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Ribosomal RNA (rRNA) carries extensive 2'-O-methyl marks at functionally important sites. This simple chemical modification is thought to confer stability, promote RNA folding, and contribute to generate a heterogenous ribosome population with a yet-uncharacterized function. 2'-O-methylation occurs both in archaea and eukaryotes and is accomplished by the Box C/D RNP enzyme in an RNA-guided manner. Extensive and partially conflicting structural information exists for the archaeal enzyme, while no structural data is available for the eukaryotic enzyme. The yeast Box C/D RNP consists of a guide RNA, the RNA-primary binding protein Snu13, the two scaffold proteins Nop56 and Nop58, and the enzymatic module Nop1. Here we present the high-resolution structure of the eukaryotic Box C/D methyltransferase Nop1 from Saccharomyces cerevisiae bound to the amino-terminal domain of Nop56. We discuss similarities and differences between the interaction modes of the two proteins in archaea and eukaryotes and demonstrate that eukaryotic Nop56 recruits the methyltransferase to the Box C/D RNP through a protein-protein interface that differs substantially from the archaeal orthologs. This study represents a first achievement in understanding the evolution of the structure and function of these proteins from archaea to eukaryotes., (© 2021 Höfler et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

U3 snoRNA and the associated Rrp9/U3-55K protein are essential for 18S rRNA production by the SSU-processome complex. U3 and Rrp9 are required for early pre-rRNA cleavages at sites A0, A1 and A2, but the mechanism remains unclear. Substitution of Arg 289 in Rrp9 to Ala (R289A) specifically reduced cleavage at sites A1 and A2. Surprisingly, R289 is located on the surface of the Rrp9 β-propeller structure opposite to U3 snoRNA. To understand this, we first characterized the protein-protein interaction network of Rrp9 within the SSU-processome. This identified a direct interaction between the Rrp9 β-propeller domain and Rrp36, the strength of which was reduced by the R289A substitution, implicating this interaction in the observed processing phenotype. The Rrp9 R289A mutation also showed strong synergistic negative interactions with mutations in U3 that destabilize the U3/pre-rRNA base-pair interactions or reduce the length of their linking segments. We propose that the Rrp9 β-propeller and U3/pre-rRNA binding cooperate in the structure or stability of the SSU-processome. Additionally, our analysis of U3 variants gave insights into the function of individual segments of the 5'-terminal 72-nt sequence of U3. We interpret these data in the light of recently reported SSU-processome structures., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Many cellular functions rely on stable protein-only or protein-RNA complexes. Deciphering their assembly mechanism is a key question in cell biology. We here focus on box C/D small nucleolar ribonucleoproteins involved in ribosome biogenesis. The mature particles contain four core proteins and a guide RNA. Despite their relatively simple composition, these particles don't self-assemble in eukaryote and the production of a native and functional particle requires a large number of transient other proteins, called assembly factors. We present here 13 C and 15 N solid-state NMR assignment of yeast 126-residue core protein Snu13 in the context of its 50 kDa pre-complex with assembly factors Rsa1p:Hit1p. In this sample, only one third of the protein is labelled, leading to a low sensitivity. We could nevertheless obtain assignment data for 91% of the residues. Secondary structure derived from our assignments shows that Snu13p overall structure is maintained in the context of the complex. Chemical shift perturbations are analysed to evaluate Snu13p conformational changes and interaction interface upon binding to its partner proteins. While indirect perturbations are observed in the hydrophobic core, we find other good candidate residues belonging to the interaction interface. We describe the role of some Snu13p N-terminal and C-terminal residues, not identified in previous structural studies. These preliminary results will serve as a basis for future interaction studies, especially by adding RNA, to decipher box C/D snoRNP particles assembly pathway.

The AAA + ATPase R2TP complex facilitates assembly of a number of ribonucleoprotein particles (RNPs). Although the architecture of R2TP is known, its molecular basis for acting upon multiple RNPs remains unknown. In yeast, the core subunit of the box C/D small nucleolar RNPs, Nop58p, is the target for R2TP function. In the recently observed U3 box C/D snoRNP as part of the 90 S small subunit processome, the unfolded regions of Nop58p are observed to form extensive interactions, suggesting a possible role of R2TP in stabilizing the unfolded region of Nop58p prior to its assembly. Here, we analyze the interaction between R2TP and a Maltose Binding Protein (MBP)-fused Nop58p by biophysical and yeast genetics methods. We present evidence that R2TP interacts largely with the unfolded termini of Nop58p. Our results suggest a general mechanism for R2TP to impart specificity by recognizing unfolded regions in its clients.

Methylation of arginine residues in proteins, an enzyme-mediated post-translational modification (PTM), is important for mRNA processing and transport and for the regulation of many protein-protein interactions. However, proteolytic peptides resulting from alternative sites of post-translational methylation have identical masses and cannot be readily separated by standard liquid chromatography-mass spectrometry. Unlike acetylation or phosphorylation, methylation of arginine does not strongly affect the charge states of peptide ions, multiple instances of methylation can occur on a single amino acid residue, and the relative mass of the modification is <1% that of the typical proteolytic peptide. High field asymmetric waveform ion mobility spectrometry (FAIMS) is an orthogonal separation method to liquid chromatography that can rapidly separate gaseous ions prior to detection by mass spectrometry. Here, we report that FAIMS can be used to separate arginine-methylated peptides that differ by the position of a single methyl group for both mono- and dimethylated variants. Although the resolution of separation for these arginine-methylated peptides improved with increasing amounts of helium in the FAIMS carrier gas as expected, we found that the site of methylation can strongly affect the dependence of the electric field used for ion transmission on the extent of helium in the carrier gas. Thus, certain isobaric peptides can be cotransmitted at high helium concentrations whereas lower concentrations can be used for successful separations of such peptide mixtures. The capability to rapidly resolve isobaric arginine-methylated peptides should be useful in the future for the detailed analysis of protein arginine methylation in biological samples.

