It has been known for several years that eukaryotic cells contain large populations of small nucleolar RNA-protein complexes, called snoRNPs, and that these complexes mediate the formation of modified nucleotides in rRNA and facilitate cleavage of rRNA precursors. Most snoRNPs fall into two large classes named for the snoRNA component, i.e., box C/D and H/ACA snoRNPs, and most snoRNPs in each family participate in nucleotide modification—synthesis of 2'-O-methylated nucleotides in the case of the C/D snoRNPs and pseudouridine in the case of H/ACA snoRNPs. In a remarkable departure from previously described reaction schemes, the modifications are targeted by guide motifs in the snoRNA and the reaction is catalyzed by a core snoRNP protein. Recent results make clear that the period of major discovery is still very much in progress. Novel snoRNA-like guide RNAs have been identified for splicing snRNAs and mRNAs, and a set of snRNA-specific guides has been discovered to reside in the Cajal bodies; the latter species are called scaRNAs and include unusual structural variants of the canonical snoRNAs. Studies of snoRNP biogenesis have characterized the major steps involved in snoRNA maturation, early events in snoRNP assembly, and shown that trafficking of new snoRNPs involves transit through Cajal bodies in animals and structurally related nucleolar bodies in yeast. Archaeal organisms have been determined to contain C/D and H/ACA guide RNAs (called sRNAs) and corresponding core proteins, indicating that the snoRNP machinery is of ancient origin; notably, Archaea contain guide RNAs for tRNA as well as rRNA. Opening the way for in-depth structure and function studies, guided modification has been achieved with archaeal and yeast cell-free systems. These and other major advances are reviewed in the present chapter.

Introduction

Nucleoli contain scores of small stable RNAs known as snoRNAs, an acronym for small nucleolar RNAs, and these RNAs exist as snoRNA:protein complexes called snoRNPs (‘snorps’, as reviewed recently).^1,2 The snoRNPs function in the maturation of ribosomal RNA (and other RNAs), by: 1) creating two types of modified nucleotides, i.e., 2'-O-methylated nucleotides (Nm) and pseudouridine (Ψ), and, 2) mediating endonucleolytic cleavages of pre-rRNA. Nearly all snoRNPs fall into two large families that are defined by pairs of conserved ‘box’ elements in the snoRNA component; the families are the box C/D and box H/ACA snoRNPs. Most box C/D snoRNPs create Nm modifications and most box H/ACA snoRNPs convert uridine to Ψ. In both types of modification, the target nucleotide is selected through base pairing of the snoRNA to the substrate (guide function), and an integral snoRNP protein catalyzes the reaction.

Cleavage of pre-rRNA requires a small number of box C/D and box H/ACA snoRNPs, and an additional snoRNP, called MRP, which does not belong to either of the box C/D or H/ACA families. The MRP snoRNP catalyzes an endonucleolytic cleavage, whereas the functions of the other processing snoRNPs are still obscure, although they also bind to rRNA. The snoRNP machines are of ancient origin, as clear orthologs are present in Protists and, strikingly, among the Archaea. The archaeal snoRNA-like RNAs are called sRNAs and the corresponding RNPs are known as sRNPs.^3,4

In addition to guiding nucleotide modification of rRNA, snoRNAs have been shown to target Nm modification in vertebrate U6 splicing snRNAs, and candidate guide snoRNAs have been discovered that are predicted to modify mRNA, in human brain and trypanosome transcripts.^5,6 In a startling recent development, a novel subset of snoRNA-like guide RNAs has been found to reside in the Cajal (coiled) bodies of animal cells; Cajal bodies occur in the nucleoplasm, often in close association with nucleoli.⁷ The new RNAs, designated scaRNAs (small Cajal body RNAs), are predicted to modify the U1, U2, U4 and U5 snRNAs, which occur transiently in this same compartment.⁸ In the archaeal kingdom, many candidate Nm guide sRNAs have been identified that are specific for tRNAs, a situation not yet known to occur in eukaryotes.^9-11

Several important new possibilities are suggested by these latter results: 1) a variety of non-rRNAs known or suspected to pass through the nucleolus could be substrates for snoRNPs; 2) the range of nuclear substrates could include many more mRNAs than have been implicated to date—as well as other RNAs, for example, the recently discovered tiny, non-coding RNAs thought to occur in vast numbers in eukaryotes;¹² 3) additional snoRNP-like machines might be discovered to function outside of the nucleolus, and; 4) the function of the snoRNPs may not be limited to modification and processing, but could also include chaperone-like functions in RNA folding or RNP assembly, or quality control.

In the present chapter, we will review the current state of knowledge about the snoRNAs and snoRNPs. Readers are also referred to other chapters in this volume on processing of rRNA (Chapters 11,12) and trafficking of other RNAs through the nucleolus (Chapter 17). In addition, several excellent reviews on the present themes are available.^1-3,13-18 Literature citations are through November 2002.

Early History

The term snoRNA was coined in 1981,^19,20 over 10 years after the first small nuclear RNAs were detected and the first nucleolar species was defined.^21,22 The U3 species was the first snoRNA described and also the first to be examined in detail. It was observed on fractionation of small RNA prepared from rat cell nuclei by gel electrophoresis.^23,24 Other RNA bands observed in the same analysis included the yet-to-be-recognized splicing snRNAs, and because all of the species detected were rich in uridine, all were given the U-designations in use today,^19,20 We now understand that U3 was the most abundant snoRNA in those preparations and that all eukaryotic cells contain scores of snoRNAs, as indicated in early reviews.^20,25 U3 is required for the first endonucleolytic cleavages of precursor rRNA²⁶ and appears to be ubiquitous among eukaryotes. Sequence analysis of the first few U3 snoRNAs isolated revealed several conserved ‘box’ sequences (A-D), and related secondary structures.^27-33 Box C/D elements were determined to be present in other small nuclear RNAs discovered some years later (1989) and this common feature was the origin of the box C/D nomenclature subsequently used to define one of the two large families of snoRNAs.³⁴

The first efforts to describe the population of nuclear small RNAs in yeast were reported in 1983 and 1988, using different gel electrophoresis fractionation strategies.^35-38 Subsequent characterization revealed the presence of splicing snRNAs and snoRNAs. Remarkably, virtually all of the several dozens of non-splicing RNAs detected³⁹ have turned out to be snoRNAs. The large family of box H/ACA snoRNAs was identified in yeast (in 1996), from comparative sequencing of nuclear small RNAs, and identified in humans soon thereafter.^39,40 Most snoRNAs described are from human and yeast cells, however, snoRNAs have been shown to occur in a variety of other metazoan organisms (e.g., mouse, rat, Xenopus and plants), and in protists as well, early in the history of snoRNA research in some cases. The cell systems featured most heavily in biosynthesis and function studies have been yeast, human, mouse and Xenopus. Excellent progress has also come from characterization of orthologous guide RNAs in archaeal cells.^9,41,42

More Recent Landmarks and Breakthroughs

Other major developments in the field include: 1) the discovery that snoRNAs can be encoded within introns of protein genes;⁴³ 2) evidence that snoRNAs are required for cleavage of pre-rRNA;^26,44 3) discovery of the guide functions in nucleotide modification;^45-47 4) revelation that the C/D and H/ACA box elements are sufficient for localization to the nucleolus; ^48-62 5) identification of the core proteins in the C/D and H/ACA families of snoRNPs;^63-72 6) the finding that at least some snoRNAs localize to the nucleolus by way of the Cajal bodies;^49,58,60,73 7) identification of brain-specific guide snoRNAs predicted to methylate mRNA, and a putative mRNA-specific Ψ guide for Trypanosome mRNA;^5,6 8) discovery of snoRNP orthologs in the Archaea;^{9,41,42,74-82} 9) good success in reconstituting core snoRNP complexes;^76,77,83-85 10) development of in vitro guided Nm and Ψ modification systems;^77,86-88 11) describing the proteins present in the MRP and U3 processing snoRNPs;^89,90 12) discovery of snoRNA-like guide RNAs that reside in mammalian Cajal bodies (called scaRNAs), which are expected to guide methylation and pseudouridylation of splicing snRNAs that also occur at this location,^8,86 and; 13) excellent progress in defining key steps in snoRNP assembly and trafficking in vivo.^{58-60,62,91-94} These and other developments are reviewed in the sections that follow, with emphasis on recent findings.

snoRNP Structure and Function

Individual snoRNPs consist of a single snoRNA molecule, a small set of stably associated, family-specific ‘core’ proteins and, in some, perhaps all cases, less tightly associated proteins. Additional proteins are known to be unique to particular processing snoRNPs involved in cleavage of pre-rRNA. In no case yet has a snoRNP been described that contains more than one snoRNA, however, remarkable exceptions to the typical snoRNA structure have been found among the snoRNA-like RNAs identified in the Cajal bodies (scaRNAs^8,86). This population includes hybrid Nm and Ψ guide RNAs, with targeting motifs for both types of modification, and covalently linked ‘twin’ snoRNAs that target a single type of modification (see below). It seems possible that novel RNA and RNP variants could also occur among the population of more conventional snoRNPs as well; variants are also known to occur in the Archaea (see below). Important progress has been made in characterizing snoRNP proteins, including: the core proteins of the C/D and H/ACA snoRNPs, and the full repertoire of proteins in two specialized snoRNPs involved in rRNA processing, the U3 and MRP snoRNPs.

