Concurrent with transcription, the ribosomal RNA precursor (pre-rRNA) is modified and associates with many of the ribosomal proteins. The modifications are guided by small nucleolar RNAs (snoRNAs), which base-pair with target sites in eukaryotic pre-rRNA and perhaps also play a role in pre-rRNA folding. A few snoRNAs are essential for rRNA processing; they appear to dock on the pre-rRNA substrate during its transcription but are not activated until the nascent transcript is completed and released from the DNA template. Pre-rRNA contains external transcribed spacers (5' ETS and 3' ETS) at both ends and internal transcribed spacers (ITS1 and ITS2) flanking either side of 5.8S RNA. The transcribed spacers may serve as docking sites for certain snoRNAs (such as U3) and their removal by processing might ensure that steps in rRNA folding cannot run in reverse. The first cleavages in metazoan pre-rRNA remove the 3'-ETS (site T1) and sometimes the 5'-end of the 5'-ETS (site A'). The next set of cleavages (sites A0, 1 and 2) require U3, U14 and U22 snoRNAs in vertebrates and give rise to 18S rRNA. In addition, E1 snoRNA (U17) is needed for cleavage at site 1 and E2 snoRNA for cleavage at site 2. A detailed mechanism of multiple base-pairings between U3 snoRNA and 18S rRNA is hypothesized to fold pre-rRNA appropriately for these cleavages and perhaps to position the cleavage factors. Site 3, which resides slightly upstream of the 5'-end of 5.8S rRNA, can be cleaved before or after sites A0, 1 and 2, depending on the system. Site 3 may link the 18S and 28S rRNA processing pathways, although each can proceed independently of the other. The late steps of rRNA processing involve cleavages of pre-rRNA at sites 3, 4', 4 and 5 to generate mature 5.8S and 28S rRNAs. These cleavages require U8 snoRNA in vertebrates, which is postulated to base-pair with the 5'-end of 28S rRNA within pre-rRNA, perhaps facilitating later base-pairing of 5.8S RNA with the 5'-end of 28S rRNA. snoRNAs and also the protein nucleolin may act as chaperones to fold rRNA. Evolutionary comparisons between kingdoms suggest that pre-rRNA base-pairing with snoRNAs in trans might have replaced intra-molecular pre-rRNA base-pairing in cis. The cleavage steps in rRNA processing could prevent rRNA folding reactions from running in reverse.

Introduction

The nucleolus has intrigued biologists for more than 200 years, since its presumed description by Fontana.¹ Although there is debate over whether Fontana really did visualize the nucleolus,² its documentation was clear by the 1830's (reviewed by refs. 2, 3). The subject of the nucleolus stimulated so much research that by 1898 Montgomery reviewed work in about 700 papers on this topic.⁴ In the past three decades, several books have been written about the nucleolus,^5-7 and the compilation of information is now brought up to date in the present book.⁸

Pioneering research in the past half century has demonstrated that the nucleolus is the site of ribosome biogenesis. Initial clues came from the cytochemical studies of Caspersson⁹ and Brachet¹⁰ showing that the nucleolus contains RNA which they speculated might be related to cytoplasmic RNA. Other cytological studies in the same era noted that the granular component of the nucleolus^11-13 looked similar to “Palade granules” (ribosomes) in the cytoplasm.^14-15 Subsequent pulse-chase experiments^16-18 and base composition comparisons¹⁹ proved that nucleolar RNA became stable, cytoplasmic RNA. Shortly thereafter, with the advent of the technique of RNA-DNA hybridization, it was shown in Drosophila and Xenopus that the nucleolus organizer region (the chromosomal locus associated with the nucleolus) contains the genes for ribosomal RNA (rRNA).^20-23 Development of the method of in situ hybridization allowed the direct visualization by light microscopy of rRNA genes within the nucleoli of amphibia^24-25 and flies.²⁶ At the same time, Miller spreads of nucleolar DNA for electron microscopy revealed active transcription of rRNA genes.²⁷ As shown in Figure 1a, these active transcription units resemble Christmas trees where each branch is a nascent rRNA precursor (pre-rRNA)²⁸ bound to the trunk of the tree (DNA main axis) by a granule of RNA polymerase I.^29-32 There is a tandem array of rRNA genes, each separated from the other by a non-transcribed spacer.

Figure 1

rRNA processing pathways in higher organisms. (a) Miller spread of rDNA transcription visualized by electron micoscopy (courtesy of Ulrich Scheer). The branches of the “Christmas tree” are nascent pre-rRNA transcripts, and a terminal ball (more...)

Internal Modifications: 2'-O-Methylation and Pseudouridylation

Either concurrently or immediately after synthesis of pre-rRNA, there are internal modifications at evolutionarily conserved regions in its 18S, 5.8S and 28S rRNA components.^33-37 In Xenopus these comprise 10 base methylations, 105 2'-O-methylations (2'-O-Me) of ribose and about 100 pseudouridines (ψ); yeast pre-rRNA has approximately half as many internal modifications (see Chapters 12 and 13). The site for each modification is determined by a small nucleolar RNA (snoRNA)(see Chapter 13). The snoRNA base-pairs with the pre-rRNA, thereby appropriately positioning on the pre-rRNA substrate the enzyme which piggybacks along on the snoRNP complex. Members of the Box C/D snoRNA family guide formation of 2'-O-Me by the associated protein Nop1p/fibrillarin which appears to be the methylase.^38-40 Similarly, members of the Box H/ACA snoRNA family guide ψ formation by the associated protein Cbf5p/dyskerin which is the pseudouridine synthase.^41-43 It is conceivable that the base-pairing of the guide snoRNAs also plays a chaperone function in the folding of rRNA. A few snoRNAs to be discussed below are needed for cleavages in the pre-rRNA instead of, or in addition to, a role as a guide snoRNA for modification; this small group of snoRNAs is also implicated in acting in a chaperone capacity.