Eukaryotic ribosome biogenesis is a highly orchestrated process involving numerous assembly factors including ATP-dependent RNA helicases. The DEAH helicase DHX37 (Dhr1 in yeast) is activated by the ribosome biogenesis factor UTP14A to facilitate maturation of the small ribosomal subunit. We report the crystal structure of DHX37 in complex with single-stranded RNA, revealing a canonical DEAH ATPase/helicase architecture complemented by a structurally unique carboxy-terminal domain (CTD). Structural comparisons of the nucleotide-free DHX37-RNA complex with DEAH helicases bound to RNA and ATP analogs reveal conformational changes resulting in a register shift in the bound RNA, suggesting a mechanism for ATP-dependent 3'-5' RNA translocation. We further show that a conserved sequence motif in UTP14A interacts with and activates DHX37 by stimulating its ATPase activity and enhancing RNA binding. In turn, the CTD of DHX37 is required, but not sufficient, for interaction with UTP14A in vitro and is essential for ribosome biogenesis in vivo. Together, these results shed light on the mechanism of DHX37 and the function of UTP14A in controlling its recruitment and activity during ribosome biogenesis., (© 2019 Boneberg et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Structural biology studies of archaeal and yeast box C/D ribonucleoprotein particles (RNPs) reveal a surprisingly wide range of forms. If form ever follows function, the different structures of box C/D small ribonucleoprotein particles (snoRNPs) may reflect their versatile functional roles beyond what has been recognized. A large majority of box C/D RNPs serve to site-specifically methylate the ribosomal RNA, typically as independent complexes. Select members of the box C/D snoRNPs also are essential components of the megadalton RNP enzyme, the small subunit processome that is responsible for processing ribosomal RNA. Other box C/D RNPs continue to be uncovered with either unexpected or unknown functions. We summarize currently known box C/D RNP structures in this review and identify the Nop56/58 and box C/D RNA subunits as the key elements underlying the observed structural diversity, and likely, the diverse functional roles of box C/D RNPs., (© 2018 Yu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Archaeal fibrillarin (aFib) is a well-characterized S -adenosyl methionine (SAM)-dependent RNA 2'- O -methyltransferase that is known to act in a large C/D ribonucleoprotein (RNP) complex together with Nop5 and L7Ae proteins and a box C/D guide RNA. In the reaction, the guide RNA serves to direct the methylation reaction to a specific site in tRNA or rRNA by sequence complementarity. Here we show that a Pyrococcus abyssi aFib-Nop5 heterodimer can alone perform SAM-dependent 2'- O -methylation of 16S and 23S ribosomal RNAs in vitro independently of L7Ae and C/D guide RNAs. Using tritium-labeling, mass spectrometry, and reverse transcription analysis, we identified three in vitro 2'- O -methylated positions in the 16S rRNA of P. abyssi , positions lying outside of previously reported pyrococcal C/D RNP methylation sites. This newly discovered stand-alone activity of aFib-Nop5 may provide an example of an ancestral activity retained in enzymes that were recruited to larger complexes during evolution., (© 2017 Tomkuvienė et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Two proteins with PIN endonuclease domains, yUtp24(Fcf1)/hUTP24 and yUtp23/hUTP23 are essential for early pre-ribosomal (r)RNA cleavages at sites A0, A1/1 and A2/2a in yeast and humans. The yUtp24/hUTP24 PIN endonuclease is proposed to cleave at sites A1/1 and A2/2a, but the enzyme cleaving at site A0 is not known. Yeast yUtp23 contains a degenerate, non-essential PIN domain and functions together with the snR30 snoRNA, while human hUTP23 is associated with U17, the human snR30 counterpart. Using in vivo RNA-protein crosslinking and gel shift experiments, we reveal that yUtp23/hUTP23 makes direct contacts with expansion sequence 6 (ES6) in the 18S rRNA sequence and that yUtp23 interacts with the 3΄ half of the snR30 snoRNA. Protein-protein interaction studies further demonstrated that yeast yUtp23 and human hUTP23 directly interact with the H/ACA snoRNP protein yNhp2/hNHP2, the RNA helicase yRok1/hROK1(DDX52), the ribosome biogenesis factor yRrp7/hRRP7 and yUtp24/hUTP24. yUtp23/hUTP23 could therefore be central to the coordinated integration and release of ES6 binding factors and likely plays a pivotal role in remodeling this pre-rRNA region in both yeast and humans. Finally, studies using RNAi-rescue systems in human cells revealed that intact PIN domain and Zinc finger motifs in human hUTP23 are essential for 18S rRNA maturation., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Box C/D small nucleolar ribonucleoparticles (snoRNPs) support 2'-O-methylation of several target RNAs. They share a common set of four core proteins (SNU13, NOP58, NOP56, and FBL) that are assembled on different guide small nucleolar RNAs. Assembly of these entities involves additional protein factors that are absent in the mature active particle. In this context, the platform protein NUFIP1/Rsa1 establishes direct and simultaneous contacts with core proteins and with the components of the assembly machinery. Here, we solve the nuclear magnetic resonance (NMR) structure of a complex resulting from interaction between protein fragments of human NUFIP1 and its cofactor ZNHIT3, and emphasize their imbrication. Using yeast two-hybrid and complementation assays, protein co-expression, isothermal titration calorimetry, and NMR, we demonstrate that yeast and human complexes involving NUFIP1/Rsa1p, ZNHIT3/Hit1p, and SNU13/Snu13p share strong structural similarities, suggesting that the initial steps of the box C/D snoRNP assembly process are conserved among species., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Zf–HIT family members share the zf–HIT domain (ZHD), which is characterized by a fold in “treble-clef” through interleaved CCCC and CCHC ZnF motifs that both bind a zinc atom. Six proteins containing ZHD are present in human and three in yeast proteome, all belonging to multimodular RNA/protein complexes involved in gene regulation, chromatin remodeling, and snoRNP assembly. An interesting characteristic of the cellular complexes that ensure these functions is the presence of the RuvBL1/2/Rvb1/2 ATPases closely linked with zf–HIT proteins. Human ZNHIT6/BCD1 and its counterpart in yeast Bcd1p were previously characterized as assembly factors of the box C/D snoRNPs. Our data reveal that the ZHD of Bcd1p is necessary but not sufficient for yeast growth and that the motif has no direct RNA-binding capacity but helps Bcd1p maintain the box C/D snoRNAs level in steady state. However, we demonstrated that Bcd1p interacts nonspecifically with RNAs depending on their length. Interestingly, the ZHD of Bcd1p is functionally interchangeable with that of Hit1p, another box C/D snoRNP assembly factor belonging to the zf–HIT family. This prompted us to use NMR to solve the 3D structures of ZHD from yeast Bcd1p and Hit1p to highlight the structural similarity in the zf–HIT family. We identified structural features associated with the requirement of Hit1p and Bcd1p ZHD for cell growth and box C/D snoRNA stability under heat stress. Altogether, our data suggest an important role of ZHD could be to maintain functional folding to the rest of the protein, especially under heat stress conditions.