C/D snoRNPs

Box C/D snoRNAs. The snoRNAs in the box C/D family contain at least one set of box C (PuUGAUGA) and box D (UCUGA) elements and commonly contain a second, degenerate pair designated boxes C' and D' (fig. 1).⁹⁵ Most C/D snoRNAs and snoRNPs function in ribose methylation of rRNA, and quite likely other RNAs in the nucleolar compartment (see below and Chapter 17). A few others are involved in processing (cleavage) of rRNA, and some of these participate in both processing and modification. The canonical C/D boxes are usually near the 5' and 3' ends of the mature snoRNA sequence, respectively (the U3 processing snoRNA is an exception), and form a characteristic helix-asymmetric bulge-helix structure.^72,96 This motif, which was first identified in the structure of human U4 snRNA, also occurs in rRNA. The classic C/D elements influence several overlapping aspects of snoRNA and snoRNP synthesis and function, including: 1) binding of core snoRNP proteins;^{15,31,72,76,77,85,100,101} 2) providing metabolic stability;^{49,56,57,102-104} 3) defining the 5' and 3' ends of the mature snoRNA;^49,105,106 4) hypermethylation of monomethylated 5' caps, where these occur;^57,59,107 5) localizing the newly synthesized snoRNA to the nucleolus and Cajal bodies,^48-62 and; 6) 2'-O-methylation activity in the case of the Nm guide snoRNAs.^45,95 These functions are almost certainly related to binding of the core proteins, and perhaps other, unidentified shared proteins.

Figure 1

Structures of the box C/D, box H/ACA and MRP snoRNAs. Consensus structures are shown for the three known classes of small nucleolar RNAs. (A) The snoRNAs in the C/D family have a characteristic C/D folding motif (kink turn, flanked by stem I and stem (more...)

The only other RNA structural feature known to be common, but not universal to all C/D snoRNAs is the presence of one or two guide sequences that target methylation (fig. 1). These are located on the 5'-side of box D or D' (1-2 nts upstream), and consist of long (9-21 nt) sequences that are complementary to the substrate region to be modified.^{45,95,108-110} Methylation occurs at a substrate site located five (and sometimes six) nucleotides upstream of box D or D', typically 4-5 nucleotides within the region of complementarity. Some C/D processing snoRNAs also contain guide elements, for example the U14 species, which is universal among eukaryotes, is needed for both processing and methylation of 18S rRNA.^111,112 The fact that two guide motifs have been identified in some snoRNAs and only one in others suggests that new, non-rRNA substrates may be found, or that non-guiding elements may be intermediates or stable end-products in snoRNA evolution. In principle, the antisense sequences could also be involved in chaperone-like functions (see below).

C/D snoRNP proteins. Four core proteins are stably associated with the C/D snoRNAs (Table 1) and these are believed to be universal among eukaryotes.^63-72 Clear orthologs of these proteins also occur among archaeal organisms as well.^{9,41,74-77,81} The eukaryotic core proteins were first identified as snoRNP components in yeast and humans, and are usually referred to in other organisms (including the Archaea) by the human designations. The yeast (and human) proteins include: Snu13p (NHPX, also 15.5 kDa); Nop1p (fibrillarin); Nop56p (hNop56p), and; Nop58p (hNop58p).

Table

Core snoRNP proteins.

Interestingly, Snu13p/NHPX also occurs in U4 snRNP complexes and in both types of RNPs interacts directly with a K-turn folding motif.^61,72,96-98 Nop56p and Nop58p are closely related, exhibiting 41% identify in yeast and 37% identity in humans, and the single archaeal variant (Nop56/58p) is closely related to both, suggesting all have a common ancestor.^67,70,75 Another distinction for the Archaea is that the counterpart of Snu13p/NHPX is a ribosomal protein (aL7a).^3,76,77

Nop1p/fibrillarin is widely accepted to catalyze the methylation reaction. Although this has not yet been demonstrated directly, the case is compelling. Key evidence includes: 1) the presence of four short sequence elements conserved among several types of methylases that utilize S-adenosylmethionine as the methyl donor;^113,114 2) results showing that point mutations in the motif-containing region of yeast Nop1p can block formation of Nm modifications in rRNA in a global manner,^114,115 and; 3) remarkable similarity of the 3-D structures of an archaeal ortholog of Nop1p (from M. jannaschii) and several methylases, over most of the length of the protein.¹¹⁴ Notably, the human autoimmune disease, scleroderma, is characterized by autoantibodies targeted to fibrillarin.¹¹⁶ Information about the assembly and interactions of the core proteins is provided below, in a section devoted to snoRNP assembly.

Properties of the C/D snoRNPs. Beyond the four core proteins, the actual numbers and types of proteins in the natural C/D snoRNPs are still obscure. It seems likely that the subset of processing and modifying snoRNPs have different compositions, reflecting their different, specialized functions, however, information on this point is also sparse. Nor is it known to what extent the individual processing or modifying snoRNPs are related at the protein level. Most information on these issues comes from characterization of the U3 box C/D snoRNP, and the MRP snoRNP (see below), which play highly specialized roles early in rRNA processing.

Early genetic and immunological studies of U3 snoRNPs identified nearly a dozen U3-associated proteins unique to the yeast or human particle, as cited elsewhere.⁹⁰ Those findings were extended very substantially by recent direct analysis of yeast U3 complexes, isolated by an affinity purification procedure.⁹⁰ More than 30 proteins associated with the snoRNP complex were detected and identified by microsequencing. In addition to the four C/D core proteins, 23 of 24 other proteins thought to be specific were found to be essential for growth and 18S rRNA production. It remains to be seen if the list of proteins is complete; three proteins that had been identified by others as U3-associated were not observed. Conversely, some proteins may not be integral snoRNP proteins, but are part of the pre-rRNP maturation complex. Key fractionation steps included enrichment of C/D snoRNPs by selection of a tagged C/D core protein, followed by selection of a tagged U3-specific protein.⁹⁰

H/ACA snoRNPs

Box H/ACA snoRNAs. Similar to the C/D snoRNAs, a few H/ACA snoRNAs are involved in processing of rRNA, however, most guide Ψ formation, and cases exist where a snoRNA has both functions. The RNAs in this family are distinguished by: 1) two types of short box elements that can vary in sequence, but are positionally conserved in the secondary structure, and; 2) a consensus secondary structure consisting of two characteristic helix-bulge-helix domains separated by a hinge region Figure 1.^39,40 The signature H (hinge) and ACA boxes occur downstream of the distinguishing folding domains. Because of the variable nature of the box elements, secondary structure analysis is an important aid in identifying snoRNAs in this class. Another complication in identifying these RNAs is the fact that the secondary structures are not limited to the consensus features and other folding domains can be present as well; this is a substantial complication for the yeast snoRNAs, which are typically much larger than those from vertebrates and other sources.

The H/ACA boxes influence synthesis and function of both the snoRNAs and corresponding snoRNPs, as is the case for the C/D boxes. In particular, the H/ACA boxes are, variously, involved in: 1) providing metabolic stability;^39,40 2) defining the ends of the mature snoRNA (ACA);^39,40 3) localization to the nucleolus and Cajal bodies,^53,54 and; 4) pseudouridylation, in the case of the Ψ guide RNAs.^46,117 These effects are believed to be a consequence of protein binding, in particular the core proteins, but perhaps others are involved as well.

The Ψ guide snoRNAs contain pairs of short guide sequences (3-10 nts) in the bulge region of one or both folded domains.⁴⁷ Site selection involves: 1) base pairing of the two guide sequences with substrate nucleotides that flank the uridine to be isomerized, and; 2) a distance measurement (14-16 nts) between the target uridine and corresponding H or ACA box element.^46,47 As for the methylation guide snoRNAs, a single Ψ guide snoRNA can use one or both of the guide domains to target modification. Time may reveal the actual modification capacity to be larger than currently understood, and that the snoRNAs have additional functions.

H/ACA snoRNP proteins. Four core proteins have also been identified for this family of snoRNPs and, these too are thought to be common to all family members (Table 1). The yeast (and human) proteins include: Cbf5p (dyskerin), Gar1p (hGar1p), Nhp2p (hNhp2p), and Nop10p (hNop10p).^118-120 Catalysis of Ψ formation is almost certainly mediated by Cbf5p/dyskerin based on: 1) the presence of three signature sequence elements conserved among known Ψ synthases,¹²¹ and; 2) global disruption of Ψ synthesis in yeast rRNA when point mutations were introduced into these elements in Cbf5p.¹²²

Properties of the H/ACA snoRNPs. Among the H/ACA snoRNP proteins, Gar1 and Nhp2p are known to interact directly with H/ACA snoRNAs.^83,123 The Nhp2p protein has the interesting property of being related to the C/D snoRNP protein Snu13p/15.5 kDa, which binds to the C/D motif.⁷² The sequences of the two yeast proteins are 38% identical and 61% similar, which could reflect the presence of common binding domains. No K-turn has yet been identified in any H/ACA snoRNA, however, similar motifs occur in Archaeal H/ACA sRNAs (see below), and are recognized by the archaeal L7 protein, which also binds to the K-turn of C/D sRNAs.^3,4,124 Interactions between the H/ACA proteins and snoRNA are described below.