Addition of Ribosomal Proteins

The predominant initial pre-rRNA ranges in size from 37S (yeast) to 40S (Xenopus) to 45S (mammals). The pre-rRNA assembles with ~80 ribosomal proteins, and the sequence of events in mammals is as follow. Immuno-electron microscopy of Miller spreads revealed that some early binding ribosomal proteins associate with nascent pre-rRNA while it is being transcribed.⁴⁴ Rapidly labeled 30S RNP isolated from nucleoli contains 45S pre-rRNA and seems to be a precursor to the 80S RNP particle.^45-47 The 80S RNP contains 45S pre-rRNA, over two thirds of the 60S ribosomal proteins and about half of the 40S ribosomal proteins.^48-53 Most of the remaining ribosomal proteins are not added until later when rRNA processing is completed. The 80S RNP is the precursor to the 55S RNP^54-55 which constitutes 70 to 80% of the nucleolar population of pre-ribosomes, while 80S RNP constitutes only 10-20%.⁷ This may reflect the fact that cleavage of the 32S pre-rRNA intermediate, which is found in the 55S RNP, is a slow step in the kinetics of rRNA processing. Ultimately, the 55S RNP is converted into the mature 60S ribosomal subunit. Although the 80S RNP is also the precursor for the 40S ribosomal subunit, it has been harder to identify a direct precursor RNP of the 40S subunit in the nucleolus of vertebrate cells; probably it is rapidly transported to the cytoplasm.

Assembly of proteins with pre-rRNA seems to have some differences between mammals (see above) and yeast (see Chapter 12). Yeast pre-rRNA assembles with numerous ribosomal and non-ribosomal proteins (presumably needed for ribosome maturation) in a 90S particle, which is subsequently processed into 66S and 43S pre-ribosomes that form the 60S and 40S ribosomal subunits. Surprisingly, the yeast 90S precursor contains few proteins to form the 60S subunit, although it has many to form the 40S subunit.^56-57 This suggests that ribosome assembly is biphasic in yeast, with formation of the 40S ribosomal subunit preceding that of the 60S subunit.

Ribosomal RNA Processing

After the addition of the 2'-O-Me and ψ internal modifications and assembly with some of the ribosomal proteins, cleavage of the rRNA precursor begins. Usually this occurs after the termination of transcription and release of the pre-rRNA. An exception to this is Dictyostelium rRNA which begins its processing during transcription, as seen by electron microscopy of Miller spreads.⁵⁸

Stepwise Order of the Universal Cleavage Steps

Early pulse-labelling experiments revealed that the 40-45S pre-rRNA is processed through various intermediates to form the mature rRNA species.^77-79 Through a series of cleavages, the external and internal transcribed spacers (ETS, ITS) are removed from the precursor to liberate the mature 18S, 5.8S and 28S rRNAs. There is some flexibility to the order of the initial steps that ultimately result in mature 18S rRNA. As shown in Figure 1b, rRNA processing in Xenopus oocytes can follow either pathway A or pathway B. In pathway A, cleavage occurs first at site 3, separating the 5.8S and 28S rRNA coding regions in 32S pre-rRNA from the 18S rRNA coding region in 20S pre-rRNA. Pathway A is taken by HeLa cells for rRNA processing.⁸⁴ Alternatively, in pathway B, cleavages at sites A0, 1 and 2 occur first to generate 18S rRNA before cleavage at site 3. Pathway B occurs in Xenopus somatic cells,^85-86 mouse L cells,⁸⁶ Drosophila⁸⁷ and yeast (see Chapter 12). Sometimes, the pathway can even vary in the same cell type.^86,88-90 In one example, a temperature-sensitive mutant of BHK cells followed pathway A at 33.5°C and pathway B at 38.5°C.⁸⁸ Xenopus oocytes have either just pathway A or both pathways A and B.⁸¹ In the latter case, both pathways can co-exist in a single cell,⁸¹ indicating that the choice of pathway does not reflect some limiting component in the cell.

rRNA processing pathway choice seems to be influenced by U3 snoRNA.⁸² U3 snoRNA is the most abundant snoRNA and it is one of a few snoRNAs that are required for rRNA processing (also see Chapters 12 and 13). Chemical mutagenesis and phylogenetic comparisons of U3 snoRNA have resulted in a recently revised universal structural model that is conserved between yeast and higher organisms and consists of two domains (I and II) separated by two single-stranded “hinge” regions (fig. 2).⁹¹ A large complex containing U3 snoRNA and 28 proteins has recently been identified in yeast;⁹⁵ most of the proteins are associated with domain II of U3. U3 snoRNA is a member of the Box C/D family of snoRNAs, but it functions solely in rRNA processing and not in 2'-O-methylation. The Box C/D signature motif is required for its nucleolar localization^96-97 after traffic through the Cajal body.^97-99 Domain II of U3 snoRNA plays a role in cleavage at site 3,^81-82 but domain I and the hinge regions are also critically important for cleavages at sites A0, 1 and 2.^82,91,100

Figure 2

Model of secondary structure of Xenopus laevis U3 snoRNA. The secondary structure drawn here for Xenopus U3 snoRNA is universally conserved between yeast and higher organisms. This model is based upon compensatory base changes in phylogenetic comparisons (more...)

