Processing of Mammalian rRNA Precursors at the 3′ End of 18S rRNA

Abstract

Molecular mechanisms involved in the nucleolytic cleavage at the 18S rRNA/internal transcribed spacer 1 (ITS 1) junction, a late step of small-subunit pre-rRNA processing in vertebrates, remain largely unknown, mostly due to the lack of faithful in vitro assays. To identify the minimal cis-acting signals required for this reaction, we studied the processing of truncated human rRNA gene transcripts transiently expressed upon transfection of rRNA minigenes into cultured mouse cells. We observed that processing at this site was faithfully reproduced with transcripts containing only 60 nucleotides of 18S rRNA and the adjacent 103 nucleotides of ITS 1, but was abolished or severely altered by further shortening of either sequence. Remarkably, this minimal transcript contains, within its 18S rRNA part, long sequences complementary to both U20 and U13 small nucleolar RNAs (snoRNAs). The cis-acting elements essential for the reaction were studied further by site-directed mutagenesis. The U20 snoRNA complementary region in 18S rRNA was not required for faithful processing at the 18S rRNA/ITS 1 junction. Also, processing at this site was not appreciably altered by random substitution of proximal ITS 1 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 (methyl)2A1850.(methyl)2A1851 doublet was not required for processing at this site. Two highly conserved 18S rRNA elements acted as major cis-acting signals for processing at the 3' end, the CAUU sequence immediately preceding the 3'-terminal nucleotide and the 3' strand of the 3'-terminal 18S rRNA helix, complementary to U13 snoRNA. Compensatory mutations, restoring the potential for helix formation, but not U13 snoRNA complementarity, did not restitute the cleavage at the 3' end of 18S rRNA. This suggests that U13 snoRNA may be a trans-acting factor in the nucleolytic cleavage at the 3' end of 18S rRNA.