Depletion of U3 small nucleolar RNA inhibits cleavage in the 5'' external transcribed spacer of yeast pre-ribosomal RNA and impairs formation of 18S ribosomal RNA.

Multiple processing events are required to convert a single eukaryotic pre-ribosomal RNA (pre-rRNA) into mature 18S (small subunit), 5.8S and 25-28S (large subunit) rRNAs. We have asked whether U3 small nucleolar RNA is required for pre-rRNA processing in vivo by depleting Saccharomyces cerevisiae of U3 by conditional repression of U3 synthesis. The resulting pattern of accumulation and depletion of specific pre-rRNAs indicates that U3 is required for multiple events leading to the maturation of 18S rRNA. These include an initial cleavage within the 5' external transcribed spacer, resembling the U3 dependent initial processing event of mammalian pre-rRNA. Formation of large subunit rRNAs is unaffected by U3 depletion. The similarity between the effects of U3 depletion and depletion of U14 small nucleolar RNA and the nucleolar protein fibrillarin (NOP1) suggests that these could be components of a single highly conserved processing complex.     

The sequence of the 5' end of the U8 small nucleolar RNA is critical for 5.8S and 28S rRNA maturation.

Ribosome biogenesis in eucaryotes involves many small nucleolar ribonucleoprotein particles (snoRNP), a few of which are essential for processing pre-rRNA. Previously, U8 snoRNA was shown to play a critical role in pre-rRNA processing, being essential for accumulation of mature 28S and 5.8S rRNAs. Here, evidence which identifies a functional site of interaction on the U8 RNA is presented. RNAs with mutations, insertions, or deletions within the 5'-most 15 nucleotides of U8 do not function in pre-rRNA processing. In vivo competitions in Xenopus oocytes with 2'O-methyl oligoribonucleotides have confirmed this region as a functional site of a base-pairing interaction. Cross-species hybrid molecules of U8 RNA show that this region of the U8 snoRNP is necessary for processing of pre-rRNA but not sufficient to direct efficient cleavage of the pre-rRNA substrate; the structure or proteins comprising, or recruited by, the U8 snoRNP modulate the efficiency of cleavage. Intriguingly, these 15 nucleotides have the potential to base pair with the 5' end of 28S rRNA in a region where, in the mature ribosome, the 5' end of 28S interacts with the 3' end of 5.8S. The 28S-5.8S interaction is evolutionarily conserved and critical for pre-rRNA processing in Xenopus laevis. Taken together these data strongly suggest that the 5' end of U8 RNA has the potential to bind pre-rRNA and in so doing, may regulate or alter the pre-rRNA folding pathway. The rest of the U8 particle may then facilitate cleavage or recruitment of other factors which are essential for pre-rRNA processing.     

A second base pair interaction between U3 small nucleolar RNA and the 5'-ETS region is required for early cleavage of the yeast pre-ribosomal RNA

In eukaryotes, U3 snoRNA is essential for pre-rRNA maturation. Its 5'-domain was found to form base pair interactions with the 18S and 5'-ETS parts of the pre-rRNA. In Xenopus laevis, two segments of U3 snoRNA form base-pair interactions with the 5'-ETS region and only one of them is essential to the maturation process. In Saccharomyces cerevisiae, two similar U3 snoRNA-5' ETS interactions are possible; but, the functional importance of only one of them had been tested. Surprisingly, this interaction, which corresponds to the non-essential one in X. laevis, is essential for cell growth and pre-rRNA maturation in yeast. In parallel with [Dutca et al. (2011) The initial U3 snoRNA:pre-rRNA base pairing interaction required for pre-18S rRNA folding revealed by in vivo chemical probing. Nucleic Acids Research, 39, 5164-5180], here we show, that the second possible 11-bp long interaction between the 5' domain of S. cerevisiae U3 snoRNA and the pre-rRNA 5'-ETS region (helix VI) is also essential for pre-rRNA processing and cell growth. Compensatory mutations in one-half of helix VI fully restored cell growth. Only a partial restoration of growth was obtained upon extension of compensatory mutations to the entire helix VI, suggesting sequence requirement for binding of specific proteins. Accordingly, we got strong evidences for a role of segment VI in the association of proteins Mpp10, Imp4 and Imp3.     