H/ACA RNA-guided ribonucleoprotein particle (RNP), the most complicated RNA pseudouridylase so far known, uses H/ACA guide RNA for substrate capture and four proteins (Cbf5, Nop10, L7Ae and Gar1) for pseudouridylation. Although it was shown that Gar1 not only facilitates the product release, but also enhances the catalytic activity, the chemical role that Gar1 plays in this complicated machinery is largely unknown. Kinetics measurement on Pyrococcus furiosus RNPs at different temperatures making use of fluorescence anisotropy showed that Gar1 reduces the catalytic barrier through affecting the activation entropy instead of enthalpy. Site-directed mutagenesis combined with molecular dynamics simulations demonstrated that V149 in the thumb loop of Cbf5 is critical in placing the target uridine to the right position toward catalytic D85 of Cbf5. The enzyme elegantly aligns the position of uridine in the catalytic site with the help of Gar1. In addition, conversion of uridine to pseudouridine results in a rigid syn configuration of the target nucleotide in the active site and causes Gar1 to pull out the thumb. Both factors guarantee the efficient release of the product., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Box C/D snoRNPs are responsible for rRNA methylation and processing, and are formed by snoRNAs and four conserved proteins, Nop1, Nop56, Nop58 and Snu13. The snoRNP assembly is a stepwise process, involving other protein complexes, among which the R(2)TP and Hsp90 chaperone. Nop17, also known as Pih1, has been shown to be a constituent of the R(2)TP (Rvb1, Rvb2, Tah1, Pih1) and to participate in box C/D snoRNP assembly by its interaction with Nop58. The molecular function of Nop17, however, has not yet been described., Results: To shed light on the role played by Nop17 in the maturation of snoRNP, here we analyzed the interactions domains of Nop58 - Nop17 - Tah1 and the importance of ATP to the interaction between Nop17 and the ATPase Rvb1/2., Conclusions: Based on the results shown here, we propose a model for the assembly of box C/D snoRNP, according to which R(2)TP complex is important for reducing the affinity of Nop58 for snoRNA, and for the binding of the other snoRNP subunits.

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoprotein particles (C/D snoRNPs) involves conserved trans-acting factors, which are proposed to facilitate the assembly of the core proteins Snu13p/15.5K, Nop58p/NOP58, Nop56p/NOP56 and Nop1p/Fibrillarin on box C/D small nucleolar RNAs (C/D snoRNAs). In yeast, protein Rsa1 acts as a platform, interacting with both the RNA-binding core protein Snu13 and protein Pih1 of the Hsp82-R2TP chaperone complex. In this work, a proteomic approach coupled with functional and structural studies identifies protein Hit1 as a novel Rsa1p-interacting partner involved in C/D snoRNP assembly. Hit1p contributes to in vivo C/D snoRNA stability and pre-RNA maturation kinetics. It associates with U3 snoRNA precursors and influences its 3'-end processing. Remarkably, Hit1p is required to maintain steady-state levels of Rsa1p. This stabilizing activity is likely to be general across eukaryotic species, as the human protein ZNHIT3(TRIP3) showing sequence homology with Hit1p regulates the abundance of NUFIP1, the Rsa1p functional homolog. The nuclear magnetic resonance solution structure of the Rsa1p317-352-Hit1p70-164 complex reveals a novel mode of protein-protein association explaining the strong stability of the Rsa1p-Hit1p complex. Our biochemical data show that C/D snoRNAs and the core protein Nop58 can interact with the purified Snu13p-Rsa1p-Hit1p heterotrimer., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Post-transcriptional modifications are essential to the cell life cycle, as they affect both pre-ribosomal RNA processing and ribosome assembly. The box C/D ribonucleoprotein enzyme that methylates ribosomal RNA at the 2'-O-ribose uses a multitude of guide RNAs as templates for the recognition of rRNA target sites. Two methylation guide sequences are combined on each guide RNA, the significance of which has remained unclear. Here we use a powerful combination of NMR spectroscopy and small-angle neutron scattering to solve the structure of the 390 kDa archaeal RNP enzyme bound to substrate RNA. We show that the two methylation guide sequences are located in different environments in the complex and that the methylation of physiological substrates targeted by the same guide RNA occurs sequentially. This structure provides a means for differential control of methylation levels at the two sites and at the same time offers an unexpected regulatory mechanism for rRNA folding.

The U3 snoRNA is required for 18S rRNA processing and small subunit ribosome formation in eukaryotes. Different from other box C/D snoRNAs, U3 contains an extra 5' domain that pairs with pre-rRNA and a unique B/C motif essential for recruitment of the U3-specific Rrp9 protein. Here, we analyze the structure and function of Rrp9 with crystallographic, biochemical, and cellular approaches. Rrp9 is composed of a WD repeat domain and an N-terminal region. The crystal structures of the WD domain of yeast Rrp9 and its human ortholog U3-55K were determined, revealing a typical seven-bladed propeller fold. Several conserved surface patches on the WD domain were identified, and their function in RNP assembly and yeast growth were analyzed by mutagenesis. Prior association of Snu13 with the B/C motif was found to enhance the specific binding of the WD domain. We show that a conserved 7bc loop is crucial for specific recognition of U3, nucleolar localization of Rrp9, and yeast growth. In addition, we show that the N-terminal region of Rrp9 contains a bipartite nuclear localization signal that is dispensable for nucleolar localization. Our results provide insight into the functional sites of Rrp9.

