Xenopus U3 snoRNA docks on pre-rRNA through a novel base-pairing interaction

Abstract

U3 small nucleolar RNA (snoRNA) is essential for rRNA processing to form 18S ribosomal RNA (rRNA). Previously, it has been shown that nucleolin is needed to load U3 snoRNA on pre-rRNA. However, as documented here, this is not sufficient. We present data that base-pairing between the U3 hinges and the external transcribed spacer (ETS) is critical for functional alignment of U3 on its pre-rRNA substrate. Additionally, the interaction between the U3 hinges and the ETS is proposed to serve as an anchor to hold U3 on the pre-rRNA substrate, while box A at the 5' end of U3 snoRNA swivels from ETS contacts to 18S rRNA contacts. Compensatory base changes revealed base-pairing between the 3' hinge of U3 snoRNA and region E1 of the ETS in Xenopus pre-rRNA; this novel interaction is required for 18S rRNA production. In contrast, base-pairing between the 5' hinge of U3 snoRNA and region E2 of the ETS is auxiliary, unlike the case in yeast where it is required. Thus, higher and lower eukaryotes use different interactions for functional association of U3 with pre-rRNA. The U3 hinge sequence varies between species, but covariation in the ETS retains complementarity. This species-specific U3-pre-rRNA interaction offers a potential target for a new class of antibiotics to prevent ribosome biogenesis in eukaryotic pathogens.

FIGURE 1.

rRNA processing in Xenopus oocytes. Processing of Xenopus pre-rRNA can occur through either pathway A or B (Savino and Gerbi 1990) with cleavages at the indicated sites. Cleavage at site A′ is not readily detectable in Xenopus oocytes (Savino and Gerbi 1991; Mougey et al. 1993a,b). U3 snoRNA is needed for cleavage at sites A0, 1, 2, and 3 (bold fonts; Savino and Gerbi 1990; Borovjagin and Gerbi 1999, 2001) to produce 18S rRNA. 19S and 18.5S pre-rRNAs are transient intermediates that generally do not accumulate in Xenopus oocytes, but can be detected after U3 snoRNA mutagenesis (Borovjagin and Gerbi 2001). Mature rRNA sequences are shown by filled boxes (black for 18S rRNA and gray for 5.8S and 28S rRNA), and horizontal lines depict the external (ETS) and internal (ITS) transcribed spacers as well as the intergenic spacer (IGS). The substitution of a sequence tag in 18S rRNA is shown by a small open box.

FIGURE 2.

Structure of U3 snoRNA. Secondary structure model of U3 snoRNA with domains I and II and the 5′ and 3′ hinge regions separating them (Borovjagin and Gerbi 2000). The evolutionarily conserved sequence motifs (GAC, boxes A′, A, C′, B, C, and D) as well as the 5′ and the 3′ hinge sequences (5′H and 3′H) are indicated. The 5′ hinge and 3′ hinge sequences of U3 snoRNA have the potential to base pair with the E2 and E1 sequences of the ETS, respectively, as shown in the drawing. The 3′H–ETS interaction is required in Xenopus (this study), whereas the 5′H–ETS interaction is required in yeast (Beltrame and Tollervey 1995).

FIGURE 3.

U3 hinge-ETS base-pairing in wild type and mutants. Putative base-pairing between the 5′ hinge (5′H) of U3 snoRNA and region E2 of the ETS or between the 3′ hinge (3′H) of U3 snoRNA and region E1 of the ETS can be destroyed by mutation in the U3 hinge and/or the ETS, and it can be restored by compensatory mutation. The wild-type nucleotides are shown in lowercase font and the substitutions are shown in bold uppercase.

FIGURE 4.

