Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs

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1 Non-coding RNAs: lessons from the small nuclear and small nucleolar RNAs A. Gregory Matera*, Rebecca M. Terns and Michael P. Terns Abstract Recent advances have fuelled rapid growth in our appreciation of the tremendous number, diversity and biological importance of non-coding (nc)rnas. Because ncrnas typically function as ribonucleoprotein (RNP) complexes and not as naked RNAs, understanding their biogenesis is crucial to comprehending their regulation and function. The small nuclear and small nucleolar RNPs are two well studied classes of ncrnps with elaborate assembly and trafficking pathways that provide paradigms for understanding the biogenesis of other ncrnps. Non-coding (nc)rna A functional RNA molecule that does not code for a protein (that is, it is not an mrna). *Department of Genetics, Case Western Reserve University, Cleveland, Ohio , USA. Departments of Biochemistry & Molecular Biology, and Genetics, University of Georgia, Athens, Georgia 30602, USA. s: [email protected]; [email protected]; [email protected] doi: /nrm2124 Less than 2% of the human genome is translated into protein, yet more than 40% of the genome is thought to be transcribed into RNA 1. The vast, untranslated fraction of the human transcriptome includes a truly remarkable number of functional non-coding (nc)rnas 2. Indeed, the ongoing discovery of new classes of ncrna (for example, micrornas, short interfering (si)rnas, repeat-associated RNAs and germline-specific RNAs) and of new members of existing classes (for example, small nucleolar (sno)rnas) underscores the breadth and depth of ncrna function. Importantly, ncrnas have emerged as key trans-acting regulators of diverse cellular activities in all three domains of life 3 7 (TABLE 1). Among the known activities of ncrnas are: endonucleolytic RNA cleavage and ligation, site-specific RNA modification, DNA methylation, DNA (telomere) synthesis and modulation of protein function. These activities are important (at many levels) for gene expression and also for genome stability (TABLE 1). In some cases, the molecular mechanisms by which ncrnas function are well understood, whereas in others they are completely unknown. ncrnas commonly function as adaptors that secure and position a nucleic-acid target molecule for enzymatic activity that is catalysed by an associated partner protein. Therefore, ncrna activity is typically driven by base pairing 8 and often involves several partner proteins; that is, the functional unit is the non-coding ribonucleoprotein (ncrnp). The steps of ncrnp assembly are often not well defined but, importantly, can be regulated to control the activity of the complex. Therefore, harnessing the tremendous biomedical and applied research potential of ncrnps (as both biomedical tools and therapeutic targets) will require a detailed understanding of their biogenesis. The small nuclear (sn)rnps and snornps are arguably the best studied examples of ncrnps, and analysis of their biogenesis has revealed an unanticipated complexity in their assembly, trafficking and mechanisms of action. This review highlights significant aspects of snrnp and snornp biogenesis that have emerged from recent studies, placing them in the broader context of earlier findings. The principles of RNP-complex formation and intracellular trafficking are probably not unique to snrnps and snornps, and will probably also govern the function of other ncrnps. Therefore, we end with a summary of the lessons learned from these RNPs that might expedite our understanding of other ncrnps. The snrnas snrnas comprise a small group of highly abundant, non-polyadenylated, non-coding transcripts that function in the nucleoplasm. The snrnas can be divided into two classes on the basis of common sequence features and protein cofactors. Sm-class RNAs are characterized by a 5 -trimethylguanosine cap, a 3 stem loop and a binding site for a group of seven Sm proteins (the Sm site) that form a heteroheptameric ring structure (FIG. 1a). Lsm-class RNAs contain a monomethylphosphate cap and a 3 stem loop, terminating in a stretch of uridines that form the binding site for a distinct heteroheptameric ring of Lsm proteins (FIG. 1b). The Sm class of snrnas is comprised of U1, U2, U4, U4 atac, U5, U7, U11 and U12, whereas the Lsm class is made up of U6 and U6 atac. With the exception of the NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 8 MARCH

2 Table 1 Functions of established and emerging non-coding RNAs ncrna Function RNA processing and modification Spliceosomal snrnas Pre-mRNA intron splicing U7 snrna Histone pre-mrna 3 -end formation C/D RNAs 2 O-methylation of rrnas, snrnas and trnas; rrna processing H/ACA RNAs Pseudouridylation of rrnas and snrnas; rrna processing RNase P RNA pre-trna maturation (5 -end cleavage) Trypanosomal guide RNAs mrna editing; production of alternative mrnas Transcription 7SK RNA P-TEFb-mediated transcription-elongation control 6S RNA Transcriptional regulation in bacteria by direct interaction with RNA polymerase Translation trna mrna translation tmrna Quality-control factor in bacteria; triggers release of ribosomes stalled in translation Protein trafficking SRP RNA Protein translocation to the endoplasmic reticulum Regulation of gene expression sirnas Gene silencing: cleavage of RNAs derived from viruses, retroelements and repeat sequences mirnas Gene silencing: translational repression or cleavage of target mrnas pirnas Gene silencing: mammalian germline silencing of repeat transcripts (chromatin modification?) rasirnas Gene silencing: Drosophila germline silencing of repeat transcripts (chromatin modification?) Eubacterial antisense RNAs Gene silencing: translational repression or cleavage of target mrnas; in rare cases, gene activation is observed Genomic stability Telomerase RNA Telomere synthesis mirna, microrna; ncrna, non-coding RNA; pirna, PIWI-interacting RNA; P-TEFb, positive transcription elongation factor-b; rasirna, repeat-associated sirna; RNase, ribonuclease; rrna, ribosomal RNA; sirna, small interfering RNA; snrna, small nuclear RNA; SRP, signal recognition particle; tmrna, transfer messenger RNA; trna, transfer RNA. U7 snrnp, which functions in histone pre-mrna 3 processing, the other