The box H/ACA RNA-guided pseudouridine synthase is a complicated ribonucleoprotein enzyme that recruits substrate via both the guide RNA and the catalytic subunit Cbf5. Structural studies have revealed multiple conformations of the enzyme, but a quantitative description of the reaction pathway is still lacking. Using fluorescence correlation spectroscopy, we here measured the equilibrium dissociation constants and kinetic association and dissociation rates of substrate and product complexes mimicking various reaction intermediate states. These data support a sequential model for substrate loading and product release regulated by the thumb loop of Cbf5. The uridine substrate is first bound primarily through interaction with the guide RNA and then loaded into the active site while progressively interacted with the thumb. After modification, the subtle chemical structure change from uridine to pseudouridine at the target site triggers the release of the thumb, resulting in an intermediate complex with the product bound mainly by the guide RNA. By dissecting the role of Gar1 in individual steps of substrate turnover, we show that Gar1 plays a major role in catalysis and also accelerates product release about 2-fold. Our biophysical results integrate with previous structural knowledge into a coherent reaction pathway of H/ACA RNA-guided pseudouridylation.

Alu repetitive sequences are the most abundant short interspersed DNA elements in the human genome. Full-length Alu elements are composed of two tandem sequence monomers, the left and right Alu arms, both derived from the 7SL signal recognition particle RNA. Since Alu elements are common in protein-coding genes, they are frequently transcribed into pre-mRNAs. Here, we demonstrate that the right arms of nascent Alu transcripts synthesized within pre-mRNA introns are processed into metabolically stable small RNAs. The intron-encoded Alu RNAs, termed AluACA RNAs, are structurally highly reminiscent of box H/ACA small Cajal body (CB) RNAs (scaRNAs). They are composed of two hairpin units followed by the essential H (AnAnnA) and ACA box motifs. The mature AluACA RNAs associate with the four H/ACA core proteins: dyskerin, Nop10, Nhp2, and Gar1. Moreover, the 3' hairpin of AluACA RNAs carries two closely spaced CB localization motifs, CAB boxes (UGAG), which bind Wdr79 in a cumulative fashion. In contrast to canonical H/ACA scaRNPs, which concentrate in CBs, the AluACA RNPs accumulate in the nucleoplasm. Identification of 348 human AluACA RNAs demonstrates that intron-encoded AluACA RNAs represent a novel, large subgroup of H/ACA RNAs, which are apparently confined to human or primate cells.

Box C/D small (nucleolar) ribonucleoproteins [s(no)RNPs] catalyze RNA-guided 2'-O-ribose methylation in two of the three domains of life. Recent structural studies have led to a controversy over whether box C/D sRNPs functionally assemble as monomeric or dimeric macromolecules. The archaeal box C/D sRNP from Methanococcus jannaschii (Mj) has been shown by glycerol gradient sedimentation, gel filtration chromatography, native gel analysis, and single-particle electron microscopy (EM) to adopt a di-sRNP architecture, containing four copies of each box C/D core protein and two copies of the Mj sR8 sRNA. Subsequently, investigators used a two-stranded artificial guide sRNA, CD45, to assemble a box C/D sRNP from Sulfolobus solfataricus with a short RNA methylation substrate, yielding a crystal structure of a mono-sRNP. To more closely examine box C/D sRNP architecture, we investigate the role of the omnipresent sRNA loop as a structural determinant of sRNP assembly. We show through sRNA mutagenesis, native gel electrophoresis, and single-particle EM that a di-sRNP is the near exclusive architecture obtained when reconstituting box C/D sRNPs with natural or artificial sRNAs containing an internal loop. Our results span three distantly related archaeal species--Sulfolobus solfataricus, Pyrococcus abyssi, and Archaeoglobus fulgidus--indicating that the di-sRNP architecture is broadly conserved across the entire archaeal domain.

The integral telomerase RNA subunit templates the synthesis of telomeric repeats. The biological accumulation of human telomerase RNA (hTR) requires hTR H/ACA domain assembly with the same proteins that assemble on other human H/ACA RNAs. Despite this shared RNP composition, hTR accumulation is particularly sensitized to disruption by disease-linked H/ACA protein variants. We show that contrary to expectation, hTR-specific sequence requirements for biological accumulation do not act at an hTR-specific step of H/ACA RNP biogenesis; instead, they enhance hTR binding to the shared, chaperone-bound scaffold of H/ACA core proteins that mediates initial RNP assembly. We recapitulate physiological H/ACA RNP assembly with a preassembled NAF1/dyskerin/NOP10/NHP2 scaffold purified from cell extract and demonstrate that distributed sequence features of the hTR 3' hairpin synergize to improve scaffold binding. Our findings reveal that the hTR H/ACA domain is distinguished from other human H/ACA RNAs not by a distinct set of RNA-protein interactions but by an increased efficiency of RNP assembly. Our findings suggest a unifying mechanism for human telomerase deficiencies associated with H/ACA protein variants.

