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.     

A temperature sensitive mutant of Saccharomyces cerevisiae defective in pre-rRNA processing.

A recessive temperature sensitive mutant has been isolated that is defective in ribosomal RNA processing. By Northern analysis, this mutant was found to accumulate three novel rRNA species: 23S', 18S' and 7S', each of which contains sequences from the spacer region between 25S and 18S rRNA. 35S pre-rRNA accumulates, while the level of the 20S and 27S rRNA processing intermediates is depressed. Pulse-chase analysis demonstrates that the processing of 35S pre-rRNA is slowed. The defect in the mutant appears to be at the first processing step, which generates 20S and 27S rRNA. 7S' RNA is a form of 5.8S RNA whose 5' end is extended by 149 nucleotides to a position just 5 nucleotides downstream of the normal cleavage site that produces 20S and 27S rRNA. 7S' RNA can assemble into 60S ribosomal subunits, but such subunits are relatively ineffective in joining polyribosomes. A single lesion is responsible for the pre-rRNA processing defect and the temperature sensitivity. The affected gene is designated RRP2.     

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.     

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

Mutations in the 5' portion of Xenopus U3 snoRNA were tested for function in oocytes. The results revealed a new cleavage site (A0) in the 3' region of vertebrate external transcribed spacer sequences. In addition, U3 mutagenesis uncoupled cleavage at sites 1 and 2, flanking the 5' and 3' ends of 18S rRNA, and generated novel intermediates: 19S and 18.5S pre-rRNAs. Furthermore, specific nucleotides in Xenopus U3 snoRNA that are required for cleavages in pre-rRNA were identified: box A is essential for site A0 cleavage, the GAC-box A' region is necessary for site 1 cleavage, and the 3' end of box A' and flanking nucleotides are required for site 2 cleavage. Differences between metazoan and yeast U3 snoRNA-mediated rRNA processing are enumerated. The data support a model where metazoan U3 snoRNA acts as a bridge to draw together the 5' and 3' ends of the 18S rRNA coding region within pre-rRNA to coordinate their cleavage.     

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.     

Two distinct recognition signals define the site of endonucleolytic cleavage at the 5''-end of yeast 18S rRNA.

Three of the four eukaryotic ribosomal RNA molecules (18S, 5.8S and 25-28S rRNA) are transcribed as a single precursor, which is subsequently processed into the mature species by a complex series of cleavage and modification reactions. Early cleavage at site A1 generates the mature 5'-end of 18S rRNA. Mutational analyses have identified a number of upstream regions in the 5' external transcribed spacer (5' ETS), including a U3 binding site, which are required in cis for processing at A1. Nothing is known, however, about the requirement for cis-acting elements which define the position of the 5'-end of the 18S rRNA or of any other eukaryotic rRNA. We have introduced mutations around A1 and analyzed them in vivo in a genetic background where the mutant pre-rRNA is the only species synthesized. The results indicate that the mature 5'-end of 18S rRNA in yeast is identified by two partially independent recognition systems, both defining the same cleavage site. One mechanism identifies the site of cleavage at A1 in a sequence-specific manner involving recognition of phylogenetically conserved nucleotides immediately upstream of A1 in the 5' ETS. The second mechanism specifies the 5'-end of 18S rRNA by spacing the A1 cleavage at a fixed distance of 3 nt from the 5' stem-loop/pseudoknot structure located within the mature sequence. The 5' product of the A1 processing reaction can also be identified, showing that, in contrast to yeast 5.8S rRNA, the 5'-end of 18S rRNA is generated by endonucleolytic cleavage.     

Trypanosoma brucei 5'ETS A'-cleavage is directed by 3'-adjacent sequences, but not two U3 snoRNA-binding elements, which are all required for subsequent pre-small subunit rRNA processing events.

Trypanosoma brucei pre-rRNA processing commences by cleavage near the 5' end of 5.8 S sequences. The 5' external transcribed spacer (5'ETS) is removed from pre-small subunit (SSU) rRNAs by sequential cleavages at internal A' and A0 sites, and A1 at the 5' end of SSU rRNA. The A' and A0 sites positionally resemble the U3 small nucleolar RNA-dependent, primary pre-rRNA cleavages of vertebrates and yeast, respectively. Uniquely in T. brucei, two U3-crosslinkable 5'ETS sites are essential for SSU rRNA production: site1b is novel in its 3' location to the A' site, and site3 lies upstream of A0 in a position analogous to the yeast U3-binding site. Here, in vivo analysis of mutated 5'ETS sequences shows that sequences 5' to the A' site are not needed for A' cleavage or SSU rRNA production. A' cleavage is linked to, but is not sufficient to trigger, downstream pre-SSU rRNA processing events. These events require an intact 11 nt sequence, 3'-adjacent to A', which directs efficient and accurate A' cleavage. Neither the A' nearby site1b nor the site3 U3-binding elements affect A' processing, yet each is required for A0 and A1 cleavage, and SSU rRNA production. The same U3 3' hinge bases evidently bind a core element, UGUu/gGGU, within site1a and site3; the U3-site1b interaction is less reliant on base-pairing than the U3-site3 interaction. As yeast U3 5' hinge bases pair to 5'ETS sequences, it is clear that distinct U3 hinge regions can interact at both novel and related 5'ETS sites to promote 3'-proximal 5'ETS processing events in diverse organisms. The T. brucei data fit a model wherein processing factors assemble at the 5'ETS site1a to affect A' cleavage and stabilize a U3-site1b complex, which may work in concert with the downstream U3-site3 complex to assist processing events leading to ribosomal SSU production.     