uridine-rich snrnps form the core of the spliceosome and catalyse the removal of introns from pre-mrna 9. Accurate removal of intronic sequences is guided by base-pairing interactions between the spliceosomal snrnas and the intron exon junctions. During the course of the splicing reaction, specific and dynamic base-pairing interactions take place among and between the snrnas themselves. More than 150 partner proteins are also involved in this process, a subject that has been extensively reviewed 10,11. Less well understood is the life history of the snrnps prior to their assembly into the spliceosome. The recent studies highlighted here indicate that transcription and 3 processing of most snrnas are coupled by a system that is parallel to, but distinct from, that which produces mrnas. Furthermore, the cellular systems that function in nuclear transport and assembly of snrnps (and probably of other ncrnps) will be described. Coupled transcription and 3 processing The Sm-class snrna genes share several common features with protein-coding genes, including the arrangement of upstream and downstream control elements (FIG. 1c). Sm-class genes are transcribed by a specialized form of RNA polymerase II (Pol II) that is functionally similar to the Pol II used by the mammalian proteincoding genes 12. Promoter-swap experiments performed more than two decades ago 13,14 indicated that factors that are important for accurate recognition of 3 -processing signals (FIG. 1c) must load onto the polymerase at the promoter; however, factors responsible for cleavage of these non-polyadenylated transcripts have only recently been identified. Baillat et al. 15 purified a complex from human cells, called the Integrator, that associates with the C-terminal domain (CTD) of Pol II and can be co-immunoprecipitated with snrna promoter sequences, but not with those of control mrna or histone genes. Furthermore, two of the purified proteins, INT9 and INT11, share significant similarity with the cleavage and polyadenylation specificity factors CPSF-100 and CPSF-73, respectively 15, which are essential for the proper cleavage and polyadenylation of the 3 ends of mrna and for the formation of non-polyadenylated histone premrna 3 ends 16,17 (FIG. 1c). Moreover, INT11 is probably the endonuclease subunit of the Integrator complex, as depletion of the endogenous protein or overexpression of a construct containing a mutation in a conserved metal-chelating residue inhibited 3 -end formation in snrna 15. Therefore, it seems that CPSF-73 has a catalytic role in two independent mrna 3 -processing complexes and that a related protein, INT11, has a similar role in cleavage of Sm-class snrna 3 ends. In metazoa, CPSF and Integrator complexes interact with the CTD of Pol II to couple transcription to downstream RNA-processing events. The role of CPSF in mediating communication between transcription and polyadenylation is conserved among eukaryotes. However, the putative role of the metazoan Integrator proteins in coupling transcription and processing seems to be carried out by the Nrd1 complex in yeast Together with its partner proteins Nab3, Sen1 and others, the Nrd1 complex binds to the Pol II CTD and directs 3 -end formation of non-polyadenylated transcripts such as snrnas and non-intronic snornas The evolutionary relationship, if any, between the Nrd1-complex proteins and those of the Integrator complex has not been established. The Lsm-class snrna genes (U6 and U6 atac ) are transcribed by Pol III using specialized external promoters 24. The run of uridines that forms the Lsmbinding site at the 3 end (FIG. 1b) also doubles as a Pol III transcription terminator. Therefore, there are few parallels between Lsm-class genes and genes that encode proteins. 210 MARCH 2007 VOLUME 8

3 Nuclear export systems In higher eukaryotes, Lsm-class snrnas never leave the nucleus, whereas the biogenesis of Sm-class snrnps is a multistep process that takes place in distinct subcellular compartments. Sm-class snrnas are exported from the nucleus for cytoplasmic maturation events. The 5 cap structure and length of the RNAs are key determinants in nuclear export, and the main components of the trafficking system have been defined. Following transcription and 3 processing in the nucleus, newly transcribed Sm-class snrnas are transported to the cytoplasm by an export complex (FIG. 2) that contains the snrna-specific export adaptor protein PHAX, the export receptor chromosome region maintenance-1 (CRM1; also known as exportin-1), a Sm-class snrna b Lsm-class snrna CH 3 CH 3 CH 3 GpppApNp 3 stem 3 stem OCH 3 Sm site MPG cap Lsm site TMG cap Consensus U7 snrna AAUUUUUGG AAUUUGUCU CH 3 pppgp UUUUU E G F D2 D1 D3 B E G F D3 B Sm core U7 core Lsm core c Generic Pol II gene Control region Coding region Cleavage site Terminator Processing signals Control region Processing signals Cleavage site Gene product Distal Proximal Upstream Downstream Site (factor) snrnas DSE PSE 3 stem? 3 box? (INT11) Histones CCPE TATA box HSL HDE CA (CPSF-73) Proteins Enhancer TATA box AAUAAA G/U rich CA (CPSF-73) Adaptor protein Bridges proteins that link specific cargoes to their cognate transport receptors. Export/import receptor Collectively called karyopherins, these proteins facilitate transport across nuclear pores. Import factors are called importins and export factors are called exportins. Receptors primarily mediate interactions with nuclear pores but can also bind cargo directly. Figure 1 Anatomical features of Sm- and Lsm-class small nuclear RNAs. a Sm-class small nuclear (sn)rnas are transcribed by RNA polymerase II and contain three important recognition elements (boxed): a 5 -trimethylguanosine (TMG) cap, an Sm-protein-binding site (Sm site) and a 3 stem loop structure. In metazoans, the Sm site and the 3 -stem elements are required for recognition by the survival motor neuron (SMN) complex for assembly into stable core ribonucleoproteins (RNPs), whereas the TMG cap and the assembled Sm core are required for recognition by the nuclear import machinery. With the exception of U7, each of the Sm-class snrnps contains a common core of seven Sm proteins and a unique set of snrnp-specific proteins. The consensus sequence of the Sm site and the sequence of the specialized Sm site of the U7 snrna are shown. The consensus Sm site