CAPturAM, a Chemo‐Enzymatic Strategy for Selective Enrichment and Detection of Physiological CAPAM‐Targets

Abstract

Modified nucleotides impact all aspects of eukaryotic mRNAs and contribute to regulation of gene expression at the transcriptional and translational level. At the 5' cap, adenosine as first transcribed nucleotide is often N 6 -methyl-2'-O-methyl adenosine (m 6 A m ). This modification is tissue dependent and reversible, pointing to a regulatory function. CAPAM was recently identified as methyltransferase responsible for m 6 A m formation, however, the direct assignment of its target transcripts proves difficult. Antibodies do not discriminate between internal N 6 -methyl adenosine (m 6 A) and m 6 A m . Here we present CAPturAM, an antibody-free chemical biology approach for direct enrichment and probing of physiological CAPAM-targets. We harness CAPAM's cosubstrate promiscuity to install propargyl groups on its targets. Subsequent functionalization with an affinity handle allows for their enrichment. Using wildtype and CAPAM À /À cells, we successfully applied CAPturAM to confirm or disprove CAPAM-targets, facilitating the verification and identification of CAPAM targets.

Trinucleotide mRNA Cap Analogue N6-Benzylated at the Site of Posttranscriptional m6Am Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo

“…It may serve as a basis for dynamic translation regulation mechanisms, relying on the addition and removal of the methyl group by m 6 A writers and erasers. , N -Methyltransferase Pcif1/CAPAM has been the only so far identified mRNA cap-specific m6 A writer, − while FTO has been identified as the m6 A eraser for both internal m6 A and m6 A m within the 5′ cap (Figure B). , However, the biological effects of m 6 A m and underlying molecular mechanisms are still under debate since no cap-specific m6 A m reader has been identified. Interestingly, the N 6-methylation of adenosine as FTN is common in all types of mammalian cells and has been found to be relatively abundant in several tissues: e.g., in mice, the fraction of N 6 - methylated 5′ terminal A reaches around 70% in the liver, 90% in the heart, and 94% in the brain. , The large differences in the m6 A m content observed in transcripts from different genes also support this modification’s putative regulatory role. , …”

“…Synthetic modifications of mRNA 5′ cap have already proven to be an effective way to modulate translation efficiency of exogenously delivered transcripts. , However, unnatural modifications of the first transcribed nucleotide have been rarely explored so far. , This is mostly because the typical protocols for the preparation of capped mRNA utilize dinucleotide cap analogues, which are not compatible with the majority of modifications at the FTN position . However, recently developed tri- and tetranucleotide capping reagents overcome these limitations. ,, Here, we aimed to develop a capping reagent introducing an m6 A m mimic at the mRNA 5′ cap that would potentially retain the majority of its properties but was resistant to demethylation by FTO.…”

Trinucleotide mRNA cap analog N6-benzylated at the site of posttranscriptionalm6Am mark facilitates mRNA purification and confers superior translational properties in vitro and in vivo

“…17, 18 The large differences in m6 Am content observed in transcripts from different genes also support this modification’s putative regulatory role. 19, 20…”

“…31, 32 However, unnatural modifications of the first transcribed nucleotide have been rarely explored so far. 19 This is mostly because the typical protocols for the preparation of capped mRNA utilize dinucleotide cap analogs, which are not compatible with the majority of modifications at the FTN position. 33 However, recently developed tri- and tetranucleotide capping reagents overcome these limitations.…”

“…17,18 The large differences in m6 Am content observed in transcripts from different genes also support this modification's putative regulatory role. 19,20 Over the past few years, the molecular effect of the m6 Am modification on mRNA properties has been the subject of a lively scientific debate, with some conflicting data often resulting from the difficulty of separating the effects of 5′ cap-adjacent m6 Am and internal m6 A modifications. Most of the researchers agree that the level of cap-adjacent m6 Am positively correlates with mRNA translation rate, 9 although that effect seems to be cell-line or tissue-dependent.…”