An enzyme-coupled high-throughput assay for screening RNA methyltransferase activity in<i>E. Coli</i>cell lysate

Schulz, Daniela; Rentmeister, Andrea

doi:10.4161/rna.19818

Cited by 18 publications

(21 citation statements)

References 39 publications

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“…The coproduct, S ‐adenosylhomocysteine (SAH), inhibits methyltransferases, but can be enzymatically degraded in the same pot . To produce the required AdoMet analogues in situ, we explored the substrate promiscuity of recombinantly produced human MATIIa I117A (MAT‐Var), which was reported to accept longer alkyl side chains bearing methionine derivatives (see Figure S1 in the Supporting Information) .…”

Section: Methodsmentioning

confidence: 99%

“…[4a,d, 13] Thec oproduct, S-adenosylhomocysteine (SAH), inhibits methyltransferases,but can be enzymatically degraded in the same pot. [14] To produce the required AdoMet analogues in situ, we explored the substrate promiscuity of recombinantly produced human MATIIa I117A (MAT-Va r), which was reported to accept longer alkyl side chains bearing methionine derivatives (see Figure S1 in the Supporting Information). [12b] Since MATs hows strong product inhibition, [8,9b] we set up an enzyme-coupled reaction to directly consume AdoMet (or its analogues) and label the mRNA 5' cap (Scheme 1).…”

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confidence: 99%

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A Biocatalytic Cascade for Versatile One‐Pot Modification of mRNA Starting from Methionine Analogues

Muttach

Rentmeister

2015

Angew Chem Int Ed

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Methyltransferases have proven useful to install functional groups site-specifically in different classes of biomolecules when analogues of their cosubstrate S-adenosyl-l-methionine (AdoMet) are available. Methyltransferases have been used to address different classes of RNA molecules selectively and site-specifically, which is indispensable for biophysical and mechanistic studies as well as labeling in the complex cellular environment. However, the AdoMet analogues are not cell-permeable, thus preventing implementation of this strategy in cells. We present a two-step enzymatic cascade for site-specific mRNA modification starting from stable methionine analogues. Our approach combines the enzymatic synthesis of AdoMet with modification of the 5' cap by a specific RNA methyltransferase in one pot. We demonstrate that a substrate panel including alkene, alkyne, and azido functionalities can be used and further derivatized in different types of click reactions.

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

A Biocatalytic Cascade for Versatile One‐Pot Modification of mRNA Starting from Methionine Analogues

Muttach

Rentmeister

2015

Angew Chem Int Ed

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“…Recombinant human TGS1 (hTGS1) was expressed and purified as previously described 56,57 . Briefly, E. coli Tuner cells transformed with pRSET-A-hTGS1 618-853 were grown at 37 °C in 2YT medium until OD 600 of 0.6 and induced with 0.2 mM IPTG.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

FTO controls reversible m6Am RNA methylation during snRNA biogenesis

et al. 2019

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Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. Here we show that snRNAs contain a regulated and reversible nucleotide modification causing them to exist as two different methyl isoforms, m 1 and m 2 , reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m 1-isoform with a single-methylated adenosine (2'-O-methyladenosine, Am), which is then converted to a dimethylated m 2-isoform (N 6 ,2'-O-dimethyladenosine, m 6 Am). The relative m 1-and m 2-isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m 2-isoform. We show FTO is inhibited by the oncometabolite D-2hydroxyglutarate, resulting in increased m 2-snRNA levels. Furthermore, cells that exhibit high m 2-snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO controls a previously unknown central step of snRNA processing involving reversible methylation, and suggest that epitranscriptomic information in snRNA may influence mRNA splicing.

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“…GlaTgs2-Var1, MTAN and LuxS were recombinantly produced and purified as previously described. 11 , 12 , 56 …”

Section: Methodsmentioning

confidence: 99%

Enzymatic modification of 5′-capped RNA with a 4-vinylbenzyl group provides a platform for photoclick and inverse electron-demand Diels–Alder reaction

2015

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An enzyme-coupled high-throughput assay for screening RNA methyltransferase activity inE. Colicell lysate

Cited by 18 publications

References 39 publications

A Biocatalytic Cascade for Versatile One‐Pot Modification of mRNA Starting from Methionine Analogues

A Biocatalytic Cascade for Versatile One‐Pot Modification of mRNA Starting from Methionine Analogues

FTO controls reversible m6Am RNA methylation during snRNA biogenesis

Enzymatic modification of 5′-capped RNA with a 4-vinylbenzyl group provides a platform for photoclick and inverse electron-demand Diels–Alder reaction

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