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. 2011:1:126.
doi: 10.1038/srep00126. Epub 2011 Oct 24.

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

Susanne Kruse  1 , Silin Zhong, Zsuzsanna Bodi, James Button, Marcos J C Alcocer, Christopher J Hayes, Rupert Fray
Affiliations

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

Susanne Kruse et al. Sci Rep. 2011.

Abstract

A method is described for the detection of certain nucleotide modifications adjacent to the 5' 7-methyl guanosine cap of mRNAs from individual genes. The method quantitatively measures the relative abundance of 2'-O-methyl and N(6),2'-O-dimethyladenosine, two of the most common modifications. In order to identify and quantitatify the amounts of N(6),2'-O-dimethyladenosine, a novel method for the synthesis of modified adenosine phosphoramidites was developed. This method is a one step synthesis and the product can directly be used for the production of N(6),2'-O-dimethyladenosine containing RNA oligonucleotides. The nature of the cap-adjacent nucleotides were shown to be characteristic for mRNAs from individual genes transcribed in liver and testis.

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Figures

Figure 1
Figure 1. Cap adjacent modification in higher Eukaryotes.
Formation of the cap1 structure takes place by 2'-O –methylation on the ribose residue on the first nucleotide adjacent to the m7G cap. The example shows a structure with adenosine as the first nucleotide, adjacent to the m7G cap (a). The cap1 structure can then be converted to a cap2 by a further 2'-O-ribose methylation on the following nucleotide (b), or converted to a cap1 m6Am by placing a further methyl group on the adenosine in the N6 position (c). The figure is a schematic representation of the mRNA cap structures. The yellow structure represents 7-methyl-guanine, the green represents adenine and the grey pentagon is ribose. On the ribose only the methylated functional groups are shown.
Figure 2
Figure 2. Relative mobility of modified adenosines using 2 dimensional thin layer chromatography.
RNA oligonucleotide fragments of known compositions were labelled at their 5' ends with32P, digested to 5' monophosphates and separated by 2 dimensional thin layer chromatography in order to establish the mobility of pm6Am relative to other nucleotides. Adenosine and its methylated derivatives are indicated by shading.
Figure 3
Figure 3. T4 polynucleotide kinase does not preferentially label m6Am or Am containing ends.
The oligonucleotides SK-526 (m6Am) and SK-524 (Am) were mixed in ratios of 5:1, 2:1, 1:1. 1:2 and 1:5, end labelled using T4 polynucleotide kinase, digested to 5' phosphate mononucleotides and separated by TLC. The relative intensities of the resulting spots were quantified using phosphorimaging. The calculated percentage of m6Am closely matched the actual m6Am percentage.
Figure 4
Figure 4. Relative abundance of modified adenosines in mRNA from different mouse organs and in individual mRNAs.
(A) 2'-O-methylated nucleotides are present in the mRNA populations from various mouse organs. Arrow indicates m6Am. Different organs have characteristic m6Am:Am ratios; brain (15:1), kidney (2.4:1), liver (2:1), testis (10:1). (B) Transcripts from individual genes have characteristic nucleotides or nucleotide modifications at the first nucleotide position; Pabpc1 (poly(A) binding protein cytoplasamic 1), Apoa1 (apolipoprotein A-I), Prm2, (protamine 2), Alb (albumin). (C) Schematic indicating the relative position of the nucleotide spots (left) and the location of the cold single stranded target sequences on the hybridisation template (right). Single stranded DNA from the yeast gene IME2 was used as a negative control DNA in the hybridisations.
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