directs assembly of the canonical heptameric protein ring (Sm core). In the U7 snrnp, two of the seven Sm proteins (SmD1 and SmD2) are replaced by LSM10 and LSM11, respectively, forming the U7 core RNP. b Lsm-class snrnas are transcribed by RNA polymerase III and contain a 5 -monomethylphosphate (MPG) cap, a 3 stem and terminate in a stretch of uridine residues (the Lsm site) that is bound by the Lsm core. U6 and U6 atac are the only known Lsm-class snrnas. c Parallels in the organization of control elements for RNA polymerase II transcription and processing of Sm-class snrna genes, cell-cycle-dependent histone genes and canonical protein-coding genes. For each of the three gene classes, the elements and factors involved in their expression are tabulated. The putative snrna-gene upstream-processing signal and the 3 -cleavage-site consensus sequence have yet to be determined. In addition to the TATA box, control regions for the cell-cycle-dependent histone genes include an upstream cell-cycle-promoter element (CCPE) and an internal element 136 located in the protein-coding region (not shown). CPSF, cleavage and polyadenylation specificity factors; DSE, distal sequence element; HSL, histone stem loop; HDE, histone downstream element; INT11, Integrator-11; PSE, proximal sequence element. NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 8 MARCH

4 CRM1 RAN GDP CBC PHAX m 7 G F E G Sm proteins D2 D3 D1 B SMN complex IGC RAN CRM1 GTP PHAX P CBC m 7 G snrna export IGC Sm-core assembly NPC CB Cap hypermethylation TGS1? m 7 G m 7 G Nucleolus Sm core SMN complex Cytoplasm Nucleus Transcription Exo 3 -end trimming IGC Imp-β SPN TMG Imp-β SPN Sm core SMN complex TMG Sm core PFs SMN complex CB snrnp maturation snrnp import Figure 2 Biogenesis of Sm-class small nuclear RNPs. The life-cycle of Sm-class small nuclear ribonucleoproteins (snrnps) includes both nuclear and cytoplasmic phases. Following transcription by a specialized form of RNA polymerase II, pre-snrna transcripts are exported to the cytoplasm, with a potential intermediate stop at Cajal bodies (CBs). The snrna-export complex consists of the export receptor chromosome region maintenance-1 (CRM1), the hyperphosphorylated form of the export adaptor PHAX, the heterodimeric cap-binding complex (CBC) and the GTP-bound form of Ran GTPase. These factors dissociate from the pre-snrna in the cytoplasm after binding by the survival of motor neuron (SMN) complex and dephosphorylation of PHAX. The SMN complex recognizes specific sequence elements in the snrnas (the Sm-protein-binding site and the 3 stem loop) and recruits a set of seven Sm proteins, three of which contain symmetrical dimethylarginine residues (orange stars), to form the Sm-core RNP. Following assembly of the Sm core, the 7-methylguanosine (m 7 G) cap is hypermethylated by trimethylguanosine synthase-1 (TGS1) to form a 2,2,7-trimethylguanosine (TMG) cap structure, and the 3 end is trimmed by a yet-to-beidentified exonuclease (Exo). The formation of the TMG cap triggers the assembly of the import complex, which includes the import adaptor snurportin-1 (SPN) and the import receptor importin-β (Imp-β). Both SPN and the SMN complex make functional contacts with Imp-β. On nuclear re-entry, the Sm-class snrnps target to Cajal bodies for snrnp maturation, which includes binding by snrnp-specific proteins and site-specific modification by small Cajal body (sca)rnps. Following discharge from Cajal bodies, the newly minted snrnps either participate in splicing at perichromatin fibrils (PFs) or are stored in interchromatin granule clusters (IGCs) for later use. NPC, nuclear pore complex; P, phosphate. IGC Cajal bodies Intranuclear structures that function as ribonucleoproteinassembly, trafficking and remodelling centres. the cap-binding complex (CBC; which consists of the protein heterodimer CBP80 CBP20) and Ran GTPase 25. The ability of PHAX to export snrnas depends on its phosphorylation status. Hyperphosphorylated PHAX is predominantly nuclear. Together with CBP80 and CBP20, PHAX forms a bridge between the snrna and CRM1 (FIG. 2). Hypophosphorylated PHAX is predominantly cytoplasmic, cannot bind to snrnas and recycles to the nucleus through the import receptor importin-β 26. The kinase(s) and phosphatase(s) that regulate this process have not been identified. However, it is not clear whether fungal snrnas are exported, as their genomes do not contain recognizable PHAX orthologues. It is also not clear how PHAX distinguishes the 5 caps of certain snrna precursors from those of bulk mrnas. A few studies have begun to shed light on this conundrum, although much work remains to be done. Ohno and colleagues showed that when it comes to RNA export, length really does matter 27,28. By inserting increasingly longer sequences at various positions in U1 snrna, they showed that export could be rerouted from the PHAX pathway to the nuclear export factor-1 (NXF1)-mediated mrna-export pathway 27,28. Conversely, intronless mrnas can be progressively rerouted from the NXF1-mediated to the PHAX-mediated pathway by successive deletions 28. Therefore, length is also an important determinant of RNA-export-pathway choice. However, RNA length and cap structure do not tell the whole story, as several snorna transcripts are of similar size and have the same 5 caps as the snrnas yet are retained in the nucleus 29,30. For example, recent evidence indicates that PHAX binds to precursor U3 snornas 31 and targets them to Cajal bodies (rather than to the cytoplasm), where they interact with the 5 -cap hypermethylase trimethylguanosine synthase-1 (TGS1) prior to their accumulation in nucleoli 30. The current view holds that assembly with core snornp proteins (see below) and hypermethylation of the 5 cap can override snornp export. U3 transcripts with mutations in conserved box C and D elements (FIG. 3) are exported to the cytoplasm 30,32,33. These results, together with the findings of Smith and Lawrence 34 indicating that U2 snrna precursors that contain 3 extensions also localize in Cajal bodies, lead us to propose the existence of a discrimination step for RNA export that takes place in and around Cajal bodies. 212 MARCH 2007 VOLUME 8