The first attempt to isolate and characterize an individual H/ACA snoRNP was made with a yeast complex required for rRNA processing (snR30); the resulting particle contained seven stably associated proteins.¹²⁵ Examining the gel pattern retrospectively, it appears that all four of the known core proteins were present, plus three others. Additional proteins may exist in the natural snoRNP, which has a sedimentation coefficient of about 12S.¹¹⁹ Electron micrographs of the isolated complex revealed a V-shaped particle, which is consistent with the bipartite consensus structure of the H/ACA snoRNAs. The dimensions of the particle were estimated at 15 nm long and 12 nm wide.¹¹⁹

The MRP snoRNP

Complexes containing this RNA were first reported to function in processing of RNA primers involved in mitochondrial DNA replication, and this is the origin of the MRP designation (mitochondrial RNA-processing). Subsequently, the RNA was determined to occur in the nucleolus ^126-128 and to be required for cleavage of pre-rRNA, in the ITS-1 segment, upstream of 5.8S rRNA^44,129-131 (also see Chapters 11,12). The MRP snoRNA lacks the box elements that distinguish the two major classes of snoRNAs, however, its secondary structure is remarkably similar to that of RNase P RNA, inferring that it may be a ribozyme.^132-134 This view was strengthened with the discovery that many of the proteins in the yeast MRP and RNase P enzyme complexes are the same, and that an isolated yeast MRP particle is able to cleave pre-rRNA in vitro, in a site-specific manner.¹³⁵ Although ribozyme activity has not been demonstrated directly, nine proteins have been identified in the yeast RNase P complex and eight of these are present in yeast MRP as well.^89,136-143 Interestingly, RNase P also occurs in the nucleolus, where it mediates processing of tRNA.^128,144-148 The MRP snoRNA has a K-turn,⁹⁸ but the signficance of this is not yet known. The MRP snoRNP has been linked to cell cycle regulation,¹⁴⁹ and implicated as the causal agent in cartilage-hair hypoplasia.^150,151 Autoantigens from certain scleroderma patients recognize MRP snoRNP proteins.^152,153

What Roles Do the C/D and H/ACA snoRNPs Play in rRNA Cleavage?

It's clear that several snoRNAs in addition to MRP are required for cleavage of rRNA transcripts, and most have been shown to interact directly with rRNA through base pairing (see Chapters 11,12). The roles of these other snoRNPs are still obscure, but possible functions include: 1) recruiting a nuclease and perhaps other factors to a cleavage site, and; 2) serving as a chaperone to organize pre-rRNA for cleavage, by either a self-cleaving mechanism or a trans-acting nuclease that does not associate with a snoRNP. The processing functions of the snoRNPs were identified first, in both animal and yeast cells, and all but one of the four yeast processing snoRNAs (U3, U14, snR30, snR10) are essential for growth; the exception is snR10.¹⁵⁴

In addition to MRP, only two other processing snoRNPs are believed to be universal among eukaryotes, the U3 and U14 complexes (both are C/D species), and it remains to be seen if these occur among the Archaea. U3 and U14 are both required for cleavages that occur early in processing, and certain cleavages require both snoRNPs (and others), arguing that one or both play organizational rather than catalytic roles;^111,155,156 as reviewed elsewhere.¹⁵⁴ Two other processing snoRNPs, U8 and U22, are common to vertebrates,157 but homologs have not yet been found in yeast. The U8 snoRNP is required for cleavages of precursor 5.8S-28S rRNA and activity appears to involve regulation of pre-rRNA folding, through binding with the U8 snoRNA.¹⁵⁸

The U3 snoRNP is believed to be the first to bind to a nascent pre-rRNA transcript, near the 5' end, and is thought to play a critical organizational role in forming an active processing complex^90,159,160 (see also Chapters 11,12). The snoRNA is more abundant and considerably larger than most other snoRNAs and has an atypical structure; in this last regard, box C is located deep in the sequence, rather than at the 5' end as with other C/D snoRNAs.^{31,49,52,53,55} As noted above, the U3 snoRNP contains many more proteins than other characterized snoRNPs and most appear to be unique to the U3 complex.⁹⁰

Progress with in vitro processing systems that feature snoRNPs is still at an early stage. Key advances include demonstrations showing that processing is disrupted in animal cell extracts when several individual snoRNAs (U3, U8, U13, U14, U17/E1, E2, E3) are depleted by RNase H or immunoprecipitation,^26,156 and that processing by the yeast MRP snoRNP can be achieved in a simple reaction mix containing in vitro transcribed yeast rRNA and affinity purified MRP complex.¹³⁵

Modification of rRNA and U6 snRNA by snoRNPs

The possibility that snoRNAs may be involved in targeting nucleotide modification was raised when long sequence elements complementary to rRNA were identified in several C/D snoRNAs.^25,108,161 Chronologically, the methylation guide function of the C/D snoRNAs was discovered initially,^{45,109,162-164} and then, the Ψ guide function of the H/ACA snoRNAs.^{46,47,162,165} It seems that the populations of the large C/D and H/ACA families are roughly comparable in size and that nearly all participate in modification. In yeast, nearly all guide snoRNAs examined - and thus, snoRNPs, are dispensable; the exceptions are dual function snoRNAs that participate in both processing and modification, i.e., the box C/D species U14 and the H/ACA species snR10. There is no evidence that snoRNAs play a role in methylation of RNA bases, which is the third major category of nucleotide modification in rRNA and other RNAs.

Several factors argue that most Nm and Ψ modifications in rRNA are formed by snoRNPs. These include: 1) the solid impression that the snoRNA populations in yeast and humans are sufficiently large to accommodate the number of modifications known to occur—knowing that many snoRNAs target two modifications, and; 2) identification of a set of C/D guide snoRNAs in yeast by a computational approach, that accounts for nearly all Nm modifications in rRNA.¹¹⁰ The number of Nm modifications in yeast and human rRNA are 55 and 105, respectively, and the number of Ψ modifications are 44 and 91.^166-168 For yeast, 20 guide snoRNAs have been identified experimentally that are able to serve 27 sites of Ψ modification. ¹⁶⁹ While in principle, all Nm and Ψ modifications in cytoplasmic rRNA could be formed by snoRNPs, exceptions can also reasonably be expected.

In a surprising exception at the time, snoRNPs were shown to also be capable of methylating the human and S. pombe U6 splicing snRNAs.^170-172 The evidence included identification of snoRNAs with U6 guide sequences^170-172 and demonstrating that one such snoRNA services both U6 and rRNA from the same guide element.¹⁷⁰ In addition, modification was observed when a U6 sequence was expressed in animal cells, as part of an rRNA minigene, and in yeast cells when an animal C/D guide RNA was targeted to yeast U6.¹⁷¹

Important progress has occurred recently in developing cell-free systems to characterize the RNA-guided modification processes. The first breakthrough was establishing a successful Nm modification system from reconstituted archaeal components.^4,77 The complex was assembled at elevated temperature, from an in vitro transcribed guide RNA and recombinant C/D core proteins, in the presence of a fragment of rRNA. The substrate rRNA was methylated at the expected site with excellent efficiency, also at elevated temperature.

Using an alternative approach, successful in vitro Nm and Ψ modification systems have been established with affinity-enriched natural snoRNPs from yeast. Success was achieved initially for Nm modification of an rRNA fragment with a preparation of bulk C/D snoRNPs,⁸⁸ and then for formation of Ψ in a fragment of rRNA with bulk H/ACA snoRNPs.⁸⁷ In both cases, modification occurred at the expected natural site. These early achievements demonstrate that characterization of the guided modification processes can now be pursued with both natural and reconstituted RNPs, important advances in the field.

Effects of the Nm and Ψ Modifications

Addition of Nm and Ψ modifications is known to alter the folding properties of RNA, in addition to the sequence. As a generalization, both types of modification enhance structural stability in a localized way, by reducing conformational flexibility.¹⁷³ Methylation blocks H-bonding potential of the ribose and also protects against hydrolysis of the internucleotide bond. Hydrogen bonding potential is also altered in Ψ formation, due to gain of an additional H-donor. Thus, depending on location, individual modifications or clusters of modifications have the potential to affect a wide variety of activities of the product RNA. Changes can be expected in rates and order of RNA folding, conformational stability of individual folding domains, and in the activity of the final RNA. Modifications in rRNA can hypothetically affect any stage of ribosome synthesis, ribosome activity, and turnover of the ribosome. Similar types of effects can be expected for other RNAs containing these modifications.

The availability of ribosome crystal structures has made it possible to place the known nucleotide modifications in rRNA in a three-dimensional context.^174-176 The 3-D maps show that ribosome regions known or predicted to be important for function are rich in modifications. In yeast, substantial numbers of modifications also occur in regions with no known direct function in translation, and the same situation is predicted for the prokaryotic archaeon Pyrococcus horikoshii.^4,174 Taken together, the patterns are consistent with modifications affecting different aspects of ribosome synthesis and activity.

Many individual Nm or Ψ modifications have been blocked in yeast by depleting cells of individual guide snoRNAs, and with no strong affect on growth. In one study, depletion of multiple Ψs in the reaction center region of the large subunit showed synergistic, negative effects on growth, and blocking formation of a single Ψ in the A-loop caused the rate of in vivo translation to drop by 20%.¹⁷⁷ Results from the various depletion analyses and previous studies in E. coli¹⁷⁶ argue that most single modifications have small, positive effects, but the full repertoire is highly beneficial. That view is supported by findings showing that disrupting Nm or Ψ modification in a global manner, with enzyme point mutations, have very strong, negative effects on cell growth rate.^115,122

Do Modifying snoRNPs Influence Other Aspects of Ribosome Synthesis?

