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The appearance of highly abundant RNAs, U2 and 7SK, in both lanes 1 and 2 (Fig. 1A) is due to their nonspecific binding. U2 snRNA is a preferred sub-strate for polyadenylate (poly(A)] polymerase, which was used for cordycepin labeling.
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13
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32P]pCp labeling but was then removed by alkaline phosphatase (see above). Thus, these degraded RNAs, together with U4atac and U6atac RNAs, were ligated to an oligonucleotide and converted into cDNA (see above).
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By both primer extension and Northern blot analyses, we observed a short form of U4atac, which lacks the first four nucleotides; its abundance is about 1/10 that of full-length U4atac. This short U4atac is precipitable by antibodies to trimethylguanosine (TMG).
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16
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85035176801
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note
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Because intramolecular RNA stems can accommodate wobble base-pairing interactions and nucleotide bulges, it is not surprising that the nucleotides are conserved only on the U6atac strand but not on the opposite U4atac strand.
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2 of U12, which were detected in lane 6, Fig. 5C, after longer exposure 176). Why additional, weakly crosslinked sites were detected in a crosslinked species is unclear.
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85035178452
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note
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Because the majority of P120 precursor RNA. 5′ exon intermediate, and product were immunoprecipitated with an antibody to TMG (16), we conclude that the active AT-AC spliceosome was selected.
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8000.
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62
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85035180906
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note
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We are grateful to R. Reddy (Baylor College of Medicine) for providing anti-meATP and C. Guthrie and D. Brow for suggestions on Rg. 6B. We thank T. Yario for technical assistance, T. McConnell for helpful suggestions on the calculation of ΔG°. and Y.-T. Yu for sharing his protocol of cDNA construction. We also thank T. Nilsen, K. Tycowski, T. McConnell, A. Parker, and Y.-T. Yu for valuable comments on the manuscript. Supported by grant GM26154 from NIH.
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