5 Cytoplasmic assembly of the core snrnp by SMN On export to the cytoplasm, assembly into stable Sm-core particles is carried out by the survival motor neuron (SMN) protein complex (FIG. 2). Evidence indicates that the SMN complex also functions in the assembly of other RNPs 35,36. Loss-of-function mutations in the human SMN1 gene cause a neurogenetic disorder called spinal muscular atrophy (BOX 1). Together with its associated factors, collectively known as Gemins, the SMN complex binds to the newly exported snrna precursors and to the seven Sm proteins that form the core of the RNP 35,37 (FIG. 2). Recent data point to a WD-repeat a C/D Target b H/ACA c U3 snorna (C/D) RNA C/D motif (k-turn) CH 3 D C 5 nt Apical loop Upper stem CAB box (Eukaryotes) k-turn (Archaea) C/D motif B C Guide elements Nψ Distance 15 nt C/D motif C D C/D motif (k-turn) Terminal stem 3 C 5 nt D 5 3 CH 3 5 Guide elements (pseudou pocket) Lower stem 5 Target RNA 5 ACA 3 or H 3 TMG d Telomerase RNA (H/ACA) 3 CAB box Ligated termini (Archaea) TMG H ACA 3 Figure 3 Anatomical features of C/D and H/ACA RNAs. a The secondary structure of an archetypical C/D 2 -O-methylation guide RNA. Conserved box C (RUGAUGA) and D (CUGA) sequence elements are tethered by the terminal stem loop and apical loop and form kink-turns (k-turns). A C/D pair is associated with an antisense element (blue) located upstream of box D that base pairs with the target RNA (red). Target RNA is methylated on the ribose of the nucleotide (nt) that is base paired with the guide RNA that is 5 nucleotides upstream of box D. b Secondary structure of an archetypical H/ACA pseudouridylation guide RNA. An imperfect hairpin is formed by a lower stem, pseudouridylation (pseudou) pocket, upper stem and an apical loop. The target RNA is bound within the pseudouridylation pocket. Box ACA is found immediately downstream of the hairpin, ~15 nucleotides from the uridine that is modified (ψ; N represents a conserved nucleotide that is located next to the target ψ). In eukaryotes, H/ACA RNAs consist of two hairpin units, separated by a single-stranded hinge that contains box H (ANANNA). The apical loop can contain a Cajal body (CAB)-box sequence element (consensus UGAG) for Cajal body retention in the subset of eukaryotic H/ACA RNAs that guide the modification of small nuclear RNAs (rather than ribosomal (r)rnas) 115. In archaea, these RNAs contain a k-turn at the junction of the upper stem and apical loop. c U3 is a C/D RNA specialized to function in pre-rrna processing. Antisense elements involved in interacting with the target RNA are located in the 5 half of the RNA. The RNA retains two C/D motifs (boxed): a canonical C/D pair and a distinct B/C pair that is involved in interacting with U3-specific proteins. d The 3 half of vertebrate telomerase RNA is a double hairpin H/ACA RNA (boxed), and the 5 half is a pseudoknot required for telomere synthesis. An antisense element that recognizes the DNA substrate is located in the pseudoknot domain. snorna, small nucleolar RNA. TMG, 2,2,7-trimethylguanosine. NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 8 MARCH

6 Box 1 Small RNPs and human disease The study of small nuclear ribonucleoproteins (snrnps) and small nucleolar (sno)rnps has contributed to our understanding of several important diseases. Spinal muscular atrophy. Mutations in the survival motor neuron-1 (SMN1) gene result in the degeneration of spinal motor neurons and severe muscle wasting 37,132. The SMN protein assembles Sm-class snrnps, and probably also snornps and other RNPs 35,36. Spinal muscular atrophy affects up to 1 in 6,000 people. Dyskeratosis congenita. Mutations in key components of H/ACA RNPs are a cause of dyskeratosis congenita, a rare syndrome that is characterized by abnormal changes in the skin, nails and mucous membranes 133. Long-term problems include bone-marrow failure and cancer 133. Apparently, the syndrome can arise from mutations in multiple genes, including dyskerin (the orthologue of centromere binding factor-5 (Cbf5)), telomerase RNA and telomerase reverse transcriptase. The involvement of proliferating tissues is consistent with a primary defect in telomere maintenance. Prader Willi syndrome. This disorder affects as many as 1 in 12,000 people and is characterized by extreme hunger, cognitive and behavioural problems, poor muscle tone and short stature. The syndrome is clearly linked to the deletion of a region of paternal chromosome 15 that is not expressed on the maternal chromosome. The deleted region includes a brain-specific C/D RNA that seems to target the serotonin-2c receptor mrna and might regulate its editing 66 or splicing 67. Patients with Prader Willi syndrome do not express the C/D RNA and express reduced amounts of the functional serotonin-2c receptor mrna. Perichromatin fibril (PF). A fine structure, which is visible only under the electron microscope, that is located adjacent to transcriptionally active chromatin and is thought to be a site of active pre-mrna processing. Interchromatin granule cluster (IGC).Corresponds to a nuclear domain visible as a speckle under the light microscope. IGCs function as storage sites for splicing factors that can be recruited to perichromatin fibrils. Peptidyl transferase centre Catalytic centre of the ribosome that forms peptide bonds during protein translation. mrna-decoding centre Region of the ribosome involved in the selection of transfer RNAs that correspond to mrna codons during protein translation. protein, Gemin-5, as the specificity factor that binds snrnas that contain a consensus Sm site 38,39. It is currently unclear how the U7 snrna, which contains an unusual Sm site (FIG. 1a), is recognized by a subset of SMN complexes that contain the U7-specific Sm-like proteins LSM10 and LSM11 (REF. 40). Additional factors in the SMN complex help to bring the Sm proteins into the fold, but the known mechanistic details are few and far between. Import and assembly of snrnp-specific factors Following the assembly of the Sm core, and in association with the SMN complex (FIG. 2), the snrna 7-methylguanosine (m 7 G) cap is hypermethylated by the TGS1 protein to form a 2,2,7-trimethylguanosine (TMG) cap 41. In addition, a few nucleotides on the 3 termini of the snrnas are trimmed away by a putative exonuclease, apparently accelerating the import kinetics of snrnps 42. Sm-class snrnps contain two nuclear localization signals: the TMG cap and the Sm core itself (FIGS 1,2). Each signal uses the import receptor importin-β to transport snrnps into the nucleus 43. However, the adaptor that specific ally recognizes the TMG cap is called Snurportin-1, whereas the SMN complex (or a subcomplex thereof) is an adaptor for the Sm core 44,45. The Sm-core and TMG-cap import signals function independently in vitro 46,47, however, it is likely that the adaptors (Snurportin-1 and the SMN complex) function synergistically in vivo. Once in the nucleoplasm, the snrnps are free to diffuse throughout the interchromatin space. Newly made RNPs transiently accumulate in Cajal bodies (FIG. 2) prior to accumulation in nuclear subdomains known as perichromatin fibrils and interchromatin granule clusters 48. Additional RNP remodelling and assembly steps are thought to take place in Cajal bodies 35, including RNA-guided modification of the spliceosomal snrnas (see below) and assembly of factors that are specific to a given species of snrnp In addition to their roles in de novo RNP assembly, Cajal bodies might also be involved in recycling and remodelling U4/U6 snrnp complexes that are disrupted during the splicing reaction 49,52. The snornas The RNAs commonly referred to as snornas comprise two families, the C/D and H/ACA RNAs. The term small nucleolar RNA was originally coined to reflect the nucleolar localization of the first members of this group relative to their nucleoplasmic cousins, the snrnas. Most C/D and H/ACA RNAs function in ribosomal (r)rna modification and processing in the nucleolus. However, the C/D and H/ACA RNAs have evolved an impressive portfolio of functions and targets as well as a corresponding range of cellular localization patterns that includes sites outside of the nucleolus (to gain access to different substrates). Therefore, as described here, the snorna families have grown to include a diversity belied by their increasingly anachronistic name. In addition, we describe the roles of essential partner proteins in the function of C/D and H/ACA RNAs, and the elaborate pathways by which functional RNPs are assembled and delivered. Functional diversity and anatomy of the RNAs Whereas eukaryotic cells contain fewer than a dozen snrna species, they contain more than 200 unique C/D and H/ACA RNAs 54,55. The C/D and H/ACA RNAs are among the most numerous and functionally diverse trans-acting ncrnas currently known Moreover, these RNAs are present in archaea as well as in eukaryotes, indicating that they arose over 2 3 billion years ago. The C/D and H/ACA RNAs are essential for major biological processes including protein translation, mrna splicing and genome stability (TABLE 1). Most known C/D and H/ACA RNAs guide the modification of other ncrnas. The two classes of RNA guide different nucleotide modifications: C/D RNAs direct 2 -O-ribose methylation and H/ACA RNAs guide pseudouridylation (conversion of uridine to pseudouridine). C/D and H/ACA RNAs function in parallel to both process and modify ribosomal RNA. These RNPs modify key regions of rrna (for example, the peptidyl transferase centre and the mrna-decoding centre), and both types of modification are essential for ribo some function 60,61. Other modification targets include snrnas in eukaryotes 62, transfer RNAs in archaea 63, spliced leader RNAs in trypanosomes 64 and perhaps at least one brain-specific mrna in mammals Spliceosome function also depends on the modification of snrnas by C/D and H/ACA RNAs 60. In addition, one H/ACA RNA, telomerase RNA, is required for telomere synthesis 68. Moreover, the existence of substantial numbers of orphan RNAs that do not apparently target established substrates (that is, targets such as rrnas and snrnas) indicates that C/D and H/ACA RNAs also function on targets and in processes that remain to be identified 69, MARCH 2007 VOLUME 8

7 Box 2 Composition and organization of C/D and H/ACA RNPs a C/D RNP Fib L7Ae D Nop56/58 C C D Nop56/58 L7Ae Fib b H/ACA RNP Gar1 C/D RNPs H/ACA RNPs Archaea Human Archaea Human Fibrillarin Fibrillarin Cbf5 Dyskerin L7Ae 15.5K/NHPX L7Ae NHP2 Nop56/58 NOP56 Gar1 GAR1 NOP58 Nop10 NOP10 Kink-turn (k-turn) motif A common RNA structural motif that is bound by a family of related proteins, including L7Ae, resulting in a sharp bend (or kink) in the RNA helix. 5 L7Ae Nop10 Cbf5 ACA 3 The core archaeal C/D ribonucleoprotein (RNP) proteins are: fibrillarin (Fib; the 2 -O-methyltransferase), L7Ae and Nop56/58. Eukaryotes have homologous proteins. The eukaryotic NOP56 and NOP58 are closely related paralogues that are both associated with C/D RNAs (one with each C/D and C /D unit 134 ). These two proteins have apparently evolved distinct, but crucial, functions in eukaryotes as both are essential. The core archaeal H/ACA RNP proteins are: centromere binding factor-5 (Cbf5; the pseudouridine synthase), L7Ae, Gar1 and Nop10. Therefore, the L7Ae protein is a common component of both C/D and H/ACA RNPs in archaea. In eukaryotes, distinct L7Ae-related paralogues (for example, human 15.5K (also known as NHPX) and NHP2) are essential components of the individual C/D and H/ACA RNPs. In the archaeal C/D RNPs, fibrillarin acquires a guide RNA through Nop56/58 and L7Ae (see part a of the figure). Cbf5 binds the guide RNA directly in the archaeal H/ACA RNP (see part b in the figure). In both RNPs, L7Ae induces essential changes in the conformation of the guide RNA. C/D and H/ACA RNAs are defined by conserved signature-sequence elements and characteristic secon dary structures (FIG. 3). The C/D and H/ACA modification-guide RNAs are the archetypes, with the simplest (and apparently most ancient) configurations. The RNAs recognize and secure target molecules through antisense elements (FIG. 3). The C/D and H/ACA RNAs that are specialized to perform other functions (including pre-rrna processing and telomere synthesis) display variations on the basic architecture (FIG. 3). All of the RNAs of each family interact with core sets of highly conserved proteins to form the C/D and H/ACA RNPs (BOX 2). In the case of the modification-guide RNPs, these proteins, along with the guide RNA, are sufficient for function in vitro Other proteins are necessary for specialized functions and for function in vivo 57,59. Architecture of the C/D and H/ACA RNPs Like other ncrnas, the C/D and H/ACA modificationguide RNAs function