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.     

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.     

Multiple ribosomal RNA cleavage pathways in mammalian cells

The sequence content of mouse L cell pre-rRNA was examined by RNA gel transfer and blot hybridization. Nuclear RNAs were separated by agarose gel electrophoresis, transferred to diazo-paper, and hybridized to twelve different restriction fragments that are complementary to various sections of 45S pre-rRNA. An abundant new 34S pre-rRNA and less abundant new 37S, 26S and 17S pre-rRNAs were detected. The presence of these new pre-rRNAs suggests the existence of at least two new pre-rRNA cleavage pathways. 34S and 26S pre-rRNAs were also detected in HeLa cells suggesting that these new cleavage pathways are characteristic of mammalian cells. Further, an abundant new 12S precursor to 5.8S rRNA was also detected and is common to all the proposed cleavage pathways. The previously identified 45S, 41S, 32S and 20S pre-rRNAs were readily detected and their general structure confirmed. The 20S pre-rRNA is characteristic of the known pathway used by HeLa and other cells, and its presence suggests that growing mouse L cells use this pre-rRNA cleavage pathway. The 36S pre-rRNA characteristic of the previously described mouse L cell cleavage pathway was not detected. In all these cleavage pathways pre-rRNA cleavage sites are apparently identical and occur at or near the termini of the mature 18S, 5.8S and 28S rRNA sequences. The pathways differ only in the temporal order of cleavage at these sites.     

Deletion of the three distal S1 motifs of Saccharomyces cerevisiae Rrp5p abolishes pre-rRNA processing at site A(2) without reducing the production of functional 40S subunits

Yeast Rrp5p, one of the few trans-acting proteins required for the biogenesis of both ribosomal subunits, has a remarkable two-domain structure. Its C-terminal region consists of seven tetratricopeptide motifs, several of which are crucial for cleavages at sites A(0) to A(2) and thus for the formation of 18S rRNA. The N-terminal region, on the other hand, contains 12 S1 RNA-binding motifs, most of which are required for processing at site A(3) and thus for the production of the short form of 5.8S rRNA. Yeast cells expressing a mutant Rrp5p protein that lacks S1 motifs 10 to 12 (mutant rrp5Delta6) have a normal growth rate and wild-type steady-state levels of the mature rRNA species, suggesting that these motifs are irrelevant for ribosome biogenesis. Here we show that, nevertheless, in the rrp5Delta6 mutant, pre-rRNA processing follows an alternative pathway that does not include the cleavage of 32S pre-rRNA at site A(2). Instead, the 32S precursor is processed directly at site A(3), producing exclusively 21S rather than 20S pre-rRNA. This is the first evidence that the 21S precursor, which was observed previously only in cells showing a substantial growth defect or as a minor species in addition to the normal 20S precursor, is an efficient substrate for 18S rRNA synthesis. Maturation of the 21S precursor occurs via the same endonucleolytic cleavage at site D as that used for 20S pre-rRNA maturation. The resulting D-A(3) fragment, however, is degraded by both 5'-->3' and 3'-->5' exonuclease digestions, the latter involving the exosome, in contrast to the exclusively 5'-->3' exonucleolytic digestion of the D-A(2) fragment. We also show that rrp5Delta6 cells are hypersensitive to both hygromycin B and cycloheximide, suggesting that, despite their wild-type growth rate, their preribosomes or ribosomes may be structurally abnormal.     

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.     

Processing of the yeast pre-rRNA at sites A(2) and A(3) is linked.

Cleavage of the yeast pre-rRNA at site A(2) in internal transcribed spacer 1 (ITS1) requires multiple snoRNP species, whereas cleavage at site A(3),located 72 nt 3' in ITS1, requires Rnase MRP. Analyses of mutations in the pre- rRNA have revealed an unexpected link between processing at A(2) and A(3). Small substitution mutations in the 3' flanking sequence at A(2) inhibit processing at site A(3), whereas a small deletion at A(3) has been shown to delay processing at site A(2). Moreover, the combination of mutations in cis at both A(2) and A(3) leads to the synthesis of pre-rRNA species with 5' ends within the mature 18S rRNA sequence, at sites between + 482 and + 496. The simultaneous interference with an snoRNP processing complex at site A(2) and an Rnase MPRP complex at site A(3) may activate a pre-rRNA breakdown pathway. The same aberantpre-rRNA species are observed in strains with mutations in the RNA component of Rnase MRP, consistent with interactions between the processing complexes. Furthermore, genetic depletion of the snoRNA, snR30, has been shown to affect the coupling between cleavage by Rnase MRP and subsequent exonuclease digestion.We conclude that an sno-RNP-dependent processing complex that is required for A(2) cleavage and that recognizes the 3' flanking sequence at A(2), interacts with the RNase MRP complex bound to the pre-rRNA around site A(3).     

