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Accurate Processing of Human Pre-rRNA In Vitro

1989; Taylor & Francis; Volume: 9; Issue: 10 Linguagem: Inglês

10.1128/mcb.9.10.4422-4431.1989

1098-5549

Gregory J. Hannon, Patricia A. Maroney, Andrea D. Branch, Bonnie J. Benenfield, Hugh D. Robertson, Timothy W. Nilsen,

RNA Research and Splicing

We report here that the mature 5' terminus of human 18S rRNA is generated in vitro by a two-step processing reaction. In the first step, SP6 transcripts were specifically cleaved in HeLa cell nucleolar extract at three positions near the external transcribed spacer (ETS)-18S boundary. Of these cleavage sites, two were major and the other was minor. RNase T1 fingerprint and secondary nuclease analyses placed the two major cleavage sites 3 and 8 bases upstream from the mature 5' end of 18S rRNA and the minor cleavage site 1 base into the 18S sequence. All three cleavages yielded 5'-hydroxyl, 2'-3'-cyclic phosphate termini and were 5' of adenosine residues in the sequence UACCU, which was repeated three times near the ETS-18S boundary. In the second step, the initial cleavage product containing 3 bases of ETS was converted to an RNA with a 5' terminus identical to that of mature 18S RNA by an activity found in HeLa cell cytoplasmic extracts.