as adaptors that link a catalytic component of an RNP to a target. Reconstitutions of functional archaeal C/D and H/ACA modificationguide RNPs 75,76 have rapidly advanced our understanding of the organization and function of these complexes. Atomic-resolution structures of all of the core components of archaeal C/D and H/ACA RNPs 82 86, including several multicomponent complexes, are also now available, providing spectacular structural insights. The C/D and H/ACA RNPs illustrate different modes for the recruitment and positioning of a partner enzyme by a guide RNA, and a range of essential functions are contributed by the other core proteins. The H/ACA RNP: direct binding of the enzyme. The simplest recruitment paradigm has been found in the H/ACA RNP, in which the protein enzyme interacts directly and specifically with the guide RNA through conserved features of the RNA 75,76,84,86. Centromere binding factor-5 (Cbf5; known as dyskerin in humans) is a pseudouridine synthase with a catalytic domain and a PUA (pseudouridine and archeosine transglycosylase) domain. Box ACA and the lower stem of the guide RNA are bound by the PUA domain of Cbf5, anchoring the antisense elements near the catalytic site 86 (BOX 2). In humans, point mutations that affect a cluster of amino acids in the PUA domain of the dyskerin protein result in dyskeratosis congenita 82 (BOX 1). This dyskeratosis congenita mutation cluster was uncovered by mapping the widely dispersed dyskeratosis congenita mutations in the context of the predicted three-dimensional structure of the human protein (modelled from the crystal structure of archaeal Cbf5) 82. The affected amino acids are in the immediate vicinity of, but do not include, those amino acids that are directly involved in guide RNA protein interactions (which presumably would significantly disrupt RNA interactions and result in more severe defects) 82,86. Consistent with the hypothesis that the disease-inducing mutations affect (but do not eliminate) RNA binding, the amounts of H/ACA RNAs, including telomerase RNA and snornas, are reduced in patients with dyskeratosis congenita and mouse models of the disease Nonetheless, there are also other viable hypotheses for the molecular basis of this disease 82,86. The other three H/ACA RNP proteins are none theless essential for the function of the complex 75. Gar1 and Nop10 each bind to distinct sites on the catalytic domain of Cbf5, whereas L7Ae interacts directly with the guide RNA 75,82 84,86 (BOX 2). Current evidence indicates that Gar1 is involved in the binding and/or release of the target RNA 82,86. Nop10 is positioned along the upper stem of the guide RNA between Cbf5 and L7Ae, and appears to interact with both L7Ae and the guide RNA (as well as Cbf5) in the context of the complex, indicating a coordinating function 86. L7Ae binds the kink-turn (k-turn) motif that is located in the upper stem apical loop of the guide H/ACA RNA (FIG. 3) and induces a major bend (or kink) in the RNA 85,86. It remains to be determined how the resulting changes might contribute to the function of the H/ACA RNP. NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 8 MARCH

8 An H/ACA RNA can have one, two or three hairpins, and the evidence indicates that each hairpin serves as a binding site for the complete set of four core H/ACA RNP proteins 75,90. In mammals, H/ACA RNAs almost exclusively consist of two hairpin units and the available evidence indicates that the organization of the RNP is similar 91,92. a Cotranscriptional assembly of inactive RNPs 5 CTD b Trafficking to Cajal bodies and assembly of active RNPs by factor exchange c Targeting to functional destination Mature H/ACA or C/D RNP Nascent H/ACA or C/D RNP Pol II Cajal body snorna snorna Nucleolus Cajal body Telomere? Figure 4 Coordinated synthesis, assembly and trafficking of C/D and H/ACA RNPs. Current information indicates an emerging model in which major steps in the production of both C/D and H/ACA ribonucleoproteins (RNPs) are coordinated. a Cotranscriptional assembly of inactive pre-rnps (early biogenesis). A subset of the core RNP proteins (red) and an exchange factor (yellow) are associated with the C-terminal domain (CTD) of RNA polymerase II (Pol II) at the guide RNA transcription site, and assemble on the nascent RNA, forming a metabolically stable, but inactive, pre-rnp. b Maturation to functional RNPs at Cajal bodies (late biogenesis and functional activation). The pre-rnp moves to Cajal bodies where final steps in the formation of functional complexes can occur, including RNA modifications and replacement of the exchange factor with a missing core protein. c Targeting to functional sites (localization). Mature RNPs move to the sites where they function, for example, to nucleoli, Cajal bodies or telomeres. snorna, small nucleolar RNA. The C/D RNP: a protein bridge to secure the enzyme. The association of the methyltransferase fibrillarin with a C/D guide RNA depends on a bridge formed by the other two core proteins, L7Ae and Nop56/58 (REFS 71,73,74). L7Ae nucleates the assembly of the C/D RNP. As in the H/ACA RNP, L7Ae binds a k-turn motif in the C/D guide RNA, in this case formed by the interaction of box C and D elements (FIG. 3; BOX 2). The resultant restructuring of the RNA seems to create a new binding site that is recognized by the Nop56/58 protein 93. Nop56/58 in turn recruits the catalytic protein fibrillarin to complete the assembly of the functional complex. A given C/D RNA can have one or two functional units (each comprised of box C, box D and an antisense element; FIG. 3). Recent results also indicate protein bridging between these functional units (BOX 2). Highresolution X-ray structures of fibrillarin and Nop56/58 reveal a fibrillarin Nop56/58 Nop56/58 fibrillarin complex in which two fibrillarin Nop56/58 heterodimers are connected through extensive coiled-coil interactions between the Nop56/58 proteins 79. This bridge complex has the potential to place Nop56/58 and fibrillarin at the second functional unit, and explains the apparent lack of requirement for L7Ae binding at the second unit in some systems 73,94. Moreover, a new structure of the complex appears to capture a different conformation, revealing a substantial hinge motion that might be involved in the proper placement of fibrillarin at