Xenopus U3 snoRNA GAC-Box A' and Box A sequences play distinct functional roles in rRNA processing.

18S, 5.8S, and 25-28S rRNAs are transcribed in the nucleolus of the eukaryotic cell by RNA polymerase I in a form of long precursor rRNA (pre-rRNA) that subsequently undergoes a number of processing cleavages to remove the external transcribed spacer sequences (ETS) and internal transcribed spacer sequences (ITS) and release the mature rRNAs. rRNA processing involves a number of small nucleolar RNAs (snoRNAs). U3 and U14 snoRNAs have been implicated in processing steps leading to 18S rRNA formation in both lower (U3 [20] and U14 [31, 32]) and higher (U3 [7, 24, 43] and U14 [27]) eukaryotes. In addition, 18S rRNA formation in vertebrates requires U22 (49), U17(E1), E2, and E3 snoRNAs (35), and in yeast it requires snR10 (48) and snR30 (36) snoRNAs. The role of snoRNAs in rRNA processing is distinct from the function of the majority of snoRNAs that serve as guide RNAs for rRNA modification (2′-O-ribose methylation and pseudouridine formation). In this paper, we investigate the role of U3 snoRNA in rRNA processing to form 18S rRNA. U3 is the most abundant snoRNA in the cell and is essential for viability, as tested in yeast (20, 42). Vertebrate U3 snoRNA appears to determine the order of cleavage events leading to 18S rRNA formation (7). In Xenopus laevis, U3 snoRNA is required for cleavage at site 1 (5′ end of 18S rRNA), site 2 (3′ end of 18S rRNA), and site 3 (5′ end of 5.8S RNA) (7, 43). In addition, U3 together with U14, U17(E1), and E3 snoRNAs are needed for cleavage near the 5′ end of the ETS in many eukaryotes (12, 24). In yeast, U3 snoRNA is needed for cleavage at site A0 (near the 3′ end of the ETS), site A1 (5′ end of 18S rRNA), and site A2 (within the ITS1) (20). What is the mechanism by which U3 snoRNA mediates these cleavages in pre-rRNA? It has been proposed that the single-stranded 5′ hinge (4) and 3′ hinge (8), which separate domains I and II of U3 snoRNA, base pair with the ETS, thus docking U3 snoRNA on the pre-rRNA substrate. These base-pairing interactions are supported by phylogenetic comparisons (8), and compensatory base changes validate the U3 5′ hinge base pairing with the ETS of pre-rRNA in yeast (5). Once U3 snoRNA has docked on the pre-rRNA, it may base pair with sequences within the 18S pre-rRNA. In doing so, U3 snoRNA could act as a chaperone to prevent premature pseudoknot formation in 18S rRNA (19), similar to comparable interactions that occur in cis in bacteria (11). In yeast, three evolutionarily conserved regions in domain I of U3 snoRNA, the GAC element, box A′, and box A (42), are complementary to sequences in 18S rRNA (19), and chemical modification supports the model that they base pair (34). Compensatory mutations validated the base pairing interaction between the 5′ portion of box A and 18S rRNA in yeast but did not confirm the interaction between the 3′ portion of box A and 18S rRNA (46). The interaction between box A′ and 18S rRNA has not yet been experimentally tested in any organism. Base pairing between the GAC element and 18S rRNA can be drawn for yeast but not for vertebrates, calling its role into question. Therefore, further investigation is needed to explore the roles of the GAC element, box A′, and box A in U3 snoRNA, and this is the focus of the present study. In order to understand the mechanism for U3 snoRNA function in rRNA processing, we have created a systematic array of mutations in the 5′ portion of the molecule, including the evolutionarily conserved GAC element, box A′, and box A, and tested their effects in a functional assay. The results reported here have uncovered a new cleavage site in the 3′ region of the ETS of vertebrate pre-rRNA, homologous with site A0 in yeast. In addition, by mutagenesis of U3 snoRNA we have uncoupled cleavage at sites 1 and 2, which normally occur in unison. Furthermore, specific nucleotides in U3 snoRNA that are required for distinct cleavages in pre-rRNA were identified: box A is essential for site A0 cleavage, the GAC-box and A′ region is necessary for site 1 cleavage, and a few nucleotides at the 3′ end of box A′ are required for site 2 cleavage. Finally, based on these data, a new model is presented in which U3 snoRNA acts as a bridge to draw together the 5′ and 3′ ends of the 18S rRNA coding region within pre-rRNA.