Box C/D and H/ACA RNPs are essential ribonucleoprotein particles that are found throughout both eukaryotes [small nucleolar RNPs (snoRNPs)] and archaea [snoRNP-like complexes (sRNPs)]. These complexes catalyze the site-specific pseudouridylation and most of the methylation of ribosomal RNA (rRNA). The numerous modifications, which are clustered in functionally important regions of the rRNA, are important for rRNA folding and ribosome function. The RNA component of the complexes [small nucleolar RNA (snoRNA) or small RNA (sRNA)] functions in substrate binding by base pairing with the target site and as a scaffold coordinating the organization of the complex. In eukaryotes, a subset of snoRNPs do not catalyze modification but, through base pairing to the rRNA or flanking precursor sequences, direct pre-rRNA folding and are essential for rRNA processing. In the last few years there have been significant advances in our understanding of the structure of archaeal sRNPs. High resolution structures of the archaeal C/D and H/ACA sRNPs have not only provided a detailed understanding of the molecular architecture of these complexes but also produced key insights into substrate binding and product release. In both cases, this is mediated by significant movement in the complexes. Advances have also been made in our knowledge of snoRNP recruitment and release from pre-ribosome complexes in eukaryotes. New snoRNA-rRNA interactions have been documented, and the roles of RNA helicases in releasing snoRNP complexes from the rRNA have been described., (Copyright © 2011 John Wiley & Sons, Ltd.)

Cbf5 is a pseudouridine synthase that usually acts in a guide RNA-dependent manner as part of H/ACA small ribonucleoproteins; however archaeal Cbf5 can also act independently of guide RNA in modifying uridine 55 in tRNA. This guide-independent activity of Cbf5 is enhanced by proteins Nop10 and Gar1 which are also found in H/ACA small ribonucleoproteins. Here, we analyzed the specific contribution of Nop10 and Gar1 for Cbf5-catalyzed pseudouridylation of tRNA. Interestingly, both Nop10 and Gar1 not only increase Cbf5's affinity for tRNA, but they also directly enhance Cbf5's catalytic activity by increasing the k(cat) of the reaction. In contrast to the guide RNA-dependent reaction, Gar1 is not involved in product release after tRNA modification. These results in conjunction with structural information suggest that Nop10 and Gar1 stabilize Cbf5 in its active conformation; we hypothesize that this might also be true for guide-RNA dependent pseudouridine formation by Cbf5.

Shq1 is a conserved protein required for the biogenesis of eukaryotic H/ACA ribonucleoproteins (RNPs), including human telomerase. We report the structure of the Shq1-specific domain alone and in complex with H/ACA RNP proteins Cbf5, Nop10 and Gar1. The Shq1-specific domain adopts a novel helical fold and primarily contacts the PUA domain and the otherwise disordered C-terminal extension (CTE) of Cbf5. The structure shows that dyskeratosis congenita mutations found in the CTE of human Cbf5 likely interfere with Shq1 binding. However, most mutations in the PUA domain are not located at the Shq1-binding surface and also have little effect on the yeast Cbf5-Shq1 interaction. Shq1 binds Cbf5 independently of the H/ACA RNP proteins Nop10, Gar1 and Nhp2 and the assembly factor Naf1, but shares an overlapping binding surface with H/ACA RNA. Shq1 point mutations that disrupt Cbf5 interaction suppress yeast growth particularly at elevated temperatures. Our results suggest that Shq1 functions as an assembly chaperone that protects the Cbf5 protein complexes from non-specific RNA binding and aggregation before assembly of H/ACA RNA.

SHQ1 is an essential assembly factor for H/ACA ribonucleoproteins (RNPs) required for ribosome biogenesis, pre-mRNA splicing, and telomere maintenance. SHQ1 binds dyskerin/NAP57, the catalytic subunit of human H/ACA RNPs, and this interaction is modulated by mutations causing X-linked dyskeratosis congenita. We report the crystal structure of the C-terminal domain of yeast SHQ1, Shq1p, and its complex with yeast dyskerin/NAP57, Cbf5p, lacking its catalytic domain. The C-terminal domain of Shq1p interacts with the RNA-binding domain of Cbf5p and, through structural mimicry, uses the RNA-protein-binding sites to achieve a specific protein-protein interface. We propose that Shq1p operates as a Cbf5p chaperone during RNP assembly by acting as an RNA placeholder, thereby preventing Cbf5p from nonspecific RNA binding before association with an H/ACA RNA and the other core RNP proteins.

Box H/ACA ribonucleoprotein particles (RNPs) mediate pseudouridine synthesis, ribosome formation, and telomere maintenance. The structure of eukaryotic H/ACA RNPs remains poorly understood. We reconstituted functional Saccharomyces cerevisiae H/ACA RNPs with recombinant proteins Cbf5, Nop10, Gar1, and Nhp2 and a two-hairpin H/ACA RNA; determined the crystal structure of a Cbf5, Nop10, and Gar1 ternary complex at 1.9 Å resolution; and analyzed the structure-function relationship of the yeast complex. Although eukaryotic H/ACA RNAs have a conserved two-hairpin structure, isolated single-hairpin RNAs are also active in guiding pseudouridylation. Nhp2, unlike its archaeal counterpart, is largely dispensable for the activity, reflecting a functional adaptation of eukaryotic H/ACA RNPs to the variable RNA structure that Nhp2 binds. The N-terminal extension of Cbf5, a hot spot for dyskeratosis congenita mutation, forms an extra structural layer on the PUA domain. Gar1 is distinguished from the assembly factor Naf1 by containing a C-terminal extension that controls substrate turnover and the Gar1-Naf1 exchange during H/ACA RNP maturation. Our results reveal significant novel features of eukaryotic H/ACA RNPs.

Small nuclear and nucleolar RNAs that program pre-mRNA splicing and rRNA processing have a signature 5'-trimethylguanosine (TMG) cap. Whereas the mechanism of TMG synthesis by Tgs1 methyltransferase has been elucidated, we know little about whether or how RNP biogenesis, structure and function are perturbed when TMG caps are missing. Here, we analyzed RNPs isolated by tandem-affinity purification from TGS1 and tgs1Δ yeast strains. The protein and U-RNA contents of total SmB-containing RNPs were similar. Finer analysis revealed stoichiometric association of the nuclear cap-binding protein (CBP) subunits Sto1 and Cbc2 with otherwise intact Mud1- and Nam8-containing U1 snRNPs from tgs1Δ cells. CBP was not comparably enriched in Lea1-containing U2 snRNPs from tgs1Δ cells. Moreover, CBP was not associated with mature Nop58-containing C/D snoRNPs or mature Cbf5- and Gar1-containing H/ACA snoRNPs from tgs1Δ cells. The protein composition and association of C/D snoRNPs with the small subunit (SSU) processosome were not grossly affected by absence of TMG caps, nor was the composition of H/ACA snoRNPs. The cold-sensitive (cs) growth defect of tgs1Δ yeast cells could be suppressed by mutating the cap-binding pocket of Cbc2, suggesting that ectopic CBP binding to the exposed U1 m(7)G cap in tgs1Δ cells (not lack of TMG caps per se) underlies the cs phenotype.