An evolutionary intra-molecular shift in the preferred U3 snoRNA binding site on pre-ribosomal RNA

Correct docking of U3 small nucleolar RNA (snoRNA) on pre-ribosomal RNA (pre-rRNA) is essential for rRNA processing to produce 18S rRNA. In this report, we have used Xenopus oocytes to characterize the structural requirements of the U3 snoRNA 3′-hinge interaction with region E1 of the external transcribed spacer (ETS) of pre-rRNA. This interaction is crucial for docking to initiate rRNA processing. 18S rRNA production was inhibited when fewer than 6 of the 8 bp of the U3 3′–hinge complex with the ETS could form; moreover, base pairing involving the right side of the 3′-hinge was more important than the left. Increasing the length of the U3 hinge–ETS interaction by 9 bp impaired rRNA processing. Formation of 18S rRNA was also inhibited by swapping the U3 5′- and 3′-hinge interactions with the ETS or by shifting the base pairing of the U3 3′-hinge to the sequence directly adjacent to ETS region E1. However, 18S rRNA production was partially restored by a compensatory shift that allowed the sequence adjacent to the U3 3′-hinge to pair with the eight bases directly adjacent to ETS region E1. The results suggest that the geometry of the U3 snoRNA interaction with the ETS is critical for rRNA processing.     

Base pairing between U3 and the pre-ribosomal RNA is required for 18S rRNA synthesis.

The nucleolus, the site of pre-ribosomal RNA (pre-rRNA) synthesis and processing in eukaryotic cells, contains a number of small nucleolar RNAs (snoRNAs). Yeast U3 snoRNA is required for the processing of 18S rRNA from larger precursors and contains a region complementary to the pre-rRNA. Substitution mutations in the pre-rRNA which disrupt this base pairing potential are lethal and prevent synthesis of 18S rRNA. These mutant pre-rRNAs show defects in processing which closely resemble the effects of genetic depletion of components of the U3 snoRNP. Co-expression of U3 snoRNAs which carry compensatory mutations allows the mutant pre-rRNAs to support viability and synthesize 18S rRNA at high levels. Pre-rRNA processing steps which are blocked by the external transcribed spacer region mutations are largely restored by expression of the compensatory U3 mutants. Pre-rRNA processing therefore requires direct base pairing between snoRNA and the substrate. Base pairing with the substrate is thus a common feature of small RNAs involved in mRNA and rRNA maturation.     

Base Pairing between U3 Small Nucleolar RNA and the 5′ End of 18S rRNA Is Required for Pre-rRNA Processing

The loop of a stem structure close to the 5' end of the 18S rRNA is complementary to the box A region of the U3 small nucleolar RNA (snoRNA). Substitution of the 18S loop nucleotides inhibited pre-rRNA cleavage at site A(1), the 5' end of the 18S rRNA, and at site A(2), located 1.9 kb away in internal transcribed spacer 1. This inhibition was largely suppressed by a compensatory mutation in U3, demonstrating functional base pairing. The U3-pre-rRNA base pairing is incompatible with the structure that forms in the mature 18S rRNA and may prevent premature folding of the pre-rRNA. In the Escherichia coli pre-rRNA the homologous region of the 16S rRNA is also sequestered, in that case by base pairing to the 5' external transcribed spacer (5' ETS). Cleavage at site A(0) in the yeast 5' ETS strictly requires base pairing between U3 and a sequence within the 5' ETS. In contrast, the U3-18S interaction is not required for A(0) cleavage. U3 therefore carries out at least two functionally distinct base pair interactions with the pre-rRNA. The nucleotide at the site of A(1) cleavage was shown to be specified by two distinct signals; one of these is the stem-loop structure within the 18S rRNA. However, in contrast to the efficiency of cleavage, the position of A(1) cleavage is not dependent on the U3-loop interaction. We conclude that the 18S stem-loop structure is recognized at least twice during pre-rRNA processing.     

The DEAH-box Helicase Dhr1 Dissociates U3 from the Pre-rRNA to Promote Formation of the Central Pseudoknot

In eukaryotes, the highly conserved U3 small nucleolar RNA (snoRNA) base-pairs to multiple sites in the pre-ribosomal RNA (pre-rRNA) to promote early cleavage and folding events. Binding of the U3 box A region to the pre-rRNA is mutually exclusive with folding of the central pseudoknot (CPK), a universally conserved rRNA structure of the small ribosomal subunit essential for protein synthesis. Here, we report that the DEAH-box helicase Dhr1 (Ecm16) is responsible for displacing U3. An active site mutant of Dhr1 blocked release of U3 from the pre-ribosome, thereby trapping a pre-40S particle. This particle had not yet achieved its mature structure because it contained U3, pre-rRNA, and a number of early-acting ribosome synthesis factors but noticeably lacked ribosomal proteins (r-proteins) that surround the CPK. Dhr1 was cross-linked in vivo to the pre-rRNA and to U3 sequences flanking regions that base-pair to the pre-rRNA including those that form the CPK. Point mutations in the box A region of U3 suppressed a cold-sensitive mutation of Dhr1, strongly indicating that U3 is an in vivo substrate of Dhr1. To support the conclusions derived from in vivo analysis we showed that Dhr1 unwinds U3-18S duplexes in vitro by using a mechanism reminiscent of DEAD box proteins.     

