Chemoenzymatic strategies for RNA modification and labeling

Mattay, Johanna; Dittmar, Maria; Rentmeister, Andrea

doi:10.1016/j.cbpa.2021.01.008

Cited by 13 publications

(14 citation statements)

References 65 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The cofactor binding guided the initial design of bisubstrate analogues as chemical tools to mimic the state at which both the substrate nucleoside and the SAM cofactor are bound in the catalytic pocket of the enzyme ( 14–17 ). In recent years, there have been significant efforts made toward the development of SAM analogues with the aim to study the transfer of moieties other than simple methyl groups ( 18 , 19 ), to obtain protein inhibition ( 20 ) or to study SAM-dependent DNA-methyltransferase mechanisms ( 21 , 22 ). Here, the aim is to facilitate protein/RNA crystallization to gain insights into currently poorly understood RNA nucleotide methylation processes.…”

Section: Introductionmentioning

confidence: 99%

Synthesis of RNA-cofactor conjugates and structural exploration of RNA recognition by an m6A RNA methyltransferase

Meynier

Iannazzo

Catala

et al. 2022

Nucleic Acids Research

View full text Add to dashboard Cite

Chemical synthesis of RNA conjugates has opened new strategies to study enzymatic mechanisms in RNA biology. To gain insights into poorly understood RNA nucleotide methylation processes, we developed a new method to synthesize RNA-conjugates for the study of RNA recognition and methyl-transfer mechanisms of SAM-dependent m6A RNA methyltransferases. These RNA conjugates contain a SAM cofactor analogue connected at the N6-atom of an adenosine within dinucleotides, a trinucleotide or a 13mer RNA. Our chemical route is chemo- and regio-selective and allows flexible modification of the RNA length and sequence. These compounds were used in crystallization assays with RlmJ, a bacterial m6A rRNA methyltransferase. Two crystal structures of RlmJ in complex with RNA–SAM conjugates were solved and revealed the RNA-specific recognition elements used by RlmJ to clamp the RNA substrate in its active site. From these structures, a model of a trinucleotide bound in the RlmJ active site could be built and validated by methyltransferase assays on RlmJ mutants. The methyl transfer by RlmJ could also be deduced. This study therefore shows that RNA-cofactor conjugates are potent molecular tools to explore the active site of RNA modification enzymes.

show abstract

Section: Introductionmentioning

confidence: 99%

Synthesis of RNA-cofactor conjugates and structural exploration of RNA recognition by an m6A RNA methyltransferase

Meynier

Iannazzo

Catala

et al. 2022

Nucleic Acids Research

View full text Add to dashboard Cite

show abstract

“…In vitro transcribed (IVT) RNAs are not only invaluable research tools but have also recently emerged as a new class of vaccines and therapeutics (Fuller and Berglund, 2020;Pardi et al, 2020;Jia and Qian, 2021;Pascolo, 2021). Recent discoveries in the field of epigenetic RNA modifications have uncovered several novel regulatory mechanisms (Wang et al, 2019;Huang et al, 2020;Netzband and Pager, 2020;Zhao et al, 2020), which have created a demand for simple methods providing access to chemically modified RNAs (Abele et al, 2020;Mattay et al, 2021;Ren et al, 2021). One important class of modified RNAs is RNAs specifically derivatized at the 5′ end.…”

Section: Introductionmentioning

confidence: 99%

Preparation of RNAs with non-canonical 5′ ends using novel di- and trinucleotide reagents for co-transcriptional capping

Depaix

Grudzien-Nogalska

Fedorczyk

et al. 2022

Front. Mol. Biosci.

View full text Add to dashboard Cite

Many eukaryotic and some bacterial RNAs are modified at the 5′ end by the addition of cap structures. In addition to the classic 7-methylguanosine 5′ cap in eukaryotic mRNA, several non-canonical caps have recently been identified, including NAD-linked, FAD-linked, and UDP-glucose-linked RNAs. However, studies of the biochemical properties of these caps are impaired by the limited access to in vitro transcribed RNA probes of high quality, as the typical capping efficiencies with NAD or FAD dinucleotides achieved in the presence of T7 polymerase rarely exceed 50%, and pyrimidine derivatives are not incorporated because of promoter sequence limitations. To address this issue, we developed a series of di- and trinucleotide capping reagents and in vitro transcription conditions to provide straightforward access to unconventionally capped RNAs with improved 5′-end homogeneity. We show that because of the transcription start site flexibility of T7 polymerase, R1ppApG-type structures (where R1 is either nicotinamide riboside or riboflavin) are efficiently incorporated into RNA during transcription from dsDNA templates containing both φ 6.5 and φ 2.5 promoters and enable high capping efficiencies (∼90%). Moreover, uridine-initiated RNAs are accessible by transcription from templates containing the φ 6.5 promoter performed in the presence of R2ppUpG-type initiating nucleotides (where R2 is a sugar or phosphate moiety). We successfully employed this strategy to obtain several nucleotide-sugar-capped and uncapped RNAs. The capping reagents developed herein provide easy access to chemical probes to elucidate the biological roles of non-canonical RNA 5′ capping.