the target modification sites (S. Oruganti, Y. Zhang, H. Li, M. T., R. T., W. Yang and H. Li, unpublished observations) and addressing a concern that arose from the analysis of intracomplex distances that assumed no movement 79. Regulated assembly and trafficking in eukaryotes The results emerging from recent studies indicate that the assembly and trafficking of C/D and H/ACA RNPs are intricately regulated in eukaryotic cells (FIG. 4). It seems that the complexes are assembled as inactive pre-rnps on nascent guide RNA transcripts at the genes. The pre-rnps are transported to Cajal bodies, where evidence indicates that they are matured to functional complexes. Last, the RNPs must be forwarded to the sites where they function, but in at least one case, delivery seems to be regulated to control the activity of the RNP. The regulation of transport and assembly seems to involve transient interactions with various factors, some of which are now known. Co-transcriptional assembly of pre-rnps. As an H/ACA RNA is transcribed, three of the four core H/ACA RNP proteins and an assembly factor, called nuclear assembly factor-1 (Naf1), associate with the RNA (FIG. 4a). The subset of three H/ACA RNP proteins Cbf5, Nop10 and Nhp2 (an L7Ae homologue) and Naf1 can be observed at transcriptionally active H/ACA genes by chromatin immunoprecipitation and other assays Naf1 also interacts with components of the Pol II transcriptional machinery, including the CTD of the polymerase large subunit, indicating intimate coupling of RNP assembly to transcription 97, MARCH 2007 VOLUME 8

9 Box 3 Genomic origins of C/D and H/ACA RNAs Exon TRAMP complex A nuclear polyadenylation complex consisting of Trf4 (a poly(a) polymerase), Air2 (a zinc-knuckle protein) and Mtr4 (an RNA helicase). TRAMP functions together with the exosome as a qualitycontrol mechanism to stimulate the degradation of various aberrant target RNAs. Exosome A complex of 3 5 exonucleases that has important roles in RNA processing and turnover. Intronic Polycistronic Monocistronic C/D and H/ACA RNAs are encoded in eukaryotic genomes in various ways, and understanding this point has facilitated the discovery and study of the biogenesis of these RNAs 54,59,135. Most mammalian and plant C/D and H/ACA RNA genes (shown as coloured arrows in the figure) are located in the introns of genes that encode proteins involved in ribosome biogenesis (top panel). Generation of functional C/D and H/ACA RNAs requires processing of the introns, which are released following pre-mrna splicing. Interestingly, in some cases, the host genes do not appear to encode proteins; the intron-encoded RNAs are the only obvious products of these genes. Other C/D and H/ACA RNAs are derived from polycistronic transcripts that can contain up to ten different RNAs (middle panel). In this case, each RNA is liberated from the precursor RNA by endonucleolytic cleavages. A few C/D and H/ACA RNAs are independently transcribed by RNA polymerase II (bottom panel). Like mrnas and small nuclear (sn)rnas, the independently transcribed C/D and H/ACA RNAs receive a monomethylated (m 7 G) 5 cap, and like the snrnas, the cap is hypermethylated to the trimethylated (m2,2,7g) form. Naf1 might ensure the assembly of a stable H/ACA pre-rnp that is inactive until Naf1 is exchanged for Gar1. Evidence indicates that Naf1 and Gar1 interact with a common site on Cbf5 in a mutually exclusive manner 82,95,97,99. Naf1 and Gar1 share a region of homology that probably mediates binding to Cbf5, but the proteins are otherwise substantially different. Gar1 is essential for the function of H/ACA RNPs 75, but is the only core protein that is not required for the accumulation of H/ACA RNAs. Naf1 is required for the accumulation of all classes of H/ACA RNA 95, However, Naf1 is not found associated with H/ACA RNPs that are functionally engaged. The exchange of Naf1 for Gar1 could be a key step in the regulated transition of pre-rnps into active H/ACA RNPs. The assembly of C/D RNPs also seems to occur co-transcriptionally and to be tightly coupled to pre-mrna splicing 106. The molecular link between these two processes has been identified as a general splicing factor, called IBP60, that binds upstream of intronic C/D RNAs and has putative helicase activity, which seems to trigger C/D RNP assembly 107. C/D RNP assembly also involves a potential exchange factor called Bcd1 (box C/D RNA). Like Naf1, Bcd1 is essential for the accumulation of all C/D RNAs 108,109, but is not a stable component of the mature RNPs. Bcd1 also seems to interact with the Pol II machinery. As the only core protein that is not required for the accumulation of C/D RNAs in eukaryotes, Nop56 might be the Bcd1-exchange partner (analogous to Gar1). The C/D and H/ACA RNPs are also subject to co-transcriptional quality control. Aberrant RNAs or RNPs seem to be targeted for destruction by a mechanism that involves nuclear polyadenylation by the TRAMP complex and degradation by the exosome 110. This nuclear surveillance system seems to be closely linked to transcription and 3 -end processing 18,21,111,112. There are several additional factors implicated in the co-transcriptional assembly of C/D and H/ACA RNPs. For example, two putative RNA or DNA helicases, called RVB1 and RVB2 (also known as TIP49a and TIP49b, or as p50 and p55), are essential for the accumulation of both C/D and H/ACA RNAs 31,113. These helicases might have a role in chaperoning RNP assembly or in releasing nascent transcripts from genes. Maturation at Cajal bodies. All C/D and H/ACA RNPs seem to be rapidly targeted to Cajal bodies where it is thought that essential maturation steps occur (FIG. 4b). As described above, PHAX might have a role in the localization of certain C/D and H/ACA RNAs to Cajal bodies. Cajal bodies are complex intranuclear structures enriched in factors involved in the modification of RNA and in the assembly of RNA protein complexes 35,117,118. In particular, these factors include TGS1, the enzyme responsible for 5 -cap hypermethylation of capped C/D and H/ACA RNAs 105, and the SMN complex. As discussed above, the SMN complex is an established snrnp-assembly factor 37,119. However, the SMN complex also associates with both C/D and H/ACA RNPs. Moreover, the SMN complex interacts with fibrillarin and Gar1 through the same domains