At the simplest level, the snoRNPs that mediate modification can be considered nano-scale machines that create modified nucleosides and have no other effect on the target RNA. However, it is also reasonable to consider that modifying snoRNPs might affect RNA folding or RNP assembly as well, either directly or indirectly (fig. 2). This notion was strengthened substantially following discovery of the first several C/D snoRNAs with antisense sequences, which were later determined to guide modification. It was suggested that such snoRNAs might function as chaperones in ribosome synthesis, to bring order to the complicated processes of RNA folding and assembly of ribosomal subunits.^25,108,161 Such effects could be relevant for only a few snoRNPs or perhaps involve a large number. This hypothesis remains very interesting and attractive, however, firm, supporting evidence is still lacking.

Figure 2

Do modifying snoRNPs influence rRNA folding and ribosome assembly? In principle, binding and release of modifying snoRNPs can promote or block specific pre-rRNA folding events, and fine-tune protein binding processes. Some snoRNPs or modifications could (more...)

Continuing in this vein, a chaperone function by a snoRNP could, in principle, be the dominant basis for selecting and maintaining a modifying snoRNP in a cell, in a situation where the resulting modification may or may not provide any benefit. A conserved snoRNP (U14) that mediates both processing and methylation has been implicated in rRNA folding, through potentially concomitant binding at two distant segments in 18S rRNA.¹⁷⁸ However, it is too soon to know if this or any other modifying snoRNP has effects beyond its modification function. For snoRNPs that mediate processing, but not modification, the evidence for roles in rRNA folding is stronger, especially for the U3 and U8 species (see also Chapters 11,12).^158,179-185

When Do snoRNPs Act?

Early studies of rRNA modification show that unprocessed precursors have a high content of 2'-O-methylations, indicating that most modification reactions occur before cleavage; this situation was observed for yeast^186-188 and animal cells^189-192 as reviewed previously.¹⁶⁸ However, it is not known if modification occurs during or after transcription. The details of this situation promise to be very interesting, as large numbers of ribosomal and non-ribosomal proteins also associate with pre-rRNA at an early stage, and binding and release of snoRNPs must be coordinated with the rRNA folding and assembly events. Good progress is being made in characterizing pre-rRNP intermediates from yeast^160,193-197 and from animal cells.¹⁹⁸ Results from protein composition analyses with yeast complexes suggest that assembly of the precursors to the small and large subunits occurs in rather distinct stages, prior to processing. From the patterns, it appears that a quite-mature form of the small subunit is created while assembly of the large subunit is still at a very early stage,¹⁶⁰ as reviewed elsewhere.¹⁹⁹

Thus far, only the U3 snoRNP has been detected in the pre-ribosomal RNP complexes analyzed, in 90S and 60S yeast complexes (with variable yields in the latter), which either reflects low abundance or absence of the other snoRNPs from these intermediates.^160,194,197 Detection of other snoRNPs may be a problem in any case, if the half-life of a snoRNP:rRNA complex is short, or the interactions are not biochemically stable. Because the total number of snoRNPs involved in modifying and processing rRNA is very substantial—probably at least 60-70 for yeast and twice as many for humans, it would seem simplest mechanistically, for most modifying snoRNPs to bind and act early during transcription, while ribosome assembly is at a very early stage. Post-transcriptional binding scenarios can be imagined as well, where snoRNP binding sites in pre-rRNA are still exposed or, less attractive, made accessible through large scale remodeling of the pre-rRNP complexes (fig. 3).

Figure 3

When do snoRNPs act? Binding of processing and modifying snoRNPs is coordinated with rRNA folding, binding of ribosomal and non-ribosomal proteins and formation of precursor ribosomal subunit complexes. The U3 processing snoRNP binds near the 5' end (more...)

The case for snoRNPs acting early is stronger yet when the relative volumes of the snoRNPs and a fully condensed ribosomal subunit are considered. Estimates of snoRNP size are still at an early stage, but the aforementioned electron microscopic analysis of the snR30 (processing) and snR42 (modifying) snoRNPs from yeast indicate these complexes have substantial volume. The dimensions of the bipartite structures translate into a volume that is roughly 15% of the 80S ribosome (6 × 10⁵ų vs. 4.3 × 10⁶ų, based on different methods).^119,200 Even if a generic modifying snoRNP is somewhat smaller, as expected, it seems likely that snoRNPs complete their tasks before the pre-rRNP substrate is very much condensed, either during or after transcription; condensation of the individual precursor rRNPs could occur after the snoRNPs have acted. In this context, snoRNPs or the resulting modified nucleotides could play roles in staging assembly events, and a subset of snoRNPs or rRNA modifications might function in quality control. Some protein-only rRNA modifying enzymes in yeast and E. coli have already been reported to have essential, non-modifying functions, i.e., the protein, but not the modification is important; the vital functions have not yet been defined^201-203(and T. Mason, personal communication).

Related RNAs and RNPs

A growing number of snoRNA-related RNAs exist that have striking similarities and striking differences to the snoRNAs that function in eukaryotic rRNA synthesis. The related species include: 1) C/D and H/ACA guide RNAs for rRNA and tRNA in the Archaeal kingdom,^9,41,42 2) familiar and new guide RNA forms that reside in Cajal bodies,⁸ 3) telomerase RNA which has features of H/ACA snoRNAs,²⁰⁴ and; 4) guide RNAs predicted to target mRNAs.^5,6

Archaeal Guide RNAs

Hints of the presence of box C/D snoRNA homologs in Archaea have been available since the discoveries that rRNA of some Archaeal species contain numerous Nm modifications^205,206 and that archaeal genomes have clear orthologs of fibrillarin and Nop56p/Nop58p.^{9,68,74,207,208} More recently, modification guide RNAs were described in Archaea. Immunoprecipitation and cloning of archaeal RNA associated with fibrillarin and Nop56p/58p orthologs,⁹ as well as computer-assisted genomic searches,^9,41,42 revealed the existence of scores of box C/D-like small RNAs. Evidence that these RNAs are actual guide RNAs came initially from documentation of Nm modifications at the expected sites in natural rRNA, and subsequently in tRNA as well^9,10,41 and reviewed elsewhere.^3,4,11 Because Archaea are thought to lack nucleoli or an equivalent structure, these RNAs are not formally snoRNAs and were thus called sRNAs.⁹

Box C/D sRNAs appear to be widespread among archaeal species, having been detected in the two phyla, Crenarchaeota and Euryarchaeota, and in a variety of species within these phyla.^9,11 It thus appears that the box C/D RNA family is extremely ancient, predating the appearence of splicing snRNAs, and may be as ancient as the tRNAs and RNAse P; in addition, this RNA family appears to be very highly conserved. The sequences of the box elements and the general fold of the RNA are nearly identical between Archaea and Eukarya, and archaeal box C/D sRNAs are assembled properly and can guide methylation when introduced into Xenopus oocytes, despite separation of approximately 2 billions years between the two organisms ²⁰⁹.

However, systematic comparison of the C/D sRNAs and snoRNAs has also revealed some characteristic features for each family.^3,9,11,41,42 First, the eukaryotic RNAs are, in general, larger than their archaeal counterparts, with sizes of about 75 and 100 nucleotides in yeast and mammals, respectively, compared to an average of 50-60 nts for Archaea. Second, only about 20% of eukaryotic snoRNAs guide ribose methylation from both boxes D and D', and in these cases the substrates are often different rRNA molecules. In contrast, the majority of archaeal RNAs appear to direct rRNA methylation from both box D and box D', and this often occurs at nearby sites in the same rRNA molecule. Interestingly, the number of C/D sRNAs among different species increases with optimal growth temperature,⁹ consistent with the notion that C/D RNPs might act as chaperones to help RNA folding and that the general role of the modifications is to stabilize active rRNA structures. Additional support for a stabilizing role for the modifications (or sRNPs) comes from a study showing that the content of Nm nucleotides in the 16S rRNA of one archaeal species (Sulfolobus solfactaricus) increases with growth temperature.²⁰⁶

Two other aspects of the archaeal C/D guide RNAs are particularly interesting. Strikingly, the C/D guide motif can occur within tRNA introns,^3,9,11,41,42 demonstrating that the link between snoRNA expression and introns is very ancient. In addition, the occurrence of archaeal C/D guides for tRNA indicates that guide RNAs target more than one type of RNA in ancient organisms. The fact that no tRNA modification guides have yet been found in eukaryotes suggests that the repertoire of RNAs altered by guided modification may have been larger in the past. Remarkably, nucleotide modification by an intronic sRNA (sR50) appears to occur in cis,¹⁰ meaning that the sRNA sequences target 2'-O-methylation of the pre-tRNA while embedded within it! This unusual situation suggests that some C/D RNAs may have evolved from tRNA introns, perhaps as a machine that modifies tRNA or helps tRNA folding. In another interesting case, a C/D RNA was found in the non-coding region of precursor rRNA,²¹⁰ suggesting that it may act in cis in rRNA synthesis. Thus, box C/D RNAs could have evolved from spacer sequences in ribosomal genes, consistent with the fact that archaeal C/D sRNAs utilize a ribosomal protein, L7a, as a core component of the RNP.^76,77

Very recently, archaeal orthologs of H/ACA snoRNAs were also described.⁴² It seems likely that these assemble with proteins similar to the eukaryotic counterparts, as orthologs of Nop10p, Gar1p and Cbf5p also occur in the Archaea.⁸² The content of pseudouridines in various archaeal rRNAs is low, and much less abundant than in eukaryotes.^176,211 Consistent with this situation, only a few archaeal H/ACA sRNAs have been identified thus far (four, in Archaeoglobus fulgidus).⁴² These RNAs appear to be true modification guides, as well, since Ψ occurs at the predicted positions in rRNA. The structures of the H/ACA sRNAs can differ significantly from those of the canonical eukaryotic guides, in some cases containing a single hairpin ending with the ACA triplet and no box H domain, or three guide-like motifs.⁴² It should be noted that the single-hairpin cases are not unprecedented as similar guide RNAs also occur in Trypanosomes, an early-branching protist.²¹² In an interesting development, the archaeal L7 protein, which binds to the K-turn in C/D sRNAs, has been shown to bind a similar motif in H/ACA sRNAs, located a short distance above the pseudouridylation pocket.¹²⁴

Taken together, the discoveries that both the C/D and H/ACA RNAs predate the split between Archaea and Eukarya, and that both show intimate relationships with tRNA and rRNA maturation, suggests that they may have originated from extremely ancient organisms, and may have facilitated RNA biogenesis as the RNA World evolved.