While plant rRNA gene organization and expression have been studied for several decades, the repetitive nature and high sequence identity between the tandemly-repeated units has prevented precise studies to determine which units are active and elucidate mechanisms of spatial and temporal transcriptional control. We have detected four variants among rRNA genes of Arabidopsis thaliana by analysis of the 3' external spacer region. Surprisingly, the most abundant variant, representing ~50% of the genes, is not expressed in wild-type plants but is transcribed in lines mutated in one of the two nucleolin genes. Analysis of Arabidopsis ecotypes indicated considerable variability in numbers and presence/absence of variants, although more closely-related ecotypes show similar profiles. Sequence analysis showed that one of the variants is not only unexpectedly located within a 5S RNA gene cluster in the pericentromeric region of chromosome 3 but is also potentially highly expressed in wild-type plants. We present a model to explain how transcription of this this unusual variant, maintained in an active state by nucleolin binding, could be involved in control of expression of the major variant, while absence of nucleolin leads to silencing of the minor variant and consequent expression of the major variant.

Box C/D ribonucleoproteins (RNP) guide the 2'-O-methylation of targeted nucleotides in archaeal and eukaryotic rRNAs. The archaeal L7Ae and eukaryotic 15.5kD box C/D RNP core protein homologues initiate RNP assembly by recognizing kink-turn (K-turn) motifs. The crystal structure of the 15.5kD core protein from the primitive eukaryote Giardia lamblia is described here to a resolution of 1.8 Å. The Giardia 15.5kD protein exhibits the typical α-β-α sandwich fold exhibited by both archaeal L7Ae and eukaryotic 15.5kD proteins. Characteristic of eukaryotic homologues, the Giardia 15.5kD protein binds the K-turn motif but not the variant K-loop motif. The highly conserved residues of loop 9, critical for RNA binding, also exhibit conformations similar to those of the human 15.5kD protein when bound to the K-turn motif. However, comparative sequence analysis indicated a distinct evolutionary position between Archaea and Eukarya. Indeed, assessment of the Giardia 15.5kD protein in denaturing experiments demonstrated an intermediate stability in protein structure when compared with that of the eukaryotic mouse 15.5kD and archaeal Methanocaldococcus jannaschii L7Ae proteins. Most notable was the ability of the Giardia 15.5kD protein to assemble in vitro a catalytically active chimeric box C/D RNP utilizing the archaeal M. jannaschii Nop56/58 and fibrillarin core proteins. In contrast, a catalytically competent chimeric RNP could not be assembled using the mouse 15.5kD protein. Collectively, these analyses suggest that the G. lamblia 15.5kD protein occupies a unique position in the evolution of this box C/D RNP core protein retaining structural and functional features characteristic of both archaeal L7Ae and higher eukaryotic 15.5kD homologues.

Cbf5 is the catalytic subunit of the H/ACA small nucleolar/Cajal body ribonucleoprotein particles (RNPs) responsible for site specific isomerization of uridine in ribosomal and small nuclear RNA. Recent evidence from studies on archaeal Cbf5 suggests its second functional role in modifying tRNA U55 independent of guide RNA. In order to act both as a stand-alone and a RNP pseudouridine synthase, Cbf5 must differentiate features in H/ACA RNA from those in tRNA or rRNA. Most H/ACA RNAs contain a hallmark ACA trinucleotide downstream of the H/ACA motif. Here we challenged an archaeal Cbf5 (in the form of a ternary complex with its accessory proteins Nop10 and Gar1) with T-stem-loop RNAs with or without ACA trinucleotide in the stem. Although these substrates were previously shown to be substrates for the bacterial stand-alone pseudouridine synthase TruB, the Cbf5-Nop10-Gar1 complex was only able to modify those without ACA trinucleotide. A crystal structure of Cbf5-Nop10-Gar1 trimer bound with an ACA-containing T-stem-loop revealed that the ACA trinucleotide detracted Cbf5 from the stand-alone binding mode, thereby suggesting that the H/ACA RNP-associated function of Cbf5 likely supersedes its stand-alone function.

Box C/D ribonucleoprotein particles guide the 2'-O-ribose methylation of target nucleotides in both archaeal and eukaryotic RNAs. These complexes contain two functional centers, assembled around the C/D and C'/D' motifs in the box C/D RNA. The C/D and C'/D' RNPs of the archaeal snoRNA-like RNP (sRNP) are spatially and functionally coupled. Here, we show that similar coupling also occurs in eukaryotic box C/D snoRNPs. The C/D RNP guided 2'-O-methylation when the C'/D' motif was either mutated or ablated. In contrast, the C'/D' RNP was inactive as an independent complex. Additional experiments demonstrated that the internal C'/D' RNP is spatially coupled to the terminal box C/D complex. Pulldown experiments also indicated that all four core proteins are independently recruited to the box C/D and C'/D' motifs. Therefore, the spatial-functional coupling of box C/D and C'/D' RNPs is an evolutionarily conserved feature of both archaeal and eukaryotic box C/D RNP complexes.