that mediate the binding and assembly of snrnps , and the accumulation of U3 C/D RNA depends on the SMN complex 31. Distribution to functional sites. Depending on their function, the C/D and H/ACA RNAs ultimately localize to nucleoli, Cajal bodies or telomeres (FIG. 4c). Nucleolar RNAs move quickly through Cajal bodies to nucleoli. Nucleolar targeting of these RNAs depends on the signature sequences (box C and D, or box H and ACA) and an adjacent stem that tethers the box elements 123,124. A subset of the C/D and H/ACA guide RNAs, called small Cajal body (sca)rnas 62, remains in Cajal bodies to modify snrnas. Interestingly, the scarnas can be hybrids that contain both C/D and H/ACA motifs 62. Retention of H/ACA scarnas in Cajal bodies is mediated by Cajal body (CAB) boxes (FIG. 3), however, the H/ACA domain (that is, the nucleolar targeting element) is also required for proper Cajal-body localization 115. Mutation of the CAB boxes results in the appearance of the RNAs in nucleoli, indicating that the CAB signal supersedes the nucleolar targeting signal 115,125. A subset of Sm proteins was recently shown to associate with the scarnas in a CAB-box-dependent manner, and it will be interesting to see whether the Sm proteins are important for Cajal-body localization 126. Recent evidence indicates that PHAX and CRM1 interact with the few C/D RNAs that are independently transcribed and m 7 G-capped (BOX 3), and have a role in trafficking to Cajal bodies and nucleoli, respectively 30,31. However, the vast majority of vertebrate NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 8 MARCH

10 C/D and H/ACA RNAs are salvaged from introns and remain uncapped (BOX 3), and the factors involved in their trafficking remain to be identified. An interesting regulatory paradigm has emerged from recent studies of telomerase: an H/ACA RNP (in vertebrates) that functions in telomere synthesis 127,128. These results indicate that access of telomerase RNA (and its protein partner, telomerase reverse transcriptase (TERT)) to its substrate is regulated as a function of the cell cycle to restrict the activity of the telomerase enzyme to S phase. In human cells, telomerase RNA is retained in Cajal bodies throughout most of the cell cycle through a CAB box 127,128. At the same time, TERT is found in distinct nuclear foci (that do not correspond to Cajal bodies, telomeres or other known structures) 127. During S phase, both telomerase RNA and TERT move to telomeres 127,128. The trafficking of both components seems to involve intermediate steps in nucleoli and in foci that arise immediately adjacent to Cajal bodies, indicating further regulation in the biogenesis and transport of this enzyme 127,128. In yeast, telomerase resembles an Sm snrnp 129 rather than an H/ACA snornp, and therefore trafficking of this enzyme is regulated by distinct pathways in different organisms. Paradigms for the biogenesis of ncrnps Over the past few decades, intensive study of snrnps and snornps has rewarded us with an understanding of RNA-mediated mechanisms of RNA modification, cleavage and splicing. The concept of targeting enzymatic activity through guide RNAs that have antisense elements (derived from studies of the snrnas and snornas) has helped pave the way for the discovery and understanding of many other ncrnas. From the snrnps and snornps, we have learned that members of a given ncrnp family can function in distinct processes. This can be accomplished by specializations in the ncrnas that result in interactions with different sets of partner proteins (as in the case of the C/D and H/ACA RNPs involved in rrna processing). In some cases (for example, as proposed for the spliceosome), enzymatic activity might be intrinsic to the RNA. Ongoing studies of snrnps and snornps have revealed unexpectedly elaborate biogenesis pathways. These RNAs can be made through interesting variations on the familiar mrna-production pathways, but can also arise from unexpected pathways such as salvage from introns (BOX 3). Indeed, the two most diverse families of ncrnas (snornas and micrornas) are predominantly encoded by introns in human cells. The biogenesis of ncrnps can even involve symbiotic relationships, wherein the biogenesis of one ncrnp family requires the function of the other, and vice versa. For example, snrnps are required to liberate intronically trapped snornas, and snrnas, in turn, require snornp-mediated post-transcriptional modifications to function. ncrnas often require partner proteins that provide various essential functions in addition to the obvious associated enzymatic activities. For example, C/D and H/ACA RNP function seems to require the adjustment of the conformation of the guide RNA by the binding of the auxiliary protein L7Ae. A common principle in the biogenesis of both snrnps and snornps is the assembly of stable, but inactive, pre- RNPs that require maturation at locations distinct from the functional sites. Assembly of a functional complex and delivery to its site of function might be regulated via progression through a dynamic series of intermediate complexes and subcellular locales. Studies of the snrnas and snornas have defined cellular systems that function in the transport and assembly of ncrnps, including PHAX, the SMN complex and Cajal bodies. 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We thank current and past members of the Matera and Terns groups who have contributed to the work in our laboratories. M.P.T. and R.M.T. are grateful to C. Glover for continued scientific discourse. This work was supported by National Institutes of Health (National Institute of Neurological Disorders and Stroke and National Institute of General Medical Sciences) and Muscular Dystrophy Association grants to A.G.M. and by grants from the National Institutes of Health (National Institute of General Medical Sciences and National Cancer Institute) and the Nora L. Redman Fund to M.P.T. and R.M.T. Competing interests statement The authors declare no competing financial interests. DATABASES The following terms in this article are linked online to: UniProtKB: Cbf5 CPSF-73 CPSF-100 dyskerin Gar1 INT11 Nab3 Nop10 Nrd1 PHAX Sen1 FURTHER INFORMATION A. Gregory Matera s homepage: The Terns laboratory homepage: Access to this links box is available online. 220 MARCH 2007 VOLUME 8