Small Cajal Body RNAs (scaRNAs)

Completion of the human genome sequence has greatly facilitated ‘RNomics’ studies aimed at characterizing the non-coding RNAs in cells. One of the outcomes of these studies has been the description of a novel family of modification guide RNAs.⁸ The first member of this family, U85,⁸⁶ has several unique features (fig. 4): 1) it is significantly longer than other vertebrate snoRNAs (330 nts versus an average of 100); 2) it contains the motifs characteristic of both the box C/D and H/ACA families—its structure corresponds to an H/ACA snoRNA embedded within a C/D snoRNA(!), and; 3) most importantly, U85 possesses guide sequences that are not complementary to rRNA, but to U5 snRNA. As predicted, an in vitro experiment showed that U85 can, indeed, target Ψ formation in U5.⁸⁶ It was later determined that U85 is the founding member of a novel family of guide RNAs involved in nucleotide modification of the snRNAs transcribed by RNA polymerase II.⁸ While some of these RNA are not chimerae, and have consensus structures of C/D or H/ACA snoRNAs, they have a second important property in common: they are absent from nucleoli, but concentrated in Cajal bodies. Hence, they were renamed scaRNAs for small Cajal body specific RNAs.

Figure 4

Structures of snoRNA-related RNAs. (A) Schematic of U85, the founding member of the scaRNA family. (B) Secondary structure of vertebrate telomerase RNA. Conserved regions are in grey, and conserved nucleotides appear as white dots. The pseudoknot region (more...)

The location of these RNAs argues that they mediate their functions in the Cajal bodies as well. Indeed, Cajal bodies have been known for several years to accumulate high levels of snRNAs.^213-217 More recent studies showed that snRNPs localize in Cajal bodies in the process of re-entering the nucleus following cytoplasmic assembly, and before distributing to speckles. ²¹⁸ This trafficking pattern is in line with recent data suggesting that snRNAs undergo nucleotide modification following nuclear re-import.²¹⁹

The U6 snRNA is an exception to this scheme of snRNA modification, as it is not exported to the cytoplasm, but instead localizes transiently to nucleoli.^56,220 Accordingly, the aforementioned modification guides for this RNA are bona-fide snoRNAs, with a predominant nucleolar localization at steady-state.^170,171 The splicing snRNAs of S. cerevisiae constitute a second exception to this rule. Cajal bodies do not exist in this organism and modification of the snRNAs appears to be catalyzed by protein-only enzymes.²²¹ Furthermore, the snRNAs of this yeast have been suggested to transit through the nucleolus to undergo maturation, instead of passing through the cytoplasm.⁵⁹ While some modifications could be guided by snoRNAs, none have been reported to date.

Telomerase RNA

As noted above, the RNA component of vertebrate telomerase RNP belongs to the family of H/ACA snoRNAs.²⁰⁴ This RNA provides docking sites for the reverse transcriptase and its substrate sequence. It is localized in the nucleolus^53,204,222 and can be immunoprecipitated with antibodies against the core proteins of the H/ACA snoRNPs.^84,223,224 These snoRNA properties appear to be peculiar to vertebrate telomerase, since yeast telomerase RNA belongs to the family of snRNAs and binds Sm proteins.²²⁵ Vertebrate telomerase RNA does not appear to catalyze nucleotide modification of rRNA or other RNA, and its secondary structure is slightly divergent from the classical H/ACA snoRNAs^226,227 (fig. 4).

These discoveries have a number of important implications. First, they show that the C/D or H/ACA motifs can be utilized as stability and localization elements, distinct from any role in RNA maturation. Second, they indicate that even though the RNP is localized in the nucleolus at steady-state, it likely performs its functions elsewhere in the nucleus, since telomeres are elieved to be elongated in the nucleoplasm. Third, these findings also suggest that localization of the RNA is regulated, possibly during the cell cycle. Consistent with this possibility, a recent localization analysis of telomerase reverse transcriptase in human nuclei showed that in primary cells the enzyme is localized in nucleoli except at the time of telomere replication.²²⁸ In addition, in transformed cells, or in primary cells expressing large T antigen, the reverse transcriptase is delocalized to the nucleoplasm, but can become re-routed to nucleoli in response to double-strand DNA breaks, most likely to block telomere elongation at these sites.²²⁸ While the absence of detailed information on the localization of telomerase RNA is clearly a gap that needs to be filled, the results of these early studies reveal a number of new possibilities for snoRNA functions in the nucleus.

Mutations in human dyskerin and in telomerase RNA itself have been correlated with a disease known as Dyskeratosis congenita.^223,229,230 Symptoms of the disease include premature aging of rapidly dividing cells, early death from bone marrow failure, increased susceptibility to cancer and other problems. The actual basis of the disease is not yet known, but in principle could reflect defects in ribosome biogenesis, in telomerase function, or both.²²³,2^29-235 Results from one study indicate that loss of telomerase function is not the primary defect in mice deficient in dyskerin, as cellular changes associated with the disease are observed well before defects in telomerase function are detected.²³⁵ These results argue that not only impaired telomerase function, but also impaired formation of Ψ in rRNA may cause the disorder, although other effects related to dyskerin function may also be possible.

Tissue-Specific, Imprinted snoRNAs

Another startling outcome of recent RNomics approaches came from characterization of brain-specific snoRNAs.^5,236 While many of the snoRNAs that were cloned by this approach fall into the canonical families of modification guide snoRNAs,²³⁷ some clearly do not guide modification of either rRNA or snRNA, as they lack sequence complementarity with those RNAs. Further analyses revealed that a few have a unique feature that is in contrast to all snoRNAs characterized to date: these RNAs are expressed only in a few tissues, and mostly in the brain or particular areas of the brain.⁵ For instance, one mouse RNA (MBI-36) is mostly expressed in the chorioid plexus. To date, brain-specific snoRNAs have been characterized mainly at two loci in mammals: 14q32 and 15q11q13 in human, and at syntenic loci in mice and rats.^5,236 The 15q11q13 locus is especially interesting because the snoRNA genes are located in a region that is often mutated in Prader-Willi and Angelman syndromes, disorders that are characterized by developmental, behavioral and mental problems. This region expresses 6 brain-specific box C/D snoRNAs that are conserved between mammals,⁵ and two of them are repeated 27 (HBII-85) and 47 times (HBII-52). All these snoRNAs are expressed from the introns of a gigantic primary transcript (fig. 4), which spans more than 460 kb!

A similar genomic organization is also found at the second locus. At the rat locus syntenic to 14q32, a gigantic primary transcript is produced that contains an intron-exon unit that is repeated about a hundred times, and the intron contains a box C/D snoRNA (RBII-36).²³⁶ The syntenic mouse and human loci also contain repeated box C/D snoRNAs, but these are quite divergent from RBII-36.²³⁸ Interestingly, the putative guide-sequence of RBII-36 is not very well conserved between the repeats, suggesting lack of strong selective pressure on these elements. This may be related to another unique characteristic of these snoRNAs, the imprinted state of their genes. Transcription of the snoRNAs at 14q32 occurs only from the maternal allele, while that of 15q11q13 occurs from the paternal one.^5,238,239 Non-coding RNAs are often found in imprinted regions,^240,241 and in some cases have been demonstrated to play a direct role in the establishment or maintenance of the imprinted state of neighboring protein genes.^242-244 Thus, this situation suggests that brain-specific snoRNAs may play a role in the imprinting process. Large amounts of RBII-36 snoRNA precursors accumulate at the transcription site (J. Cavaillépersonal communication), and it's possible that these transcripts provide docking sites for chromatin remodeling factors that could repress expression of the neighboring genes.

Remarkably, some of these brain-specific snoRNAs have the potential to target methylation to certain mRNAs that are also expressed in a tissue specific manner. Indeed, the guide sequence of one (MBII-52) is complementary to 18 nucleotides in 5-HT2c mRNA (fig. 4)⁵, an isoform of the serotonin receptor mRNA that is highly expressed in the chorioid plexus. While this correlation may be fortuitous, several facts suggest that the complementarity is functionally significant.⁵ First, the guide sequence of the snoRNA and its putative target sequence in HT2c mRNA is conserved between human and mouse. Second, this sequence is located just a few nucleotides upstream of an alternative splice site in the HT2c mRNA, in a region that is heavily edited in vivo. In fact, the nucleotide that is potentially methylated corresponds precisely to one of the edited nucleotides. Since 2'-O-methyl groups have been shown to efficiently inhibit editing in vitro,²⁴⁵ these results suggest a possible role for 2'-O-methylation in mRNA regulation. Finally, the HT2c pre-mRNA itself contains an H/ACA snoRNA in its first intron, and has a long 5'UTR that might function as an internal ribosome entry site. This unusual structure suggests that this mRNA could follow a special trafficking pathway in neural cells. However, despite these fascinating observations, it is not yet known if the HT2c mRNA is methylated in vivo.

