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

To show that T4 polynucleotide kinase does not preferentially label m 6 Am or Am, the RNA oligonucleotides SK-524 and SK-526 were mixed in different ratios, end labelled, digested with P1 nuclease and separated by TLC. The spots corresponding to pm 6 Am and pAm were then quantified using phosphorimaging. The results demonstrated that both nucleotides are labelled with equal efficiency by T4 polynucleotide kinase (Fig. 3) Labelling and analysis of the first cap adjecent nucleotide. To label the first nucleotide following the m 7 G, poly(A) RNA was prepared from various mouse organs then digested with tobacco acid pyrophosphatase to remove the m 7 G cap. The exposed 5' ends were dephosphorylated with alkaline phosphatase and after phenol/chloroform extraction and ethanol precipitation, the mRNA transcripts were radiolabelled at their 5' end using T4 polynucleotide kinase in the presence of .20 fold excess of [c-32 P] ATP. The labelled RNA was digested to monophospho-nucleotides by P1 nuclease prior to TLC separation. Using this method, new spots corresponding to the 2'-O methylated nucleotides are apparent in the labelled samples after cap removal (Fig 4A) . A spot corresponding to pm 6 Am is readily detectable in all mRNA samples tested, and with pm 6 Am:pAm ratios of between 15:1 (brain) and 2:1 (liver) it appears that m 6 Am is more prevalent at the cap1 than is Am (Fig. 4A ). Under these labelling conditions, where both ATP and polynucleotide kinase are in excess, the intensity of the spots corresponding to the unmodified Analysis of cap structures for mRNA transcripts from individual genes. In order to assay modifications on the first transcribed nucleotide for messages from individual genes, mRNA from liver and testis was de-capped and end-labelled as described above then fragmented to ,120 nt. This was then hybridised to single stranded DNA targets corresponding to the 5' region of selected messages. These DNA targets were first cross-linked to 2 mm 3 2 mm teeth cut from a Hybond N 1 membrane. After hybridization and washing, the membrane was subjected to phosphorimaging (Fig. 4B) . The individual teeth containing the labelled mRNA gene-specific fragments were then removed and digested to nucleotide 5' monophosphates using P1 nuclease. These samples were individually spotted onto TLC plates and developed as described (Fig. 4B ). Four mRNAs were chosen for analysis, apolipoprotein A-I (Apoa1, BC012253), albumin (Alb, BC024643), protamine 2 (Prm2, BC049612) and poly(A) binding protein cytoplasmic 1 (Pabpc1, BC046233). Alb and Apoa1 are predominantly liver expressed messages, whereas Prm2 shows testis specific expression and Pabpc1 is expressed highly in both organs. Pabpc1 may be subjected to translational regulation, but unlike Pabpc2, it is present in actively translating polyribosomes of mouse testicular cells 31 . For both Alb and Apoa1, the labelled nucleotide in the cap adjacent position included A, G, U and C, as well as m 6 Am and Am. This is consistent with multiple alternative transcription start sites at and around the Inr 32 . In both cases, Am and m 6 Am appeared as the major nucleotide modifications; unlike the mixed mRNA starting material, 2'-O-methylcytosine was not present (compare Fig. 4A, B) . The m 6 Am:Am ratios were 1.65:1 and 1.4:1 for Apoa1 and Alb respectively, which is only slightly lower compared to that seen for the liver mRNA population as a whole. The Prm2 mRNAs from testis tissues almost exclusively had