A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA

Kruse, Susanne; Zhong, Silin; Bodi, Zsuzsanna; Button, James; Alcocer, Marcos J. C.; Hayes, Christopher J.; Fray, Rupert

I n most eukaryotes, polymerase II (Pol II) dependent transcripts are modified co-transcriptionally at their 5'end by the addition of a 7-methyl guanosine (m 7 G) cap to the first nucleoside of the nascent transcript. If no additional modifications are made to the cap-adjacent nucleotides, the structure is referred to as a cap0. In yeast and plants, only cap0 structures are found, however, in animals, modifications of the two nucleotides adjacent to the m 7 G are possible The methylation of the first nucleotide on the ribose residue ( Fig. 1a) will form a cap1 structure 1 . Cap1 messages can be converted to cap2 structures if a further 2'ribose methylation takes place on the next nucleotide following the cap1 (Fig. 1b) . These methylation steps are sequential and carried out by nuclear located methylases [1] [2] [3] . In a transcript where the first nucleotide is an adenosine in a cap1 structure, a further methylation of the 2'-O-dimethyladenosine (Am) at the N 6 position of the adenine can take place to give N 6 ,2'-O-dimethyladenosine (m 6 Am) (Fig. 1c) 4 . The methyltransferase that carries out this modification has been partially characterized and appears to be predominantly located in the cytoplasm 5 . Transcription start sites of focused promoters are usually contained within an initiator motif (Inr) of general sequence YYANWYY (Y5pyrimidine, N-any nucleotide, W5A or T) where the A is the principal start site and neighbouring nucleotides may be used to varying degrees 6 . Since, A is often the first nucleotide after the m 7 G cap, its modification may have a functional role. In order to study the effect of m 6 Am present in cap1 messages, the chemical synthesis of RNA oligonucleotide sequences that contain m 6 Am in well-defined positions, is necessary. A number of methods exist in the literature for the preparation of N 6 -methylated adenosine derivatives, with the Dimroth rearrangement being perhaps the most well known [7] [8] [9] . This transformation relies upon an initial N1-methylation of the adenine ring followed by an alkali-mediated rearrangement to give the N6-methylated adenine product. An alternative method for accessing N6-alkylated adenines has been developed, which involves the nucleophilic aromatic substitution of adenine derivitives, that are activated at the 6-position, with amine nucleophiles (i.e. MeNH 2 ) [10] [11] [12] [13] . In order to obtain the N6-methylated phosphoramidite reagents from these adenine derivatives, a number of additional synthetic steps are required, which results in a 6 to 8 step synthesis being needed to produce each phosphoramidite reagent [10] [11] [12] [13] . Such approaches are not realistic other than in synthetic chemistry laboratories.