The two domains of U3 appear to compete with one another for the first cleavage step in rRNA processing.⁸² When domain I of U3 snoRNA is removed in Xenopus oocytes, cleavage at sites A0, 1 and 2 is prevented and all U3 activity is channeled into its domain II function to cleave site 3 as the first processing step. Conversely, when domain II function is impaired, then cleavage at sites A0, 1 and 2 is preferred as the initial event. These results suggest that pathway choice is stochastic and depends upon the orientation of U3 snoRNP when it lands on the pre-rRNA substrate—if the conformation favors domain II action, then pathway A will result, and if the conformation favors domain I action, then pathway B will result. Why do some frogs have just pathway A, whereas most have pathways A + B? There are about 20 copies of the U3 snoRNA gene per genome of Xenopus, and sequence microheterogeneity exists between the gene copies.¹⁰¹ It could be that one variant of U3 is expressed more in frogs with just pathway A, and other U3 variants are expressed in frogs with pathways A + B.⁸²

Formation of 18S rRNA

Although a few cleavages of pre-rRNA can be carried out in a test tube with nuclear extract, it has not yet been possible to recapitulate all the steps of rRNA processing in a sequential fashion in a cell-free system. Moreover, many ribosomal proteins are already assembled on pre-rRNA when processing occurs, yet in vitro reconstitution of rRNA with ribosomal proteins to form ribosomes has not yet been accomplished in eukaryotic systems. Therefore, much of what we know about rRNA processing mechanisms comes from in vivo studies in systems that can be experimentally manipulated—either yeast where genetic depletion and transformation with mutant forms of pre-rRNA and rRNA processing factors are possible (see Chapter 12) or Xenopus oocytes where endogenous snoRNA can be disrupted by RNase H after injection of antisense oligonucleotides, as first done for Xenopus U3 snoRNA,⁸¹ and subsequently mutated forms of the snoRNA can be injected.⁸²

A detailed description follows of the role of U3 snoRNA in rRNA processing to form 18S rRNA, and a cartoon of the processing steps is depicted in Figure 3, with enhanced detail in the figures that follow.

Figure 3

Xenopus U3 snoRNA and 18S rRNA processing. In the free form of U3 snoRNP, the 5' hinge and 3' hinge are single-stranded (panel 1). Compensatory base change experiments indicate that they base-pair with regions E2 and E1 of the ETS, respectively as shown (more...)

snoRNA Docking on the External Transcribed Spacer

U3 snoRNP associates with pre-rRNA primarily through protein-protein interactions¹⁰² but also through base-pairing.^103-104 In sedimentation ultracentifugation, U3 snoRNA is found over a broad range (ca 10S to 80S),¹⁰⁵ suggesting its association with both the initial rRNA precursor and various pre-rRNA intermediates. Chemical modification of accessible sites suggests that most U3 snoRNP in Xenopus is free,⁹² in contrast to U3 snoRNP in yeast that is primarily associated with pre-rRNA.¹⁰⁶ The association of U3 with pre-rRNA must be more stable or longer-lived than that of the other snoRNAs, as it is the only snoRNA that has been recovered by immunopreciptation of rRNA processing complexes.^56-57

U3 snoRNA Hinge Base-Pairing with the ETS

In Xenopus, proper docking on pre-rRNA that will allow subsequent U3 function in processing requires base-pairing (proven by compensatory base changes) between the 3'-hinge region of U3 and complementary sequences in the 5'-ETS (hereafter called simply ETS), while base-pairing between the 5'-hinge of U3 and the ETS is auxiliary but not essential (fig. 4).¹⁰⁷ Recall that the two hinge regions of U3 are single-stranded in the free U3 snoRNP (fig. 2) and therefore are available for base-pairing with the ETS. The 3'-hinge interaction with the ETS also has been validated in trypanosomes by cross-links¹¹¹ and ETS deletions that obliterated 18S rRNA production.¹¹² Interestingly, in trypanosomes there are two regions of the ETS (region 1b just downstream of cleavage site A' and region 3 upstream of cleavage site A0) that have been cross-linked with U3 snoRNA¹¹¹ and are both complementary to the 3'-hinge of U3.^112-113 Similar to the case in Xenopus, the ETS docking sites for the U3 3'-hinge are important for 18S rRNA production.¹¹³ In contrast, in budding yeast base-pairing between the 5'-hinge of U3 and the ETS is required.¹⁰⁸ In both Xenopus and yeast, base-pairing with the ETS is possible for the 5'-hinge and the 3'-hinge, but which hinge in U3 is preferred as more important for association with the ETS varies.¹⁰⁷ Compensatory base changes seem to have occurred during evolution, as base-pairing potential is maintained between the U3 hinges and the ETS although the sequence itself varies between distant species.⁹¹ Nonetheless, in closely related species, such as Xenopus laevis and Xenopus borealis, a few tracts of conserved sequences occur in the ETS, and these include the regions of the ETS that can base-pair with the U3 5'-hinge and 3'-hinge (tracts 5 and 3, respectively).^114-115

Figure 4

U3 snoRNA docking on pre-rRNA ETS. See Figure 3 and text for details.

