Accurate processing of human pre-rRNA in vitro.

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Abstract
We report here that the mature 59 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 59 end of 18S rRNA and the minor cleavage site 1 base into the 18S sequence. All three cleavages yielded 59-hydroxyl, 29-39-cyclic phosphate termini and were 59 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 59 terminus identical to that of mature 18S RNA by an activity found in HeLa cell cytoplasmic extracts. Images