Accurate processing of human pre-rRNA in vitro.
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.
Hannon
G J
GJ
Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Maroney
P A
PA
Branch
A
A
Benenfield
B J
BJ
Robertson
H D
HD
Nilsen
T W
TW
D001483:Base Sequence; D002466:Cell Nucleolus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005095:Exoribonucleases; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009710:Nucleotide Mapping; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012335:RNA, Ribosomal / Q000096:biosynthesis*; D012337:RNA, Ribosomal, 18S / Q000096:biosynthesis*
D012322:RNA Precursors; D012335:RNA, Ribosomal; D012337:RNA, Ribosomal, 18S; D005095:Exoribonucleases; C042081:exoribonuclease T