Nucleolin Helps U3 snoRNP to Dock on Pre-rRNA

The abundant phosphoprotein nucleolin (also called C23 or 100 kD protein) binds pre-rRNA as soon as it is transcribed¹¹⁶ and can be detected by electron microscopy of Miller spreads.¹¹⁷ Nucleolin is needed for traffic of U3 from Cajal bodies to the nucleolus⁹⁸ and for the docking of U3 on the ETS.109 Interestingly, nucleolin promotes nucleic acid annealing,^118-119 and therefore may facilitate the base-pairing between the U3 snoRNA hinges and the ETS. In fact, nucleolin binds to UCGA,¹²⁰ located in an 11 nt (13 nt in mammals) evolutionarily conserved motif (ECM) GAUCGAUGUGG^121-122 that is found in a single-stranded region of the ETS.¹²³ This nucleolin binding site is near the ETS sequence that base-pairs with the 3'-hinge of U3 (fig. 5). The N-terminal end of nucleolin, containing multiple phosphorylation sites, is required for its interaction with U3 snoRNP, presumably through protein-protein contacts.¹⁰⁹ The nucleolin-mediated base-pairing of the vertebrate U3 3'-hinge with the ETS is not the only contact that holds U3 snoRNP on pre-rRNA, as U3 snoRNP is still found associated with pre-rRNA in sucrose gradients of RNP particles after disruption of the U3-ETS base-pairing by mutagenesis (Borovjagin and Gerbi, unpublished observations), although U3 cannot function in rRNA processing after 3'-hinge mutation because it is not properly positioned on the pre-rRNA substrate.¹⁰⁷ Moreover, U3 snoRNP cannot be recovered with a fragment of the ETS containing the consensus sequence and ~200 nucleotides downstream,¹²⁴ suggesting that additional contacts between U3 snoRNP and pre-rRNA are needed.

Figure 5

Conserved features of the ETS. An evolutionarily conserved motif (ECM; bold nt) contains the UCGA motif required for nucleolin binding. The ECM is downstream of the A' cleavage site (horizontal bars) in pre-rRNA. Nucleolin is required to load U3 snoRNA (more...)

Nucleolin plays still other roles in ribosomal biogenesis besides helping U3 snoRNP to dock on pre-rRNA. The central region of nucleolin has four RNA binding domains (RBD) which interact with pre-rRNA.^116-117 The nucleolin recognition element (NRE) is a consensus sequence (U/G)CCCG(A/G) in a stem-loop structure with at least 4 bp in the stem and 7-14 nt in the loop.^117,125 There are 33 putative NRE sites in human pre-rRNA, including 11 in the ETS.¹²⁵ NMR spectroscopy suggested that two RBDs of nucleolin bind on opposite sides of the loop, forming a molecular clamp which brings the ends together to form the stem.^126-127 This observation agrees with the earlier finding that nucleolin promotes annealing and the formation of helices.^118-119 Thus, it may act as a chaperone to fold nascent pre-rRNA.¹²⁶ Additionally, the N-terminal domain of nucleolin can interact with ribosomal proteins,^128-129 and it is tempting to speculate that it may help to load them on pre-rRNA.

Besides help by nucleolin, it has been suggested from data in yeast that the protein complex of Imp3p, Imp4p and Mpp10p helps to guide or facilitate docking of U3 through the hinge-ETS base-pairing.¹³⁰ Unlike most of the other proteins in the U3 snoRNP, the Imp3p, Imp4p and Mpp10p complex does not associate with domain II of U3. Instead, it has been proposed that it associates with the stem between the 5'-hinge and 3'-hinge (fig.2), since deletion (though, surprisingly, not sequence substitution) of this entire stem abolishes its binding to U3.¹³¹ The location between the hinge regions would position the complex favorably for its hypothesized role to facilitate the hinge-ETS base-pairing, but this proposal remains to be tested directly.

Cleavage at Sites A0, 1 and 2 to Form 18S rRNA

Cleavage at sites A0, 1 (= A1 in yeast) and 2 (or the different site A2 in yeast) requires U3 snoRNA.^82,100,141 In addition, cleavage at these sites require U14 snoRNA (in yeast^149-150 and in Xenopus^82,151), snR10¹⁵² and snR30¹⁵³ in yeast and U22 snoRNA in Xenopus.¹⁵⁴ Moreover, in higher eukaryotes, U17 snoRNA (=E1), is needed for site 1 cleavage and E2 for site 2 cleavage.¹⁵⁵ Mechanistic details of how these snoRNA work remain unknown, and there is no evidence if they work together with U3 in a hypothetical processing particle containing several snoRNAs^141,156 and dubbed the “processome”¹⁵⁷ similar to the splicosome that contains several snRNAs for splicing, or if the snoRNAs work independently of one another (as in the newer use of the term “small subunit processome” meaning the large complex of U3 snoRNA and its associated proteins⁹⁵). Normally, the cleavages at sites A0, 1 and 2 are coordinated, resulting in the production of mature 18S rRNA. However, recently it has been possible to uncouple these events in vivo by mutagenesis of U3 snoRNA in Xenopus.¹⁰⁰ Previous studies using mini-substrates of pre-rRNA revealed that site 1 cleavage does not require the 3'-end of 18S rRNA¹⁴⁴ and site 2 cleavage does not need the 5'-end of 18S rRNA,¹⁵⁸ thus supporting the idea that cleavage at these sites can occur independently even though they usually occur close in time to each other in vivo.

Cleavage at Site A0

Site A0 has only recently been discovered in higher organisms.¹⁰⁰ It reflects cleavage after A491 and A494 in the ETS and is 218 or 221 nt upstream of site 1 in Xenopus pre-rRNA,¹⁰⁰ comparable to the position of site A0 in budding yeast (90 nt upstream of site A1)¹⁴¹ and trypanosomes (116 nt upstream of site A1).¹¹² In all three organisms, site A0 is found near the base of a long stem and is opposite site 1 (or A1)(fig. 6). Due to this secondary structure, it was proposed that RNase III cleaved the stem at sites A0 and A1,¹⁶⁰ but this idea was subsequently disproven in yeast.¹⁶¹

Figure 6

Pre-rRNA cleavage at sites A0 and 1. Bases that form the pseudoknot in mature 18S rRNA are indicated by asterisks; U3 snoRNA is believed to prevent premature pseudoknot formation during ribosome biogenesis.^,, U3 Box A' is required for site 1 cleavage (more...)