At least one of the brain specific snoRNAs (RBII-36) accumulates in nucleoli, based on in situ hybridization results.²³⁶ Importantly, nucleolar localization does not preclude a role for these RNAs in the nucleoplasm, as exemplified by the behavior of telomerase RNA.^222,246 Indeed, studies of the mobility of both fibrillarin and Snu13p/15.5kD within live cell nuclei showed that despite nucleolar localization at steady-state, these box C/D core proteins exchange rapidly between the nucleolus and the nucleoplasm.^61,247 The mean residency time in nucleoli is only about one minute, suggesting that the snoRNP is able to explore the entire volume of the nucleus in relatively short times.

Just What Is a snoRNA, Anyway?

The occurrence of these different types of box C/D and H/ACA RNAs challenges the present system of defining snoRNAs and predicting roles of snoRNA-like RNAs in the cell. In addition, the existence of these RNAs clearly demonstrates that while they may have originated from chaperone or nucleotide modification devices acting in cis, they have taken up a number of new functions during evolution. Indeed, the C/D and H/ACA RNA folds are simple and could provide powerful means to process, stabilize and localize RNAs, distinct from their functions in nucleotide modification. It seems all but certain that future studies will reveal an even greater diversity of roles and functions for these classes of small (and not-so-small) RNAs.

Biogenesis of snoRNPs

Production of snoRNPs is a complex matter, involving intricate control of many synthesis and assembly reactions, and trafficking of proteins and nascent RNP complexes through different sub-cellular compartments, as reviewed recently1 and see below. The snoRNAs are transcribed in the nucleoplasm, typically from coding sequences within introns, and the proteins are imported from the cytoplasm. One or more snoRNP core proteins are believed to bind to the pre-snoRNA molecule, followed by nucleolytic processing by a variety of enzymes. At least some and possibly all of the nascent snoRNPs then move to specialized nuclear bodies, i.e., the Cajal bodies in animal cells and functionally related nucleolar bodies (NBs) in yeast.

The scaRNPs remain in the Cajal bodies, to function there, and the nucleolar species move on to that structure.^8,86 At nuclear locations not yet defined, snoRNAs also undergoes nucleotide modification, and the final assembly steps of the mature snoRNP take place. Residency in the nucleolus may be temporary as well, as results suggest that snoRNPs may cycle between this structure and the Cajal bodies, as demand for nucleolar function waxes and wanes. Some of these processes are tightly coupled and, not surprisingly, production of snoRNPs is linked to production of ribosomes, as reviewed previously.^{1-3,17,154,248-250}

Synthesis of snoRNAs

The coding sequences for snoRNAs occur in a variety of genomic arrangements, and the types and relative abundance varies among organisms, as reviewed earlier.^1,3,249 In vertebrates, most snoRNAs are derived from coding units that are embedded within introns of protein genes; early lists appear elsewhere.^{25,40,43,45,108,164,237,251-254} However, other snoRNAs are generated from independent genes, such as U3; pseudogenes for U3 also exist.²⁵⁵ Processing snoRNAs are encoded in both arrangements, whereas the guide snoRNAs appear to be largely, if not exclusively specified by intronic coding units. Notably, some intronic snoRNAs are encoded in mRNA-like transcripts that are not translatable.^157,256-261 The aforementioned class of brain-specific snoRNAs characterizes yet another arrangement, where intronic coding units are organized in large, tandem repeats.

The situation is different in yeast, where a small minority of snoRNAs is encoded in introns, and the others are transcribed from genes that produce snoRNAs only.^25,254 This last class consists of both monocistronic and polycistronic coding schemes, and includes multimeric precursors containing from two to seven snoRNAs, as cited for representative cases.^{37,110,169,262} Plants have all of these arrangements and polycistronic coding units seem to be common.^263-270 Remarkably, one plant transcription unit encodes a tRNA in addition to several copies of a single snoRNA.²⁷¹

Among the Archaea, most coding units for the known and predicted C/D guide sRNAs occur in intergenic regions, and a few overlap with an ORF, raising questions about how the latter snoRNAs would be produced3 ,^9,11,41,42 As noted above, coding sequences for guide sRNAs also occur in tRNA introns and, in transcribed spacers of rRNA; the latter encoded as half-molecules, which are joined after cleavage of the pre-rRNA.^10,210 If the coding units for the C/D RNAs arose from a single ancestral source, ancient coding units for pre-tRNA and rRNA must be viewed as strong, logical contenders. Speculating about the origin of the H/ACA snoRNAs is more difficult, because of a much smaller genomic database.

Following transcription, precursor snoRNAs undergo nucleolytic processing by a variety of endo- and exonucleases.^272-277 Processing of intronic snoRNAs appears to occur in most cases after the intron has been excised and debranched, with 5' and 3' exonucleases carrying out the final trimming reactions.^274,278,279 However, production can also occur when debranching is blocked, mediated by endonucleolytic cutting followed by exonucleolytic trimming. In one gene system, alternative pathways exist, in which processing yields either mRNA or snoRNA.²⁷⁷ For the C/D snoRNAs in mammals, the distance of the intron from the branch point (k70 nts) is important, implying that interference can occur between formation of the splicing and snoRNA maturation complexes.¹⁰⁶ Besides end-processing, snoRNAs also undergo nucleotide modification, however, the relationship of processing and modification is still obscure.^25,35,280 Moreover, the identities of the factors that catalyze modification of the snoRNAs themselves are not known. Are they protein-only factors or do dedicated RNPs perform these tasks? Does this occur in the nucleolus or in another nuclear compartment such as the Cajal bodies?

For precursors generated from mono- or polycistronic operons, processing involves the concerted action of endonucleases, which liberate smaller precursors and these, in turn, are trimmed as needed by exonucleases. Exceptions occur for a subset of snoRNAs with monomethylated caps at the 5' end, such as U3; in these cases the cap becomes trimethylated and trimming is limited to the 3' end.^{60,104,107,281} Yeast U3 is unusual in that it contains an intron that is also removed during maturation.²⁸² It seems clear that binding of proteins to the precursor snoRNAs is essential for proper processing and for metabolic stability. For the C/D and H/ACA snoRNAs, these properties are believed to result from binding of core proteins to the corresponding motifs, but other, non-snoRNP proteins may also be involved, for example, in RNP assembly, nuclease recruitment and quality control (see below).

Excellent progress has been made in defining processing pathways for yeast snoRNAs and the events and activities seem largely conserved in vertebrates. Key steps and enzymes involved include: 1) the endonuclease Rnt1p, a homolog of the bacterial RNase III enzyme, which cleaves hairpin duplexes that precede or follow a snoRNA;^272,283,284 2) two 5'-> 3' exonucleases, Rat1p and Xrn1p,^274,279 and; 3) 3'-> 5' exonucleases that are part of the exosome, including Rrp6p.275,276 In some cases, trimming of 3' trailer sequences is initiated not by Rnt1p, but by an endonucleolytic cleaving activity that also cleaves pre-mRNA, at the site where poly-A formation takes place.^277,285

snoRNP Assembly

Core proteins. Assembly of box C/D snoRNPs occurs as a series of ordered events. The first protein to bind is Snu13p/NHPX, which recognizes specifically and directly the K-turn formed by the non-canonical GA.AG base-pairs in the box C/D motif.⁷² The crystal structure of human NHPX complexed with the U4 snRNA 5' stem-loop shows that its binding induces a 150° bend in the phosphodiester backbone.⁹⁶ Results from in vitro binding studies with snoRNAs demonstrate that this protein is absolutely required for assembling the other C/D core proteins, fibrillarin, Nop56p, and Nop58p, suggesting that the RNA conformational change induced by Snu13p/NHPX binding is essential to create the other binding sites.

A recent study using crosslinking agents inserted at specific locations showed that the U.U pair in internal stem II directly binds snoRNP proteins; the U in box C contacts Nop58p, and the one in box D is in contact with fibrillarin.²⁸⁶ Importantly, the C'/D' motif contained by most box C/D snoRNAs is much less conserved than the box C/D motif,⁹⁵ and is thus unlikely to bind NHPX independently in a stable manner. The cross-linking results further showed that the C/D motif in the RNP complex was in proximity to fibrillarin and Nop58p, while the C'/ D' motif associates with fibrillarin and Nop56p. Thus, the eukaryotic box C/D snoRNPs could adopt a pseudo-symmetrical structure, with NHPX, fibrillarin and Nop58p at the box C/D motif, and fibrillarin and Nop56p at the box C'/D' motif. To compensate for the absence of Snu13p/NHPX at the box C'/D' motif, binding of fibrillarin and Nop56p could be stabilized by protein-protein interaction with partners located at the box C/D motif. This model is consistent with effects of point mutations in stem II of the C/D motif, which suggest that Nop56p binds together with fibrillarin and independent of Nop58p.¹⁰¹ The results are also in agreement with in vivo studies of box C/D snoRNP assembly in yeast, which showed that depletion of fibrillarin prevented binding of Nop56p, but not Nop58p.⁹⁴

The box C/D motif thus appears as a bipartite structure, with the K-turn formed by the GA.AG base pairs binding NHPX, and specific, conserved nucleotides in stem II assembling with fibrillarin and Nop58p. The box C'/D' motif likely provides a secondary site to assemble with Nop56p and a second molecule of fibrillarin. A pseudo-symmetrical structure for the C/D snoRNA would be consistent with both the structure of the archaeal box C/D sRNAs, which contain a much better conserved C'/D' motif and have a single homolog of Nop56p and Nop58p,^9,41 and with the fact that both the box C/D and C'/D' motifs can direct methylation of target sequences (but see below).

