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

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.     

Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.

The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.     

Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.

The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.     

Identification and functional analysis of two U3 binding sites on yeast pre-ribosomal RNA.

It has long been known that U3 can be isolated hydrogen bonded to pre-ribosomal RNAs, but the sites of interaction are poorly characterized. Here we show that yeast U3 can be cross-linked to 35S pre-rRNA both in deproteinized extracts and in living cells. The sites of cross-linking were localized to the 5' external transcribed spacer (ETS) and then identified at the nucleotide level. Two regions of U3 near the 5' end are cross-linked to pre-rRNA in vivo and in vitro; the evolutionarily conserved box A region and a 10 nucleotide (nt) sequence with perfect complementarity to an ETS sequence. Two in vivo cross-links are detected in the ETS, at +470, within the region complementary to U3, and at +655, close to the cleavage site at the 5' end of 18S rRNA. A tagged rDNA construct was used to follow the effects of mutations in the ETS in vivo. A small deletion around the +470 cross-linking site in the ETS prevents the synthesis of 18S rRNA. This region is homologous to the site of vertebrate ETS cleavage. We propose that this site may be evolutionarily conserved to direct the assembly of a pre-rRNA processing complex required for the cleavages that generate 18S rRNA.     

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

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.     

The yeast Hansenula wingei U3 snoRNA gene contains an intron and its coding sequence co-evolved with the 5' ETS region of the pre-ribosomal RNA.

The 5' external transcribed spacer (ETS) region of the pre-rRNA in Saccharomyces cerevisiae contains a sequence with 10 bp of perfect complementarity to the U3 snoRNA. Base pairing between these sequences has been shown to be required for 18S rRNA synthesis, although interaction over the full 10 bp of complementarity is not required. We have identified the homologous sequence in the 5' ETS from the evolutionarily distant yeast Hansenula wingei; unexpectedly, this shows two sequence changes in the region predicted to base pair to U3. By PCR amplification and direct RNA sequencing, a single type of U3 snoRNA coding sequence was identified in H. wingei. As in the S. cerevisiae U3 snoRNA genes, it is interrupted by an intron with features characteristic of introns spliced in a spliceosome. Consequently, this unusual property is not restricted to the yeast genus Saccharomyces. The introns of the H. wingei and S. cerevisiae U3 genes show strong differences in length and sequence, but are located at the same position in the U3 sequence, immediately upstream of the phylogenetically conserved Box A region. The 3' domains of the H. wingei and S. cerevisiae U3 snoRNAs diverge strongly in primary sequence, but have very similar predicted secondary structures. The 5' domains, expected to play a direct role in pre-ribosomal RNA maturation, are more conserved. The sequence predicted to base pair to the pre-rRNA contains two nucleotide substitutions in H. wingei that restore 10 bp of perfect complementarity to the 5' ETS. This is a strong phylogenetic evidence for the importance of the U3/pre-rRNA interaction.     

Partially Processed pre-rRNA Is Preserved in Association with Processing Components in Nucleolus-derived Foci during Mitosis

Previous studies showed that components implicated in pre-rRNA processing, including U3 small nucleolar (sno)RNA, fibrillarin, nucleolin, and proteins B23 and p52, accumulate in perichromosomal regions and in numerous mitotic cytoplasmic particles, termed nucleolus-derived foci (NDF) between early anaphase and late telophase. The latter structures were analyzed for the presence of pre-rRNA by fluorescence in situ hybridization using probes for segments of pre-rRNA with known half-lives. The NDF did not contain the short-lived 5′-external transcribed spacer (ETS) leader segment upstream from the primary processing site in 47S pre-rRNA. However, the NDF contained sequences from the 5′-ETS core, 18S, internal transcribed spacer 1 (ITS1), and 28S segments and also had detectable, but significantly reduced, levels of the 3′-ETS sequence. Northern analyses showed that in mitotic cells, the latter sequences were present predominantly in 45S-46S pre-rRNAs, indicating that high-molecular weight processing intermediates are preserved during mitosis. Two additional essential processing components were also found in the NDF: U8 snoRNA and hPop1 (a protein component of RNase MRP and RNase P). Thus, the NDF appear to be large complexes containing partially processed pre-rRNA associated with processing components in which processing has been significantly suppressed. The NDF may facilitate coordinated assembly of postmitotic nucleoli.     

Preribosomal RNA processing in Xenopus oocytes does not include cleavage within the external transcribed spacer as an early step.

Recently it has been reported that U3 snRNA is necessary for: (a) internal cleavage at +651/+657 within the external transcribed spacer (ETS) of mouse precursor ribosomal RNA (pre-rRNA); and (b) cleavage at the 5' end of 5.8S rRNA in Xenopus oocytes. To study if U3 snRNA plays a role at more than one processing site in the same system, we have investigated whether internal cleavage sites exist within the ETS of Xenopus oocyte pre-rRNA. The ETS of Xenopus pre-rRNA contains the consensus sequence for the mammalian early processing site (+651/+657 in mouse pre-rRNA), but freshly prepared RNA from Xenopus oocytes has no cuts in this region. The only putative cleavage sites we found in the ETS of Xenopus oocyte pre-rRNA are a cluster further downstream of the mouse early processing site consensus sequence. This cluster is not homologous to the mouse +651/+657 sites because unlike the latter it is (a) not abolished by disruption of U3 snRNA, (b) not cleaved during early steps of pre-rRNA processing, and (c) lacks sequence similarity to the +651/+657 consensus. Therefore, pre-rRNA of Xenopus oocytes does not cleave within the ETS as an early step in rRNA processing. We conclude that cleavage within the ETS is not an obligatory early step needed for the rest of rRNA maturation.