Depletion of U3 snoRNA in yeast revealed that cleavage at site A0 is U3-dependent.¹⁴¹ A cross-link has been made in budding yeast between U3 snoRNA and nt 655 in the yeast ETS,¹³³ which is situated near the top of the stem that has sites A0 (nt 610) and site A1 (nt 699) at its base,¹⁶² further implicating U3 snoRNA for cleavage at sites A0 and A1. However, the portion of U3 snoRNA responsible for site A0 cleavage in yeast remains unknown, unlike the case for metazoa. Recently we showed that cleavage at site A0 in Xenopus pre-rRNA requires Box A of U3 snoRNA.¹⁰⁰ When Box A of U3 is mutated and site A0 cleavage is inhibited, 20S pre-rRNA accumulates but is not processed into 18S rRNA.¹⁰⁰ In this case, cleavage at sites 1 and 2 also is inhibited, suggesting that cleavage at site A0 precedes that at sites 1 and 2.¹⁰⁰ Consistent with this idea, inhibition of cleavage at sites 1 and 2 by mutation of the GAC-box A' element in Xenopus allows cleavage at site A0; in this situation, a novel 19S pre-rRNA intermediate is found which extends from site A0 to site 3 (fig. 1b).¹⁰⁰ Similarly, a novel 22S pre-rRNA intermediate is found in yeast when cleavage occurs at site A0 but not at sites A1 and A2.¹⁴⁰ This circumstance can be created in yeast by mutation of Box A of U3 snoRNA,¹⁵⁹ truncation of the U3-associated protein Mpp10p,¹⁶³ or depletion of the U3-associated helicase Dhr1p.¹⁶⁴

We suggest that Box A helps to position U3 snoRNP properly on pre-rRNA to allow U3-dependent cleavage at site A0 in Xenopus pre-rRNA. Intriguingly, bases at the 3'-end of Box A of U3 are complementary to bases ₄₈₂AGAAA₄₈₆ in the Xenopus ETS (fig. 6), and mutation of these bases in Xenopus U3 snoRNA inhibits site A0 cleavage.¹⁰⁰ Site A0 has not yet been mapped in other metazoa besides Xenopus, but in trypanosomes where its position is known,¹¹² it is also 8 nt downstream from 5 nt in the ETS that have the potential to base-pair with the same 3' portion of U3 Box A. Although this interaction cannot be drawn for yeast, it is noteworthy that mutation of U3 Box A does not inhibit site A0 cleavage in yeast,¹⁵⁹ unlike the case in Xenopus.¹⁰⁰ Regardless of whether this hypothesized base-pairing occurs between Box A and the ETS, Box A is positioned close to the A0 cleavage site by base-pairing of its 5'-end with a sequence near the 5'-end of 18S rRNA (fig. 6) to be discussed below.

Cleavage at Site 1

Most people believed that snoRNAs utilized for rRNA processing would base-pair with the transcribed spacers to assist in cleavage events. Therefore, it was a novel idea that Box A of U3 snoRNA might base-pair with sequences within the 18S rRNA portion of pre-rRNA,¹⁵⁹ as depicted in Figure 6. This concept was stimulated by the observations that the snoRNAs that guide modifications base-pair with sequences within 18S and 28S of the pre-rRNA. It was proposed that the 5'-end of U3 Box A base-pairs with sequences in the loop of stem adjacent to the 5'-end of 18S rRNA while it is part of the rRNA precursor.¹⁵⁹ In yeast, this interaction has been supported by relative sensitivities to chemical modification¹⁰⁶ and has been proven by compensatory base changes.¹¹⁰ Furthermore, it was hypothesized that the 3'-end of U3 Box A base-pairs with internal sequences in the 18S coding region of pre-rRNA.¹⁵⁹ In mature 18S rRNA, these internal sequences base-pair with the 5'-proximal loop to form a pseudoknot (fig. 7), and it was suggested that U3 Box A base-pairs with both of these regions to prevent premature pseudoknot formation.¹⁵⁹ Thus, U3 snoRNA would act as a chaperone and the pseudoknot would not form until U3 snoRNA departs at the end of rRNA processing. Although the proposed interactions of U3 Box A with 18S rRNA in the rRNA precursor are evolutionarily conserved,¹⁵⁹ compensatory mutations failed to prove the U3 base-pairing with the internal sequences in 18S.¹¹⁰ One possibility is that the 3'-end of U3 Box A might undergo a molecular switch in its pairing partners—pairing first with sequences in the ETS just upstream of the A0 cleavage site and subsequently pairing with the 18S internal sequences (fig. 5). In such a case, it would be necessary to create compensatory mutations at all three sites to restore 18S rRNA formation. Alternatively, base-pairing of the 5'-end of Box A with the experimentally proven 5'-proximal loop in the 18S region of the pre-rRNA would be sufficient to block premature pseudoknot formation, and the interaction of the 3'-end of U3 Box A with internal sequences in 18S might not exist. Additional experiments with compensatory mutations are needed to test these possibilities and to validate if they are applicable to higher eukaryotes.

Figure 7

Pre-rRNA cleavage at site 2. The 3'-end and flanking nt of U3 Box A' is required for cleavage at site 2 in Xenopus; base-pairing (dotted lines) is hypothesized to position it close to site 2. U3 snoRNA dissociates from pre-rRNA after cleavages at sites (more...)