show abstract

“…This provides a reactive handle on the nascent RNA for subsequent chemical modification. 12 Methytransferases (MTases) are one such class of enzymes, which transfer a methyl group from S-adenosyl-L-methionine (SAM) to nucleophilic sites on RNA. Several MTases have been reported to accept modified SAM analogues for transfer of chemical handles at different positions such as 2′-OH of all nucleosides, 15,16 N 6 of adenosine, 17 and N 2 of guanosine 18 in RNA (Figure 2A).…”

Section: Post-transcriptional Rna Labelingmentioning

confidence: 99%

“…This can be achieved by the incorporation of a chemical handle onto nascent RNA, either by post-transcriptional approaches using RNA processing enzymes or cotranscriptionally by incorporation of modified nucleosides into RNA by RNA polymerases. 12 In both approaches, the incorporated chemical handle and subsequent conjugation enable selective labeling of nascent RNA. Since these methods rely on the endogenous metabolic machinery of the cells for labeling of nascent RNA, they are called RNA metabolic labeling methods.…”

Section: Introductionmentioning

confidence: 99%

Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides

2022

View full text Add to dashboard Cite

Conspectus The discovery of previously unknown functional roles of RNA in biological systems has led to increased interest in revealing novel RNA molecules as therapeutic targets and the development of tools to better understand the role of RNA in cells. RNA metabolic labeling broadens the scope of studying RNA by incorporating of unnatural nucleobases and nucleosides with bioorthogonal handles that can be utilized for chemical modification of newly synthesized cellular RNA. Such labeling of RNA provides access to applications including measurement of the rates of synthesis and decay of RNA, cellular imaging for RNA localization, and selective enrichment of nascent RNA from the total RNA pool. Several unnatural nucleosides and nucleobases have been shown to be incorporated into RNA by endogenous RNA synthesis machinery of the cells. RNA metabolic labeling can also be performed in a cell-specific manner, where only cells expressing an essential enzyme incorporate the unnatural nucleobase into their RNA. Although several discoveries have been enabled by the current RNA metabolic labeling methods, some key challenges still exist: (i) toxicity of unnatural analogues, (ii) lack of RNA-compatible conjugation chemistries, and (iii) background incorporation of modified analogues in cell-specific RNA metabolic labeling. In this Account, we showcase work done in our laboratory to overcome these challenges faced by RNA metabolic labeling. To begin, we discuss the cellular pathways that have been utilized to perform RNA metabolic labeling and study the interaction between nucleosides and nucleoside kinases. Then we discuss the use of vinyl nucleosides for metabolic labeling and demonstrate the low toxicity of 5-vinyluridine (5-VUrd) compared to other widely used nucleosides. Next, we discuss cell-specific RNA metabolic labeling with unnatural nucleobases, which requires the expression of a specific phosphoribosyl transferase (PRT) enzyme for incorporation of the nucleobase into RNA. In the course of this work, we discovered the enzyme uridine monophosphate synthase (UMPS), which is responsible for nonspecific labeling with modified uracil nucleobases. We were able to overcome this background labeling by discovering a mutant uracil PRT (UPRT) that demonstrates highly specific RNA metabolic labeling with 5-vinyluracil (5-VU). Furthermore, we discuss the optimization of inverse-electron-demand Diels–Alder (IEDDA) reactions for performing chemical modification of vinyl nucleosides to achieve covalent conjugation of RNA without transcript degradation. Finally, we highlight our latest endeavor: the development of mutually orthogonal chemical reactions for selective labeling of 5-VUrd and 2-vinyladenosine (2-VAdo), which allows for potential use of multiple vinyl nucleosides for simultaneous investigation of multiple cellular processes involving RNA. We hope that our methods and discoveries encourage scientists studying biological systems to include RNA metabolic labeling in their toolkit for studying RNA and its role in biological...

show abstract

Chemoenzymatic strategies for RNA modification and labeling

Cited by 13 publications

References 65 publications

Synthesis of RNA-cofactor conjugates and structural exploration of RNA recognition by an m6A RNA methyltransferase

Synthesis of RNA-cofactor conjugates and structural exploration of RNA recognition by an m6A RNA methyltransferase

Preparation of RNAs with non-canonical 5′ ends using novel di- and trinucleotide reagents for co-transcriptional capping

Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides

Contact Info

Product

Resources

About