1150A. Five confirmed inhibitors were selected for follow-up, which included a radiolabeled validation assay, surface plasmon resonance studies, methyltransferase profiling, and histone methylation in cells. The identification of NSD2 inhibitors that bind the catalytic SET domain and demonstrate activity in cells validates the workflow, providing a template for identifying selective NSD2 inhibitors.

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Biokatalytische C3‐Indol‐Methylierung – ein nützliches Werkzeug für die naturstoffinspirierte stereoselektive Synthese von Pyrroloindolen

et al. 2021

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zu finden. 2021 Die Autoren. AngewandteChemie verçffentlichtv on Wiley-VCH GmbH. Dieser Open Access Beitrag steht unter den Bedingungen der Creative Commons AttributionN on-CommercialN o-Derivs License, die eine Nutzung und Verbreitung in allen Medien gestattet, sofern der ursprüngliche Beitrag ordnungsgemäßzitiert und nicht fürkommerzielle Zwecke genutzt wird und keine ¾nderungen und Anpassungen vorgenommenwerden.

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Methyltransferase-Glo: A Universal, Bioluminescent and Homogenous Assay for Monitoring all Classes of Methyltransferases

Cited by 83 publications

References 41 publications

METTL5, an 18S rRNA-specific m⁶A methyltransferase, modulates expression of stress response genes

METTL5, an 18S rRNA-specific m⁶A methyltransferase, modulates expression of stress response genes

Small Molecule Inhibitors of the Human Histone Lysine Methyltransferase NSD2 / WHSC1 / MMSET Identified from a Quantitative High-Throughput Screen with Nucleosome Substrate

Biokatalytische C3‐Indol‐Methylierung – ein nützliches Werkzeug für die naturstoffinspirierte stereoselektive Synthese von Pyrroloindolen

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Methyltransferase-Glo: A Universal, Bioluminescent and Homogenous Assay for Monitoring all Classes of Methyltransferases

Cited by 83 publications

References 41 publications

METTL5, an 18S rRNA-specific m6A methyltransferase, modulates expression of stress response genes

METTL5, an 18S rRNA-specific m6A methyltransferase, modulates expression of stress response genes

Small Molecule Inhibitors of the Human Histone Lysine Methyltransferase NSD2 / WHSC1 / MMSET Identified from a Quantitative High-Throughput Screen with Nucleosome Substrate

Biokatalytische C3‐Indol‐Methylierung – ein nützliches Werkzeug für die naturstoffinspirierte stereoselektive Synthese von Pyrroloindolen

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METTL5, an 18S rRNA-specific m⁶A methyltransferase, modulates expression of stress response genes

METTL5, an 18S rRNA-specific m⁶A methyltransferase, modulates expression of stress response genes