The position of U3 Box A near site 1 is consistent with the observation in yeast that Box A is needed for site A1 cleavage.¹⁵⁹ In Xenopus, mutation of U3 Box A inhibits cleavage at site A0 and the subsequent cleavages at sites 1 and 2,¹⁰⁰ so it cannot be determined if the role of Box A on site 1 cleavage is direct or indirect for higher organisms. In addition, the GAC-Box A' element of Xenopus U3 snoRNA is required for cleavage at site 1.¹⁰⁰ A novel 19S pre-rRNA is found, extending from site A0 to site 3, when cleavage at sites 1 and 2 are prevented by mutation in Xenopus GAC-Box A'.¹⁰⁰ The sequences of Xenopus U3 Box A' are complementary to a region just downstream of the 5'-proximal stem of 18S rRNA (fig.5), and base-pairing would nicely position U3 Box A' close to site 1 where it is required for cleavage.¹⁰⁰ Base-pairing between the GAC element of U3 snoRNA and 18S rRNA that has been hypothesized for yeast¹⁵⁹ cannot be drawn for Xenopus, and the molecular interaction involving the GAC element, which is required for site 1 cleavage in metazoa, remains to be determined in higher eukaryotes.

The mechanism by which U3 snoRNA acts for cleavage at site 1 (and at other U3-dependent sites) is not yet clear. One possibility is that it is an RNA-based cleavage, with U3 snoRNA acting as a ribozyme. Another possibility is that a protein responsible for the cleavage is associated with U3, analogous to modification enzymes that piggyback with the guide snoRNA to be correctly positioned for action. A variant of this possibility is that the endoribonuclease may use U3 snoRNP as a landing pad. An appealing candidate for the latter is Rcl1p which associates transiently with U3 snoRNP in yeast,¹⁶⁵ perhaps through association with the G protein Bms1p.^166-167 Rcl1p is a member of the 3'-phosphate cyclase family that catalyze formation of a 2',3'-cyclic phosphodiester. This is extremely intriguing when considering the earlier report that in vitro cleavage at site 1 generates a 2',3'-cyclic phosphate as a first step and trimmed 3 nucleotides as a second step to generate the mature 5'-end of 18S rRNA.^145,168

It is apparent from the model in Figure 6 that spacing between various functional elements in U3 snoRNA and in its pre-rRNA substrate should be critical to allow the hand and glove fit between U3 snoRNA and the pre-rRNA. Consistent with this, a specified distance of the 5'-proximal stem to site A1 is required for site A1 cleavage in yeast.^169-170 Similarly, in Xenopus the distance between the 5'-hinge and 3'-hinge, between Box A and the 5'-hinge and the distance between domain I and domain II of U3 snoRNA is extremely important for 18S rRNA production.⁸² This suggests that the hinge-ETS base-pairing might be maintained while GAC-Box A' and Box A base-pair with sequences in the 18S region of pre-rRNA.⁸²

Formation of 5.8S and 28S rRNA

Cleavage at Site 3

Although site 3 is generally shown abutting the 5'-end of 5.8S RNA in maps of pre-rRNA from higher organisms, in fact cleavage seems to occur somewhat upstream (fig. 1b). S1 nuclease mapping demonstrated that site 3 is 161-163 nt upstream of the 5'-end of rat 5.8S RNA.¹⁷⁶ Similarly, site 3 cleavage in Xenopus pre-rRNA occurs about 100 nt before the 3'-end of ITS1,^100,154 refining earlier results.¹⁷⁷ Xenopus 20S, 19S and 18.5S pre-rRNAs all share the same 3' terminus located ~100 nt before the 3'-end of ITS1.¹⁰⁰ The data are consistent with a model where endonucleolytic cleavage occurs at this position and subsequent trimming by a 5'-exonuclease results in the true 5'-end of 5.8S RNA indicated as site 3' in Figure 1b, comparable to Rat1p and/or Xrn1p exonuclease trimming in yeast (see Chapter 16). Site 3' is found as the 5'-end of 32S pre-rRNA in mouse,¹⁷⁸ rat¹⁷⁶ and Xenopus,¹⁵⁴ but the presumed exonucleolytic degradation of the ITS1 tail between sites 3 and 3' seems by be slowed down when U22 snoRNA is disrupted.¹⁵⁴

Cleavage at site 3 is dependent on several snoRNAs. The first one to be discovered was U3 snoRNA, where its disruption decreased (but did not eliminate) cleavage at site 3 and consequently there were reduced levels of 20S and 32S pre-rRNAs.^81-82 The effect is dependent on U3 snoRNA, since efficient site 3 cleavage can be rescued by injection of U3 snoRNA.⁸² Domain II of U3 snoRNA is sufficient for its role in site 3 cleavage.⁸² As discussed above, U3 snoRNA is required for 18S rRNA formation and does not play a direct role in 28S rRNA production. This raises the possibility that U3-dependent site 3 cleavage in Xenopus may be analogous to U3-dependent site A2 cleavage in yeast which occurs within ITS1 and is part of the processing pathway to form 18S rRNA.¹⁴¹ In addition, E3 snoRNA is required for site 3 cleavage.¹⁵⁵ Moreover, U8 snoRNA is required for site 3 cleavage in Xenopus pre-rRNA.^177,179 In contrast to U3 snoRNA, U8 snoRNA is required for 5.8S and 28S rRNA formation but not for 18S rRNA.^177,179

Because of its apparent link between the 18S and 28S rRNA processing pathways in Xenopus, it is plausible that site 3 in higher organisms is a composite of sites A2 and A3 in yeast (fig. 1b). Site A3 is downstream of site A2 in the yeast ITS1, and cleavage at site A3 requires the MRP snoRNA in vivo^180-182 and in vitro.¹⁸³ Endonucleolytic cleavage at site A3 is followed by 5'-exonucleolytic digestion of 76 nt of ITS1 until the 5'-end of a short form of 5.8S RNA which predominates in wild type yeast. However, when MRP is depleted in yeast, the end point of exonucleolytic digestion differs and a longer form of 5.8S RNA predominates, which has 7 nt more at its 5'-end.^180-182 Long and short forms of 5.8S RNA also exist in vertebrates, differing by 6-7 nt at the 5'-end with the short form predominating¹⁷⁸ just as in yeast. Although MRP snoRNA exists in higher organisms, efforts by several groups to disrupt it by injection of antisense oligonucleotides into Xenopus oocytes have been unsuccessful, and thus its putative function for site 3 cleavage has not yet been established for metazoan systems.

