The spacing between functional cis-elements of U3 snoRNA is critical for rRNA processing

Abstract

The sequences and structural features of Xenopus laevis U3 small nucleolar RNA (snoRNA) necessary for pre-rRNA cleavage at sites 1 and 2 to form 18 S rRNA were assayed by depletion/rescue experiments in Xenopus oocytes. Mutagenesis results demonstrated that the putative stem of U3 domain I is unnecessary for 18 S rRNA processing. A model consistent with earlier experimental data is proposed for the structure of domain I when U3 is not yet bound to pre-rRNA. For its function in rRNA processing, a newly discovered element (5′ hinge) was revealed to be important but not as critical as the 3′ hinge region in Xenopus U3 snoRNA for 18 S rRNA formation. Base-pairing is proposed to occur between the U3 5′ hinge and 3′ hinge and complementary regions in the external transcribed spacer (ETS); these interactions are phylogenetically conserved, and are homologous to those previously described in yeast (5′ hinge-ETS) and trypanosomes (3′ hinge-ETS). A model is presented where the base-pairing of the 5′ hinge and 3′ hinge of U3 snoRNA with the ETS of pre-rRNA helps to correctly position U3 boxes A′ + A for their function in rRNA processing. Like an earlier proposal for yeast, boxes A′ and A of Xenopus may base-pair with 18 S sequences in pre-rRNA. We present the first direct experimental evidence in any system that box A′ is essential for U3 snoRNA function in 18 S rRNA formation. The analysis of insertions and deletions indicated that the spacing between the U3 elements is important, suggesting that they base-pair with the ETS and 18 S regions of pre-rRNA at the same time.