Distinct from the assembly situation for the animal snoRNPs, an in vitro study of archaeal sRNP complex formation revealed a simpler pattern of protein binding (E.S. Maxwell, personal communication). The smaller set of core proteins bound to both the C/D and C'/D' motifs in a symmetrical manner, with all three proteins present at both sites. In vitro methylation assays showed that each individual RNP domain is active with adjoining guide sequences, but maximal activity required the presence of both the C/D and C'/D' RNP complexes. The asymmetric protein binding patterns observed for the eukaryotic snoRNP could reflect increased specialization of the larger set of binding proteins, and possible divergence of the structures of the C/D and C'/D' binding domains. It seems possible that some eukaryotic guide RNAs might have the same symmetry as archaeal sRNAs, in particular snoRNAs that use both the C/D and C'/D' motifs for methylation reactions.

An important crystal structure has been reported for an archaeal fibrillarin-Nop56/58 complex (with S-adenosyl methionine). The results show direct interaction of the proteins in a pre-formed heterodimer, and association of these complexes to form a dimeric heterodimer. The heterodimers are linked through interaction of extended coiled-coil elements at the carboxy end of Nop56/58, and fibrillarin protein interacts with Nop56/58 at its amino end. In the homodimeric heterodimer the fibrillarin molecules are located at opposite ends of the extended 4-protein complex. This structure provides a model for placement of methylase proteins at both the C/D and C'/D' motifs.²⁸⁷

Box H/ACA snoRNPs contain one protein related to NHPX, i.e., Nhp2p.^119,120 This protein also binds RNA, however, the binding site in the snoRNP is not yet defined precisely. Results from in vivo studies in yeast have established that Nhp2p, Nop10p, and Cbf5p are all required for metabolic stability of the snoRNA, in contrast to Gar1p, which is required for noRNP function, but not stability. This situation suggests that Nhp2p, Nop10p and Cbf5p may form a complex that collectively generates RNA binding specificity.

As noted above, the only homolog of Nhp2p in the Archaea is the ribosomal protein L7, and recent work suggests that it is part of the archaeal box H/ACA snoRNPs.¹²⁴ The archaeal RNAs differ from the eukaryotic counterparts by having a kink-turn motif placed 5-11 nt above each pseudouridylation pocket, which provides the docking site for L7. Thus, similar to the case for the box C/D snoRNAs, L7 may play a key role in nucleating snoRNP assembly of archaeal box H/ACA snoRNAs. In eukaryotes, the lack of stringent specificity of Nhp2p binding may be compensated by a more strict arrangement of boxes H and ACA. Consistent with this possibility, the RNA folding models and electron microscopy images of a yeast box H/ ACA snoRNP (snR30) showed a bipartite structure, which is presumed to correspond to proteins bound to the two hairpins in the RNA.¹¹⁹ In Archaea (and Trypanosomes), this bipartite structure is not conserved, and a variety of arrangements that contain from 1 to 3 hairpins have been found.⁴²

SnoRNP assembly factors. A number of proteins have been shown recently to function as snoRNP assembly factors. Interestingly, several of these are involved in production of both C/D and H/ACA snoRNPs.¹ The most conserved are the p50/p55 proteins (Tip48, Tip49 in humans, Rvb1p, Rvb2p in yeast), which are inter-related and known to have DNA helicase activity. These proteins are found in Archaea, yeast, and humans, and they have been linked to a number of processes, including DNA repair and transcription, as cited elsewhere.^75,288 While p50/p55 are not present in the mature snoRNP and are not localized in nucleoli, results from a genetic depletion study in yeast demonstrate that they are required for the biogenesis of both C/D and H/ACA snoRNAs.²⁸⁸ Importantly, the levels of the mRNAs that encode snoRNAs within introns are not affected at the time of snoRNA loss, pointing to a post-transcriptional role for these proteins.

In vitro studies demonstrated that p50 and p55 assemble onto a model C/D snoRNA.^75,85 Complex formation requires Snu13p/15.5 kDa binding, specific nucleotides in the box C/D motif, and appears to occur when Nop58p joins the snoRNP complex.¹⁰¹ Both p50 and p55 possess ATPase consensus sequences, and mutations in these elements lead to both temperature sensitive growth, and a concomitant mis-localization of C/D snoRNAs at the non-permissive temperature. Several possible roles can be envisioned for these proteins, for example, they could chaperone snoRNP assembly, carry assembled snoRNPs to nucleoli, or alternatively, prevent nucleolar localization until needed.

Another important protein recently suggested to function in the assembly of both box H/ ACA and C/D snoRNPs is SMN, the survival of motor neuron protein.^289-291 SMN is an essential protein that is conserved in many species, including fission yeast, invertebrates and vertebrates, but which is absent from S. cerevisiae.²⁹² SMN is an oligomeric protein that has been shown to be essential for cytoplasmic assembly of splicing snRNPs, where it functions as a chaperone to mediate assembly of the heptameric ring of Sm proteins around the Sm binding site in the snRNA.²⁹³ The SMN protein is also present in the nucleus in structures called gems, which often overlap with Cajal bodies.^294,295 Recently, it was shown that SMN binds in vitro and in vivo with both fibrillarin and Gar1p, through domains containing RG repeats.^289,290 In addition, a dominant-negative mutant of SMN was shown to block the nucleolar accumulation of human U3 snoRNA,^289,290 providing good support to the idea that SMN plays a role in snoRNP biogenesis, possibly by providing a platform to assemble snoRNP proteins onto the RNA.

Two other proteins, Naf1p and Shq1p, are also essential for the biogenesis of yeast H/ACA snoRNPs.^91-93 However, in contrast to p50/p55 and SMN, these proteins do not affect accumulation of C/D snoRNAs (or other stable RNAs). Naf1p and Shq1p form a complex and they interact in vitro and in two-hybrid assays with at least two protein components of the H/ACA snoRNPs, i.e., Nhp2p and Cbf5p. Naf1p also binds in vivo to H/ACA snoRNP proteins and several H/ACA snoRNAs. However, only small amounts of these components were recovered in co-immunoprecipitation assays, suggesting that they are not part of the mature snoRNP. In agreement, the major portions of these proteins localize to the nucleoplasm, and small amounts detected in the nucleolus are excluded from the dense fibrillar compartment, the site where mature H/ACA snoRNPs accumulate and are thought to function. Naf1p has the interesting property of binding to the phosphorylated CTD of RNA polymerase II and binds RNA through a domain similar to that of Nrd1p, an RNA binding protein required for 3'-end formation of snRNAs and snoRNAs in yeast.²⁸⁵

These data suggest a model in which Naf1p and Shq1p recruit Nhp2p and Cbf5p to the transcribing RNA polymerases, bind newly synthesized RNA and assist snoRNP assembly. A role in the transport of the snoRNP toward the nucleolus is also possible, given that a small fraction of these proteins is nucleolar. It is also of interest that Shq1p interacts with Rnt1p in two-hybrid assays, suggesting a possible role in coupling snoRNA processing with RNP assembly. Moreover, Rnt1p interacts with Gar1p in vivo and in vitro, and is required for its nuclear import²⁹⁶ providing further evidence of possible links between RNA processing, RNP assembly and transport. Clearly, these intricate, coupled processes need to be dissected in detail. It should be noted that Naf1p and Shq1p have homologs in many species, including mammals, raising the possibility that the roles of these proteins in H/ACA snoRNP biogenesis is widespread in metazoans.

Trafficking and Localization

Localization pathways. Early studies of snoRNA trafficking showed that the box C/D and H/ACA motifs are sufficient to target a reporter RNA to the nucleoli of yeast, Xenopus oocytes, and mammalian cells.^48,49,53,54 More recently, the roles of individual proteins in snoRNA trafficking have been analyzed in genetic studies in yeast.⁵⁸ Depletion of each of the C/D core snoRNP proteins leads to a partial mislocalization of snoRNAs to the nucleoplasm. This finding suggests that the putative nucleolar localization signal is split and that the snoRNP is stabilized in the nucleolus by multiple weak interactions, consistent with the absence of highly efficient nucleolar localization signals in the snoRNP proteins themselves. However, other interpretations are also possible, for example, quality control mechanisms could block mis-assembled snoRNPs from entering the nucleolus.

Interestingly, in vertebrates, reporter RNAs containing C/D or H/ACA motifs are also localized to Cajal bodies,^48,49,53,54 a structure that is biochemically and spatially related to nucleoli.^297,298 Time-course experiments following injection in Xenopus oocytes showed that box C/D snoRNAs first localize in Cajal bodies, and only later to nucleoli.⁷³ This important result demonstrated that snoRNAs follow a specific intranuclear route. This was the second evidence for trafficking pathways within the nucleus, following the demonstration that splicing snRNAs localize in Cajal bodies after entering the nucleus, and before they become distributed in speckles.²¹⁸ The fact that the trafficking of these two RNA species flows through the same compartment suggests an important role for Cajal bodies in sorting molecules within the nucleus. In the case of the H/ACA snoRNAs, it is not clear if localization in Cajal bodies reflects a localization pathway, as they seem to reach nucleoli and Cajal bodies at about the same time.⁵³ In this case, the flow of RNA molecules may be too fast to be resolved. It is also possible that these RNAs follow a more complex route, or simply do not follow a single pathway, but have two destinations.