Consistent with the hypothesis that site 3 might link the 18S and 28S rRNA processing pathways in higher organisms, these two pathways are linked in yeast by the protein Rrp5p^184-188 whose dual function is reflected in its bipartite structure (see Chapter 12). In yeast, Rrp5p genetically interacts with snR10,¹⁸⁹ but the homologous snoRNA has not yet been identified in metazoa.

U8 snoRNA Is Required for 5.8S and 28S rRNA Formation

Formation of 5.8S and 28S rRNA is impaired by disruption of U8 snoRNA in Xenopus oocytes.¹⁷⁷ This is the only snoRNA found so far that is needed for the 28S rRNA processing pathway, and yeast seems to lack a homologous snoRNA. Like U3 snoRNA, U8 snoRNA also is required for cleavage at several sites in pre-rRNA—specifically, sites 3, 4, 4', 5 and T1.¹⁷⁷ The 5' domain of U8 snoRNA is important for its function in rRNA processing.¹⁷⁹

The first of the U8-dependent cleavages is at site T1, and it occurs shortly after cleavage at site A' in mammals.¹⁹² rRNA transcription terminates at site T2 (or even further downstream at site T3 in Xenopus¹⁹³) and is rapidly processed to site T1 at the 3'-end of the 28S rRNA coding region in pre-rRNA. The sequence between sites T1 and T2 is called the 3'-ETS and ranges in length from 210 nt in yeast^194-195 to 235 nt in Xenopus193 to 565 nt in mouse.^196-197 The initial step in 3'-end formation of 28S rRNA in mouse involves removal of 10 nt just upstream of site T2¹⁹⁷ in pre-rRNA prior to subsequent events that result in T1 as the 3'-terminus. 3'-end processing has been carried out in vitro with yeast cell extracts and synthetic rRNA substrate, and micrococcal nuclease sensitive RNA is not required.¹⁹⁸ However, recall that yeast lacks U8 snoRNA.

The next U8-dependent cleavage occurs at site 3 near the end of ITS1 to form 32S pre-rRNA which is a long-lived intermediate. 32S pre-rRNA then undergoes cleavages to form 5.8S and 28S rRNA. U8 snoRNA is needed to form not only the 5'-end but also the 3'-end of 5.8S RNA. It is well documented in many organisms that cleavage within ITS2 (site 4' in fig. 1b) produces a precursor of 5.8S RNA that is longer at its 3'-end (yeast,¹⁹⁹ Drosophila,²⁰⁰ mouse,^90,178 rat^201-203). This 5.8S precursor is 12S in size in Xenopus oocytes and contains ca. 200 nt of ITS2.¹⁷⁷ Cleavage at site 4' fails to happen when U8 snoRNA is disrupted,¹⁷⁷ and it is possible that failure to observe cleavage at sites 4 and 5 reflects inhibition of cleavage at site 4' when U8 snoRNA is not present. Sites 4 and 5 may be formed by exonucleolytic trimming away from site 4' as in yeast rRNA processing (see Chapter 12), or by subsequent endonucleolytic cleavages at sites 4 and 5. Many of these exonucleases have been identified in yeast and include the exosome and additional proteins to form the 3'-end of 5.8S RNA (see Chapter 12) and Rat1p and/or Xrn1p to form the 5'-end of 28S rRNA.²⁰⁴ Up to 502 nt 28S rRNA sequences at its 5'-end are needed for site 5 cleavage, as studied in mammalian rDNA minigenes.¹⁷³ This span of 28S sequence includes areas complementary to both the 5' and 3'-ends of 5.8S RNA, but truncating the 28S further to only 217 nt abolishes cleavage at site 5.¹⁷³ Moreover, when a stable 2'-O-methyl oligoribonucleotide complementary to the 3'-end of 5.8S RNA is injected into Xenopus oocytes, production of 28S rRNA is inhibited.¹⁴⁷ Both of these results suggest that pairing between 5.8S and 28S rRNA may be required for cleavage at site 5 for rRNA processing. Surprisingly, and in contrast to this idea, site 5 cleavage occurs in mammalian rDNA minigenes in the absence of 5.8S sequence.¹⁷³ However, up to 326 nt of mammalian ITS2 is required,¹⁷³ and since this probably includes site 4', it suggests that site 5 cleavage may be dependent upon site 4'. The 5'-end of mammalian nuclear 28S rRNA is heterogeneous, extending a few nucleotides beyond the mature 5'-terminus.^178,205