Box C/D small nucleolar and Cajal body ribonucleoprotein particles (sno/scaRNPs) direct site-specific 2'-O-methylation of ribosomal and spliceosomal RNAs and are critical for gene expression. Here we report crystal structures of an archaeal box C/D RNP containing three core proteins (fibrillarin, Nop56/58, and L7Ae) and a half-mer box C/D guide RNA paired with a substrate RNA. The structure reveals a guide-substrate RNA duplex orientation imposed by a composite protein surface and the conserved GAEK motif of Nop56/58. Molecular modeling supports a dual C/D RNP structure that closely mimics that recently visualized by electron microscopy. The substrate-bound dual RNP model predicts an asymmetric protein distribution between the RNP that binds and methylates the substrate RNA. The predicted asymmetric nature of the holoenzyme is consistent with previous biochemical data on RNP assembly and provides a simple solution for accommodating base-pairing between the C/D guide RNA and large ribosomal and spliceosomal substrate RNAs., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The H/ACA motif of human telomerase RNA (hTR) directs specific pathways of endogenous telomerase holoenzyme assembly, function, and regulation. Similarities between hTR and other H/ACA RNAs have been established, but differences have not been explored even though unique features of hTR H/ACA RNP assembly give rise to telomerase deficiency in human disease. Here, we define hTR H/ACA RNA and RNP architecture using RNA accumulation, RNP affinity purification, and primer extension activity assays. First, we evaluate alternative folding models for the hTR H/ACA motif 5' hairpin. Second, we demonstrate an unanticipated and surprisingly general asymmetry of 5' and 3' hairpin requirements for H/ACA RNA accumulation. Third, we establish that hTR assembles not one but two sets of all four of the H/ACA RNP core proteins, dyskerin, NOP10, NHP2, and GAR1. Fourth, we address a difference in predicted specificities of hTR association with the holoenzyme subunit WDR79/TCAB1. Together, these results complete the analysis of hTR elements required for active RNP biogenesis and define the interaction specificities and stoichiometries of all functionally essential human telomerase holoenzyme subunits. This study uncovers unexpected similarities but also differences between telomerase and other H/ACA RNPs that allow a unique specificity of telomerase biogenesis and regulation.

Small nucleolar ribonucleoproteins (snoRNPs) are widely studied and characterized as guide RNAs for sequence-specific 2'-O-ribose methylation and psuedouridylation of ribosomal RNAs. In addition, snoRNAs have also been shown to interact with some tRNAs and direct alternative splicing in mRNA biogenesis. Recent advances in bioinformatics have resulted in new algorithms able to rapidly identify noncoding RNAs generally and snoRNAs specifically in genomic and metagenomic sequences, resulting in a rapid increase in the number and diversity of identified snoRNA sequences. The snoRNP database is a web-based collection of snoRNA and snoRNA-associated protein sequences from a wide range of species. The database currently contains 8994 snoRNA sequences from Bacteria, Archaea, and Eukaryotes and 589 snoRNA-associated protein sequences. The snoRNP database can be found at: http://evolveathome.com/snoRNA/snoRNA.php.

H/ACA RNAs form ribonucleoprotein complex (RNP) with proteins Cbf5, Nop10, L7Ae, and Gar1 and guide site-specific conversion of uridine into pseudouridine in cellular RNAs. The crystal structures of H/ACA RNP with substrate bound at the active site cleft reveal that the substrate is recruited through sequence-specific pairing with guide RNA and essential protein contacts. Substrate binding leads to a reorganization of a preset pseudouridylation pocket and an adaptive movement of the PUA domain and the lower stem of the H/ACA RNA. Moreover, a thumb loop flips from the Gar1-bound state in the substrate-free RNP structure to tightly associate with the substrate. Mutagenesis and enzyme kinetics analysis suggest a critical role of Gar1 and the thumb in substrate turnover, particularly in product release. Comparison with tRNA Psi55 synthase TruB reveals the structural conservation and adaptation between an RNA-guided and stand-alone pseudouridine synthase and provides insight into the guide-independent activity of Cbf5.

The structural basis for accurate placement of substrate RNA by H/ACA proteins is studied using a nonintrusive fluorescence assay. A model substrate RNA containing 2-aminopurine immediately 3' of the uridine targeted for modification produces distinct fluorescence signals that report the substrate's docking status within the enzyme active site. We combined substrate RNA with complete and subcomplexes of H/ACA ribonucleoprotein particles and monitored changes in the substrate conformation. Our results show that each of the three accessory proteins, as well as an active site residue, have distinct effects on substrate conformations, presumably as docking occurs. Interestingly, in some cases these effects are exerted far from the active site. Application of our data to an available structural model of the holoenzyme, enables the functional role of each accessory protein in substrate placement to come into view.

The kink turn (K-turn) is a common motif in RNA structure, found in many RNA species important in translation, RNA modification and splicing, and the control of gene expression. In general the K-turn comprises a three nucleotide bulge followed by trans sugar-Hoogsteen G.A pairs. The RNA adopts a tightly kinked conformation, and is a common target for binding proteins, exemplified by the L7Ae family. We have measured the rates of association and dissociation for the binding of L7Ae to the Kt-7 kink turn, from which we calculate an affinity of K(D)=10 pM. This high affinity is consistent with the role of this binding as the first stage in the assembly of key functional nucleoproteins such as box C/D snoRNP. Kink-turn RNA undergoes a two-state transition between the kinked conformation, and a more extended structure, and folding into the kinked form is induced by divalent metal ions, or by binding of proteins of the L7Ae class. The K-turn provides an excellent, simple model for RNA folding, which can be dissected at the atomic level. We have analyzed the contributions of the hydrogen bonds that form the G.A pairs to the ion- and protein-induced folding of the K-turn. We find that all four hydrogen bonds are important to the stability of the kinked form of the RNA, and we can now define all the important hydrogen bonding interactions that stabilize the K-turn. The high affinity of L7Ae binding is coupled to the induced folding of the K-turn, allowing some sub-optimal variants to adopt the kinked geometry. However, in all such cases the affinity is lowered, and the results underline the importance of both G.A pairs to the stability of the K-turn.

The H/ACA RNP pseudouridylases function on a large number of extraordinarily complex RNA substrates including pre-ribosomal and small nuclear RNAs. Recent structural data show that H/ACA RNPs capture their RNA substrates via a simple one-sided attachment model. However, the precise placement of each RNA substrate into the active site of the catalytic subunit relies on the essential functions of the RNP proteins. The specific roles of each H/ACA RNP protein are being elucidated by a combination of structural and biochemical studies.