Mutations in E1 and E2 of the ETS impair 18S rRNA production. The schematic drawings show the potential base-pairing between the 5′ hinge (5′H) and 3′ hinge (3′H) of U3 with regions E2 and E1, respectively, of the ETS. Wild-type hinge sequences are depicted by filled boxes and mutations are indicated by open boxes. Autoradiographs assayed the production of mature tagged 18S in Xenopus oocytes after injection and expression of plasmid containing an entire rDNA repeat unit with an RNA Pol I promoter and tag in the 18S rRNA coding region, followed by Northern blot hybridization using a tag-specific oligonucleotide probe. The rDNA plasmid contained either wild-type ETS sequences (WT, lanes 4,7) or mutations in the E1 or E2 regions of the ETS to destroy base-pairing with the U3 hinges. The potential ability of perfect duplex formation by base-pairing between U3 hinge regions and the ETS is indicated above each lane by a + or − for 5′H–E2 or 3′H–E1 interactions. 18S rRNA production requires the 3′H–E1 duplex, but not 5′H–E2. The sizes of various rRNA precursors are indicated. Generally, 40S rRNA levels are greater when rRNA processing is blocked and it accumulates. The band below 40S pre-rRNA may be 38S pre-rRNA (some/all ETS removed from 40S pre-rRNA); like 40S, it accumulates when processing is blocked.

FIGURE 5.

Compensatory mutations between the 3′ hinge of U3 snoRNA and region E1 of the ETS rescue 18S rRNA formation. U3 snoRNA carrying a compensatory (3′H_s1 or 3′H_s2) or noncompensatory (3′H_Ct1) mutation was coinjected with plasmid of 18S-tagged rDNA mutated in the E1 region of the ETS (E1_s1 or E1_s2). The compensatory mutations restore 18S rRNA formation. Other details as in Figure 4 ▶.

FIGURE 6.

The 3′H–E1, but not 5′H–E2 base-pairing is sufficient alone to restore 18S rRNA formation. U3 transcripts carrying the 3′H_s2 mutation were coinjected with the 18S-tagged rDNA plasmid containing the double mutation E2_s/E1_s2 and were able to restore production of tagged 18S rRNA (left). In contrast, U3 transcripts carrying the 5′H_s mutation were unable to rescue production of tagged 18S rRNA from the rDNA plasmid carrying the double mutation E2_s/E1_s1 (right). U3 transcripts with the double compensatory mutation 5′H_s/33H_s1 rescued rRNA processing of 18S-tagged pre-rRNA with the double mutation E2_s/E1_s1 (middle). Other details as in Figure 4 ▶.

FIGURE 7.

Partial base-pairing between the 3′ hinge of U3 and region E1 of the ETS results in partial restoration of 18S rRNA production. Transcripts of U3 carrying a mutation in the 3′ hinge (3′H_s1 or 3′H_s2) were coinjected into oocytes with Pol I-driven 18S-tagged rDNA containing the ETS double mutation E2_s/E1_s2 or E2_s/E1_s1. The combinations of U3 snoRNA and pre-rRNA that had no base pairs between the 5′ hinge of U3 and region E2 of the ETS and only four base pairs (when slipped; arrow) between the 3′ hinge of U3 and region E1 of the ETS (signified by a smaller plus symbol above lanes 2,3,14) gave partial restoration of 18S rRNA production. Other details as in Figure 4 ▶.

FIGURE 8.

Conserved features of the pre-rRNA ETS. An evolutionarily conserved motif (ECM; nucleotides in bold font) contains the UCGA sequence (shaded background) required for nucleolin binding (Ginisty et al. 2000). The ECM is downstream of the A′ cleavage site (horizontal bars) in pre-rRNA. Nucleolin is required to load U3 snoRNA on the ETS (Ginisty et al. 1998), and consistent with this role, the ECM is close to region E1 of the ETS that base pairs with the 3′ hinge of U3 snoRNA (nucleotides shown in italics). The binding sites for the nucleolin counterparts in yeast (gar2 in Schizosaccharomyces pombe and Nsr1p in Saccharomyces cerevisiae; Lege-Silvestre et al. 1997) on the ETS of pre-rRNA have not been identified yet (Ginisty et al. 1999). See text for further details.