PROCESSING OF MAMMALIAN RIBOSOMAL-RNA PRECURSORS AT THE 3'-END OF 18SRIBOSOMAL-RNA - IDENTIFICATION OF CIS-ACTING SIGNALS SUGGESTS THE INVOLVEMENT OF U13 SMALL NUCLEOLAR RNA

Molecular mechanisms involved in the nucleolytic cleavage at the 18S r RNA/internal transcribed spacer 1 (ITS 1) junction, a late step of sma ll-subunit pre-rRNA processing in vertebrates, remain largely unknown, mostly due to the lack of faithful ia vitro assays. To identify the m inimal cis-acting signals required for this reaction, we studied the p rocessing of truncated human rRNA gene transcripts transiently express ed upon transfection of rRNA minigenes into cultured mouse cells. We o bserved that processing at this site was faithfully reproduced with tr anscripts containing only 60 nucleotides of 18S rRNA and the adjacent 103 nucleotides of ITS 1, but was abolished or severely altered by fur ther shortening of either sequence. Remarkably, this minimal transcrip t contains, within its 18S rRNA part, long sequences complementary to both U20 and U13 small nucleolar RNAs (snoRNAs). The cis-acting elemen ts essential for the reaction were studied further by site-directed mu tagenesis. The U20 snoRNA complementary region in 18S rRNA was not req uired for faithful processing at the 18S rRNA/ITS 1 junction. Also, pr ofessing at this site was not appreciably altered by random substituti on of proximal ITS I sequences (including the 5' terminal nucleotide) or of the terminal nucleotide of mature 18S rRNA. Substitutions in the four-nucleotide loop of the 18S rRNA 3'-terminal stem-loop, including the two adenosine residues substrates of dimethylation, did not alter appreciably the formation of the 18S rRNA 3' end, showing that the (m ethyl)(2)A1850 .(methyl)(2)A1851 doubler was not required for processi ng at this site. Two highly conserved 18S rRNA elements acted as major cia-acting signals for processing at the 3' end, the CAUU sequence im mediately preceding the 3'-terminal nucleotide and the 3' strand of th e 3'-terminal 18S rRNA helix, complementary to U13 snoRNA. Compensator y mutations, restoring the potential for helix formation, but not U13 snoRNA complementarity, did not restitute the cleavage at the 3' end o f 18S rRNA. This suggests that U13 snoRNA may be a terms-acting factor in the nucleolytic cleavage at the 3' end of 18S rRNA.