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.     

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.     

Effects of alpha-amanitin, cycloheximide, and thioacetamide on low molecular weight nuclear RNA.

Studies were made on the effects of alpha-amanitin, cycloheximide, and thioacetamide on synthesis and content of low molecular weight nuclear RNA. Cycloheximide, an inhibitor of protein synthesis and the synthesis of 45S pre-rRNA and 5S RNA, also inhibited synthesis of nuclear U1 and U3 RNAs. alpha-Amanitin, an inhibited the synthesis of U1 and U2 low molecular weight nuclear RNA. Thioacetamide, which induces nucleolar hypertrophy and increased nucleolar RNA polymerase activity, markedly increased synthesis of 5.8S RNA and U3 RNA. These results show that syntheses of individual low molecular weight nuclear (LMWN) RNAs are controlled by different regulatory mechanisms. In particular, there appears to be a specific relationship between U3 RNA and functional states of the nucleolus.     

Nucleolin functions in the first step of ribosomal RNA processing.

The first processing step of precursor ribosomal RNA (pre-rRNA) involves a cleavage within the 5' external transcribed spacer. This processing requires sequences downstream of the cleavage site which are perfectly conserved among human, mouse and Xenopus and also several small nucleolar RNAs (snoRNAs): U3, U14, U17 and E3. In this study, we show that nucleolin, one of the major RNA-binding proteins of the nucleolus, is involved in the early cleavage of pre-rRNA. Nucleolin interacts with the pre-rRNA substrate, and we demonstrate that this interaction is required for the processing reaction in vitro. Furthermore, we show that nucleolin interacts with the U3 snoRNP. Increased levels of nucleolin, in the presence of the U3 snoRNA, activate the processing activity of a S100 cell extract. Our results suggest that the interaction of nucleolin with the pre-rRNA substrate might be a limiting step in the primary processing reaction. Nucleolin is the first identified metazoan proteinaceous factor that interacts directly with the rRNA substrate and that is required for the processing reaction. Potential roles for nucleolin in the primary processing reaction and in ribosome biogenesis are discussed.     

RNA B is the major nucleolar trimethylguanosine-capped small nuclear RNA associated with fibrillarin and pre-rRNAs in Trypanosoma brucei.

RNA B is one of three abundant trimethylguanosine-capped U small nuclear RNAs (snRNAs) of Trypanosoma brucei which is not strongly identified with other U snRNAs by sequence homology. We show here that RNA B is a highly diverged U3 snRNA homolog likely involved in pre-rRNA processing. Sequence identity between RNA B and U3 snRNAs is limited; only two of four boxes of homology conserved between U3 snRNAs are obvious in RNA B. These are the box A homology, specific for U3 snRNAs, and the box C homology, common to nucleolar snRNAs and required for association with the nucleolar protein, fibrillarin. A 35-kDa T. brucei fibrillarin homolog was identified by using an anti-Physarum fibrillarin monoclonal antibody. RNA B and fibrillarin were localized in nucleolar fractions of the nucleus which contained pre-rRNAs and did not contain nucleoplasmic snRNAs. Fibrillarin and RNA B were precipitated by scleroderma patient serum S4, which reacts with fibrillarins from diverse organisms; RNA B was the only trimethylguanosine-capped RNA precipitated. Furthermore, RNA B sedimented with pre-rRNAs in nondenaturing sucrose gradients, similarly to U3 and other nucleolar snRNAs, suggesting that RNA B is hydrogen bonded to rRNA intermediates and might be involved in their processing.     

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.     

The Putative RNA Helicase Dbp4p Is Required for Release of the U14 snoRNA from Preribosomes in Saccharomyces cerevisiae

Around 70 yeast snoRNAs guide rRNA modification, frequently forming base-paired interactions predicted to be very stable at physiological temperatures. Eighteen putative RNA helicases are required for ribosome synthesis, but their actual substrates were not known. We report that depletion of the DEAD box helicase Dbp4p dramatically increased cosedimentation of the snoRNAs U14 and snR41 with preribosomes. Cosedimentation was maintained after deproteinization by proteinase K, indicating that the snoRNAs remained base paired to the pre-rRNA. Affinity purification showed that U14 was strongly accumulated in early 90S preribosomes and depleted from later pre-40S complexes. U14 is required for pre-rRNA processing, and depletion of Dbp4p caused a very similar pre-rRNA processing defect, perhaps due to the reduced pool of free U14. Point mutations in helicase motifs I and III of Dbp4p blocked release of U14 from preribosomes. We conclude that the helicase activity of Dbp4p is required to unwind U14 and snR41 from the pre-rRNA.