Bypassing the rRNA processing endonucleolytic cleavage at site A2 in Saccharomyces cerevisiae

Rrp5p is the only ribosomal RNA processing trans-acting factor that is required for the synthesis of both 18S and 5.8S rRNAs in Saccharomyces cerevisiae. Mutational analyses have characterized modified forms of Rrp5p that either affect formation of 18S rRNA by inhibiting cleavage at sites A0/A1/A2, or synthesis of 5.8S rRNA by inhibiting cleavage at site A3. Here, we examine the rRNA maturation process associated with a RRP5 bipartite allele that codes for two noncontiguous parts of the protein. This slow-growing bipartite mutant has a unique rRNA-processing phenotype that proceeds without endonucleolytic cleavage at site A2. In wild-type cells, the A2 cleavage takes place on the 32S pre-rRNA and is responsible for the formation of 20S and 27SA2 species, the precursors of mature 18S and 5.8S/25S rRNAs, respectively. In the bipartite strain, such precursors were not detectable as judged by Northern analysis or in vivo labeling. They were replaced by the aberrant 21S species and the bypassing 27SA3 precursor, both descended from direct cleavage of 32S pre-rRNA at site A3, which provides an alternative rRNA maturation pathway in this strain. The 21S pre-rRNA is the sole detectable and most likely available precursor of 18S rRNA in this particular strain, indicating that 18S rRNA can be directly produced from 21S. Furthermore, 21S species were found associated with 43S preribosomal particles as similarly observed for the 20S pre-rRNA in the wild-type cells.     

Yeast Rnt1p is required for cleavage of the pre-ribosomal RNA in the 3' ETS but not the 5' ETS.

We have reexamined the role of yeast RNase III (Rnt1p) in ribosome synthesis. Analysis of pre-rRNA processing in a strain carrying a complete deletion of the RNT1 gene demonstrated that the absence of Rnt1p does not block cleavage at site A0 in the 5' external transcribed spacers (ETS), although the early pre-rRNA cleavages at sites A0, A1, and A2 are kinetically delayed. In contrast, cleavage in the 3' ETS is completely inhibited in the absence of Rnt1p, leading to the synthesis of a reduced level of a 3' extended form of the 25S rRNA. The 3' extended forms of the pre-rRNAs are consistent with the major termination at site T2 (+210). We conclude that Rnt1p is required for cleavage in the 3' ETS but not for cleavage at site A0. The sites of in vivo cleavage in the 3' ETS were mapped by primer extension. Two sites of Rnt1p-dependent cleavage were identified that lie on opposite sides of a predicted stem loop structure, at +14 and +49. These are in good agreement with the consensus Rnt1p cleavage site. Processing of the 3' end of the mature 25S rRNA sequence in wild-type cells was found to occur concomitantly with processing of the 5' end of the 5.8S rRNA, supporting previous proposals that processing in ITS1 and the 3' ETS is coupled.     

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.     

Intranuclear maturation pathways of rat liver ribosomal ribonucleic acids.

The maturation of pre-rRNA (precursor to rRNA)in liver nuclei is studied by agar/ureagel electrophoresis, kinetics of labelling in vivo with [14C] orotate and electron-microscopic observation of secondary structure of RNA molecules. (1) Processing starts from primary pre-rRNA molecules with average mol. wt. 4.6X10(6)(45S) containing the segments of both 28S and 18S rRNA. These molecules form a heterogeneous peak on electrophoresis. The 28S rRNA segment is homogeneous in its secondary structure. However, the large transcribed spacer segment (presumably at the 5'-end) is heterogeneous in size and secondary structure. A minor early labelled RNA component with mol.wt. about 5.8X10(6) is reproducibly found, but its role as a pre-rRNA species remains to be determined. (2) The following intermediate pre-rRNA species are identified: 3.25X10(6) mol.wt.(41S), a precursor common to both mature rRNA species ; 2.60X10(6)(36S) and 2.15X10(6)(32S) precursors to 28S rRNA; 1.05X10(6) (21S) precursor to 18S rRNA. The pre-rRNA molecules in rat liver are identical in size and secondary structure with those observed in other mammalian cells. These results suggest that the endonuclease-cleavage sites along the pre-rRNA chain are identical in all mammalian cells. (3) Labelling kinetics and the simultaneous existence of both 36S and 21S pre-rRNA reveal that processing of primary pre-rRNA in adult rat liver occurs simultaneously by at least two major pathways: (i) 45S leads to 41S leads to 32S+21S leads to 28S+18S rRNA and (ii) 45S leads to 41S leads to 36S+18S leads to 32S leads to 28S rRNA. The two pathways differ by the temporal sequence of endonuclease attack along the 41 S pre-rRNA chain. A minor fraction (mol.wt.2.9X10(6), 39S) is identified as most likely originating by a direct split of 28S rRNA from 45S pre-rRNA. These results show that in liver considerable flexibility exists in the order of cleavage of pre-rRNA molecules during processing.