Cajal bodies and snoRNP assembly. The transient localization of C/D snoRNAs to Cajal bodies suggests that some steps of snoRNA maturation take place there. A recent study that focused on U3 snoRNA maturation showed that this is likely to be the case.⁶⁰ Similar to the situation in yeast, mammalian U3 is synthesized as a precursor that contains a 3' extension of a few nucleotides and a monomethylated cap. Interestingly, this precursor is assembled with Snu13p/15.5 kDa, but not with fibrillarin or Nop58p. Furthermore, this precursor is present at the snoRNA transcription site and within Cajal bodies, but is excluded from nucleoli. In contrast, mature U3 RNA, which bears a trimethyl cap structure, is detected both in Cajal bodies and in nucleoli. Since all C/D core snoRNP proteins are present in Cajal bodies, these data suggest that U3 snoRNPs are preferentially assembled in Cajal bodies, consistent with the concentration of SMN in this structure and its possible role as a chaperone in snoRNP assembly (fig. 5).

Figure 5

A model for the trafficking and biogenesis of box C/D snoRNAs in yeast and human cells. See text.

A role for Cajal bodies in box C/D snoRNP assembly received further support from the finding that Snu13p/15.5kD and fibrillarin follow very distinct localization pathways.⁶¹ Indeed, when entering the nucleus, fibrillarin localizes rapidly to Cajal bodies and to nucleoli, while Snu13p/15.5kD is first distributed in speckles and Cajal bodies, and is detected in nucleoli only after a lag of about 30 minutes. This situation may indicate that Snu13p/15.5kD first binds intronic snoRNA precursors in speckles, and then accompanies them toward nucleoli. In contrast, fibrillarin may join pre-assembled complexes at the level of Cajal bodies and may thus reach the nucleoli more rapidly.^60,61

Finally, characterization of the enzyme responsible for cap hypermethylation also supports a role for the Cajal body in snoRNP maturation and assembly. The yeast enzyme, Tgs1p, was shown to hypermethylate the caps of snRNAs, and box C/D and H/ACA snoRNAs, where these exist.⁵⁹ The human genome encodes only one homolog of the enzyme, and the protein has a localization pattern similar to that of SMN: it is present both in the cytoplasm and in Cajal bodies,⁶⁰ where it could modify the cap structure of U3. Importantly, the enzyme does not bind RNA directly, but docks on the basic C-termini of Cbf5p (dyskerin), Nop56p and Nop58p,⁵⁹ providing another line of evidence that snoRNP assembly takes place in Cajal bodies.

Yeast nucleolar bodies. S. cerevisiae does not have Cajal bodies, and some evidence suggests that in this organism the nucleolus, or a specific nucleolar sub-domain, may provide some of its functions. First, Tgs1p, the cap hypermethylase, localizes in the nucleolus.⁵⁹ Remarkably, it has been shown that when yeast are grown on solid medium, a fraction of the cells concentrate Tgs1p in a dot-like structure that is within the nucleolar territory;⁶⁰ however, factors actively involved in rRNA biogenesis such as Snu13p are excluded from this structure. Second, U3 precursors that are not assembled with fibrillarin, Nop56p, and Nop58p distribute throughout the nucleus including nucleoli, and can also be found in the Tgs1p-enriched nucleolar domain. ⁶⁰ Over-expression of certain box C/D snoRNAs also triggers its assembly.⁵⁸ These results indicate that this domain is not likely a storage area for excess Tgs1p enzyme, but rather, is involved in the biogenesis of small RNAs. Electron-microscopy images show that when induced by snoRNA over-expression, the Tgs1p-rich domain has a round shape, is connected to the dense fibrillar component of the nucleoli, and strikingly, has a similar appearance.⁵⁸ Consistent with these properties this structure has been named the ‘nucleolar body’.

Because the human homolog of Tgs1p localizes in Cajal bodies,⁶⁰ where it is likely involved in U3 biogenesis, it appears that the nucleolar body is related to Cajal bodies, and may share with it at least certain functions. It is also remarkable that in many cells Cajal bodies are often physically linked with nucleoli, and that in certain cell lines they are even located within nucleoli, similar to the location of the yeast nucleolar body.²⁹⁸ While it appears that the nucleolar body forms only in certain growth conditions, its existence nevertheless shows that the nucleolar activities involved in the biogenesis of small RNAs can self-assemble and segregate from those involved in ribosome production. These properties are likely to be ancient, and they may have been at the basis of the formation of modern Cajal bodies as an independent structure.

Exploiting snoRNPs

Two major properties of snoRNAs have been harnessed for novel in vivo applications. One is using snoRNA localization elements to deliver new RNA sequences to the nucleolus. The other is guiding Nm and Ψ modifications to new sites in rRNA by expressing snoRNAs with novel guide sequences. The number of reports is still small, however good success has been achieved and additional applications seem certain to follow.

Transport of RNA aptamers and ribozymes. RNA sequences have been directed to the nucleolus (and Cajal) bodies by incorporating the experimental sequence into an internal position in a natural snoRNA or by engineering smaller snoRNA variants in which the localization signals have been added to the ends of the RNA of interest. One study designed to explicitly test this potential demonstrated that expressed polylinker sequences could be targeted to the nucleolus by simply inserting the experimental coding sequence between the terminal box C and box D elements.⁴⁹

More recently, snoRNAs have been used as vehicles to deliver a protein-binding signal to the nucleoli of mammalian cells^299,300 and a hammerhead ribozyme to the nucleoli of yeast and human cells.^301,302 The protein binding sites are from the HIV genome and known to bind the Rev and Tat proteins. These proteins are required for virus production and expression of the binding elements in a C/D snoRNA context altered snoRNA localization (Rev) or virus yield (Tat) in cultured cells. The results demonstrate the feasibility of localizing aptamers by this approach.

The ability to deliver a ribozyme to the nucleolus was first demonstrated in yeast, with remarkable results.³⁰¹ A hammerhead ribozyme was carried to the nucleolus by the U3 snoRNA and cleaved another U3 variant with nearly perfect efficiency (>95%). The hybrid snoRNA-ribozyme, called a ‘snorbozyme’, was shown to localize to the nucleolus. Building on this strategy, a hammerhead snorbozyme specific for HIV RNA was expressed in virus-infected cells, shown to localize to nucleoli and Cajal bodies, and to inhibit virus infection.³⁰²

Targeting nucleotide modification to novel RNA sites. This capability was established when the guide functions were identified^{45-47,109,162} and has since been demonstrated in two contexts. One is to first deplete a guide snoRNA for a natural modification and then introduce the modification at the same or nearby sites, by expressing a new guide RNA.^45,162 The other application is functional mapping, where creating modifications at novel sites in an RNA impairs activity.³⁰³ Nm modifications have been introduced at many new sites,^{45,109,162,303} whereas success in targeting Ψ modifications to new rRNA sites has been achieved in only a few cases.^46,117 Engineering new Ψ guide snoRNAs has been more difficult, most likely due to more stringent structural requirements for achieving good activity. Creating new Ψ guides involves inserting two guide sequences into a highly folded secondary structure domain versus placing a single Nm guide sequence in a RNA region that appears to have little or no secondary folding.

The most striking example of a positional effect seen with a natural modification, is for a Ψ in the yeast large subunit rRNA that occurs in the A-loop of the peptidyltransferase center. This loop interacts with the —CCA end of aminoacyl-tRNA and the Ψ modification is conserved among eukaryotes.¹⁷⁵ Blocking Ψ formation caused a 20% loss in the rate of in vivo protein synthesis activity. Remarkably, shifting the modification to an adjacent uridine caused a severe slow-growth phenotype (R. McCully, T. King and M.J.F., unpublished).

Excellent success has been obtained in creating interference function maps of yeast rRNA by conditional expression of novel Nm guide snoRNAs (from an inducible promoter). Modifications have been targeted to individual pre-selected sites in 18S and 25S rRNAs, and, with a library strategy, to all sites in an 800-nucleotide segment of 25S rRNA that encompasses the peptidyl transferase center (PTC)³⁰³ (and B. Liu and MJF, unpublished). Transformants are screened for growth defects, and the guide snoRNAs of interest identified by sequencing the novel snoRNA gene. Strong growth defects have been observed for several rRNA sites, whereas most new guide snoRNAs have no effect, consistent with the introduction of point mutations. The sensitive nucleotides include several sites known or predicted to be important for ribosome activity. Interestingly, the most toxic snoRNAs have been determined to interfere with ribosome biogenesis or ribosome activity (B. Liu and MJF, unpublished).

Taken together, these early results indicate that snoRNPs can be effectively exploited to manipulate RNA levels and activity in vivo. As our understanding of snoRNP trafficking and substrates advances, the potential for creating powerful and important new tools increases as well.

Acknowledgments

We are grateful to: Wayne Decatur, Ben Liu, Denis Lafontaine and E.S. Maxwell for critically reading the manuscript, W. Decateur and B. Liu for important help with literature citations, and to W. Decatur for excellent help in preparing figures. We also thank E.S. Maxwell for advice and new results, and other colleagues who also shared unpublished information. This work was supported by grants from the AFM, CNRS (ACI), EMBO YIP program (to EB), and the U.S. National Institutes of Health (GM3951 to MJF).

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