E. coli pre-rRNA lacks an ITS2, and the 5'-end of 23S rRNA is homologous to 5.8S RNA.^76,206-207 Thus, the ITS2 appears to be an insertion that breaks the large subunit RNA into two pieces (5.8S and 28S rRNA). Ultimately, both ends of 5.8S RNA base-pair with the 5' area of 28S rRNA,^208-210 thereby holding these two fragments together in eukaryotes after the ITS2 has been removed by processing. It has been proposed that U8 snoRNA acts as a chaperone to prevent premature base-pairing between 5.8S and 28S rRNA,¹⁹⁰ as diagrammed in Figure 8. In this model,¹⁹⁰0 0the 5'-end of U8 base-pairs with the 5'-end of 28S rRNA, preventing this region from base-pairing with 5.8S RNA. The potential base-pairing between U8 snoRNA and 28S rRNA is necessary but not sufficient for rRNA processing, and human-Xenopus chimeric U8 constructs indicate that sequences beyond the 5'-end of U8 snoRNA also appear to be necessary for processing.190 An evolutionarily conserved bulge in 28S rRNA that is not paired with U8 might act as a nucleation site for initiation of base-pairing with the 3'-end of 5.8S RNA.190 This model remains to be proven by compensatory base changes, but, if correct, it would position U8 very close to cleavage sites 4 and 5 which are dependent on U8.

Figure 8

U8 snoRNA-mediated 5.8S/28SrRNA processing. The5'-end of U8 snoRNAcan base-pair with the5'-end of the 28S cod-ing region in pre-rRNA, thus preventing the latter from prematurelypairing with 5.8SRNA. A bulge in 28Sthat is not paired withU8 snoRNA might (more...)

Evolution of rRNA Processing

Pre-rRNA is found in all three kingdoms of life. Analogy exists between these pre-rRNAs. For example, RNase P is used to remove the intergenic tRNA from bacterial pre-rRNA, and its close relative, MRP snoRNA, is required for cleavage at site A3 in the yeast ITS1.²¹¹ There are other structural similarities between pre-rRNAs from the various kingdoms (fig. 9). In eubacteria, exemplified by E. coli, 16S and 23S rRNA each reside at the top of long base-paired stems where processing begins by RNase III cleavage.^212-213 The two stems are also found in pre-rRNA from archaebacteria,²¹⁶ but are considerably shorter in yeast²¹⁷ and do not exist in Xenopus pre-rRNA. It has been hypothesized that base-pairing between sequences in cis in eubacterial pre-rRNA has been replaced in eukaryotes by base-pairing in trans between snoRNAs and the termini of the rRNA coding regions in pre-rRNA.^83,211,214 As discussed above, U3 snoRNA might act as a molecular bridge to bring together the two ends of the 18S coding region in pre-rRNA.¹⁰⁰ Similarly, U8 snoRNA can base-pair with the 5'-end of the 28S coding region in pre-rRNA.¹⁹⁰ Recently it was reported that cleavage and ligation of the stem below 16S rRNA in archaebacteria results in the formation of a Box C/D-like snoRNA from the base of the stem and a covalently closed circular 16S rRNA.²¹⁵ Similarly, a small RNA, though lacking snoRNA motifs, is produced from the base of the stem leading to 23S rRNA in archaebacteria. Thus, in two kingdoms Box C/D snoRNAs are implicated in bringing together the 5' and 3'-ends of rRNA in the precursor.

Figure 9

Evolution of pre-rRNA. Pre-rRNA is compared between the three kingdoms of life. In eubacteria, represented by E. coli, pre-rRNA has two long stems which are cleaved by RNase III to generate 16S and 23S rRNA.^- In higher eukaryotes, such as Xenopus, pre-rRNA (more...)

It is not yet clear why all three kingdoms bother to have pre-rRNA, rather than just transcribing the mature forms of rRNA. The transcribed spacers seem to have some role. In fact, deletion of ITS2 is lethal in yeast.²¹⁸ We propose that the transcribed spacers are required for the proper folding of rRNA. In eukaryotes they are the docking site for snoRNAs like U3 that fulfill chaperone functions. In Schizosaccharomyces pombe they are also the binding site for various polypeptides that are part of the “Ribosome Assembly Complex” (“RAC”)^219-220 that might be involved in assembly and folding of the pre-ribosome before cleavages begin, reminiscent of the binding and putative function of nucleolin. RAC protein also directs the removal of the 3' ETS in S. pombe.²²¹ In archaebacteria, transcribed spacers in pre-rRNA are used instead of U3 snoRNA to base-pair with 16S rRNA and prevent premature pseudoknot formation.²²²

Several questions remain. Do the snoRNAs that guide modifications in pre-rRNA also act as chaperones for rRNA folding? Do the snoRNAs required for rRNA processing simply fold the rRNA precursor appropriately so that exposed regions are cleaved by exogenous nucleases? Or, do the nucleases piggyback along with the snoRNAs (or use the snoRNAs as a landing pad), analogous to the modification enzymes that piggyback along with the guide snoRNAs? Is there a required order for folding rRNA in the precursor, suggesting that snoRNAs should act as an ordered series? What are the roles in ribosome biogenesis of the numerous nucleolar proteins identified by proteomics, some of which are associated with the pre-ribosome?^{56-57,223-227} Why do some proteins that affect ribosome biogenesis also affect the cell cycle^228-234 including DNA replication?^235,236

The ribosome appears to function through a series of conformational changes. Thus, it is crucial that its RNA component be properly folded—a task that appears to be carried out by snoRNAs, ribosomal proteins and other proteins. Successful completion of the various folding steps may be demarcated by cleavage events to prevent the folding reaction from running in reverse. In this view, rRNA processing exists to ensure correct folding of an amazing macomolecular machine, the ribosome.

Acknowledgements

This chapter is dedicated to James T. McIlwain upon his retirement, with respect, affection and gratitude for his continuous support and forbearance. We thank Elaine Butler for her outstanding help with the references, and NIH for grants GM20261 (previously) and GM61945 (currently) supporting our studies on rRNA.

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