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KMID : 0578319960060040436
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Molecules and Cells
1996 Volume.6 No. 4 p.436 ~ p.443
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U3 RNA Cap Structure, m32,2,7 GpppAmpA(m)pApG, of Novikoff Hepatoma Cell
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Ro-Choi Tae Suk
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Abstract
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U3 RNA 5´ cap structure has been analyzed from snRNA of rat Novikoff hepatoma cells. The snRNA was labeled by sodium periodate oxidation and [³H] potassium borohydride reduction, and then was subjected to preparative gel electrophoresis to purify U3 RNA. After alkaline hydrolysis/enzymatic digestions, the labeled nucleosides of U3 RNA were identified as m₃^(2,2,7)G´ (50.6%), A´ (26.6%), U´ (11.2%), G´ (4.8%) and C´ (6.8%). RNase A digestion of U3 RNA produced 3 peaks (A, C and D) by DEAE- Sephadex column chromatography. Peak D contained [³H]-m₃^(2,2,7)G´ (Tritium labeled trialcohol derivative of trimethylguanosine) indicating 3´ cis-diol is free in the intact molecule. T₁RNase digestion of peak D released one nucleotide indicating guanosine nucleotide was present prior to pyrimidine nucleotide. By high pressure liquid column chromatography, nucleoside composition of the oligonucleotide obtained by complete RNase A and T ₁digestion showed m₃^(2,2,7)G, Am, A and G in a molar ratio of 1.0:1.7:1.1:1.0. In addition, nucleases (T₂and U₂RNases) resistant 5´ fragments obtained from ^(32) P-labeled U3 RNA showed two spots on two dimensional electrophoretic separations. These nucleases (T₂and U₂RNases and alkaline phosphatase) resistant 5´-oligonucleotides were identified as cap I (llB, -3 charges) and cap II (llA, -4 charges) on DEAE-Shephadex. Since 2´-o-methylated nucleotides are resistant to enzymatic digestion, cap type I (llB) is m₃^(2,2,7)GpppAmpA and cap type II (llA) is m₃^(2,2,7)GpppAmpAmpA. From these experimental results, cap structure of U3 RNA has been deduced as m₃^(2,2,7)GpppAmpA(m)pApG.
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