Small nucleolar RNA molecules (snoRNA) comprise a special kind of non-coding RNAs involved in the maturation process of rRNAs, snRNAs, tRNAs and mRNAs. Traditionally, these molecules have been divided into two families depending on the type of conserved boxes that they harbour: box C/D and H/ACA snoRNAs. Both types of snoRNAs are found associated with proteins forming a complex called snoRNP. Although some of the snoRNPs of each family mediate endonucleolytic cleavages of pre-rRNA, most of them participate in nucleotide modification: 2'-O- methylated nucleotides in the case of C/D snoRNPs and pseudouridine in the case of H/ACA snoRNPs. Based on published patents, the purpose of this review is to show the biotechnological impact of these molecules, which rely on their special features: participation in the functionality of ribosome, specific location on cell, and abnormal expression in some diseases like cancer.

The most complex RNA pseudouridylases are H/ACA ribonucleoprotein particles, which use a guide RNA for substrate capture and four proteins (Cbf5, Nop10, Gar1 and L7Ae/NHP2) for substrate modification. Here we report the three-dimensional structure of a catalytically deficient archaeal enzyme complex (including the guide RNA and three of the four essential proteins) bound to a substrate RNA. Extensive interactions of Cbf5 with one guide-substrate helix and a guide RNA stem shape the forked guide–substrate RNA complex structure and position the substrate in proximity of the Cbf5 catalytic center. Our structural and complementary fluorescence analyses also indicate that precise placement of the target uridine at the active site requires a conformation of the guide–substrate RNA duplex that is brought about by the previously identified concurrent interaction of the guide RNA with L7Ae and a composite Cbf5-Nop10 surface, and further identify a residue that is critical in this process.

Naf1 is an essential protein involved in the maturation of box H/ACA ribonucleoproteins, a group of particles required for ribosome biogenesis, modification of spliceosomal small nuclear RNAs and telomere synthesis. Naf1 participates in the assembly of the RNP at transcription sites and in the nuclear trafficking of the complex. The crystal structure of a domain of yeast Naf1p, Naf1Delta1p, reveals a striking structural homology with the core domain of archaeal Gar1, an essential protein component of the mature RNP; it suggests that Naf1p and Gar1p have a common binding site on the enzymatic protein component of the particle, Cbf5p. We propose that Naf1p is a competitive binder for Cbf5p, which is replaced by Gar1p during maturation of the H/ACA particle. The exchange of Naf1p by Gar1p might be prompted by external factors that alter the oligomerisation state of Naf1p and Gar1p. The structural homology with Gar1 suggests that the function of Naf1 involves preventing non-cognate RNAs from being loaded during transport of the particle by inducing a non-productive conformation of Cbf5.

During the biogenesis of eukaryotic ribosomal RNA (rRNA) and spliceosomal small nuclear RNA (snRNA), uridines at specific sites are converted to pseudouridines by H/ACA ribonucleoprotein particles (RNPs). Each H/ACA RNP contains a substrate-specific H/ACA RNA and four common proteins, the pseudouridine synthase Cbf5, Nop10, Gar1, and Nhp2. The H/ACA RNA contains at least one pseudouridylation (psi) pocket, which is complementary to the sequences flanking the target uridine. In this article, we show structural evidence that the psi pocket can form the predicted base pairs with substrate RNA in the absence of protein components. We report the solution structure of the complex between an RNA hairpin derived from the 3' psi pocket of human U65 H/ACA small nucleolar RNA (snoRNA) and the substrate rRNA. The snoRNA-rRNA substrate complex has a unique structure with two offset parallel pairs of stacked helices and two unusual intermolecular three-way junctions, which together organize the substrate for docking into the active site of Cbf5. The substrate RNA interacts on one face of the snoRNA in the complex, forming a structure that easily could be accommodated in the H/ACA RNP, and explains how successive substrate RNAs could be loaded onto and unloaded from the H/ACA RNA in the RNP.

Eukaryotes and archaea use two sets of specialized ribonucleoproteins (RNPs) to carry out sequence-specific methylation and pseudouridylation of RNA, the two most abundant types of modifications of cellular RNAs. In eukaryotes, these protein-RNA complexes localize to the nucleolus and are called small nucleolar RNPs (snoRNPs), while in archaea they are known as small RNPs (sRNP). The C/D class of sno(s)RNPs carries out ribose-2'-O-methylation, while the H/ACA class is responsible for pseudouridylation of their RNA targets. Here, we review the recent advances in the structure, assembly and function of the conserved C/D and H/ACA sno(s)RNPs. Structures of each of the core archaeal sRNP proteins have been determined and their assembly pathways delineated. Furthermore, the recent structure of an H/ACA complex has revealed the organization of a complete sRNP. Combined with current biochemical data, these structures offer insight into the highly homologous eukaryotic snoRNPs.

Pseudouridine synthases are the enzymes responsible for the most abundant posttranscriptional modification of cellular RNAs. These enzymes catalyze the site-specific isomerization of uridine residues that are already part of an RNA chain, and appear to employ both sequence and structural information to achieve site specificity. Crystallographic analyses have demonstrated that all pseudouridine synthases share a common core fold and active site structure and that this core is modified by peripheral domains, accessory proteins, and guide RNAs to give rise to remarkable substrate versatility.

The k-turn-binding protein 15.5K is unique in that it is essential for the hierarchical assembly of three RNP complexes distinct in both composition and function, namely, the U4/U6 snRNP, the box C/D snoRNP, and the RNP complex assembled on the U3 box B/C motif. 15.5K interacts with the cognate RNAs via an induced fit mechanism, which results in the folding of the surrounding RNA to create a binding site(s) for the RNP-specific proteins. However, it is possible that 15.5K also mediates RNP formation via protein-protein interactions with the complex-specific proteins. To investigate this possibility, we created a series of 15.5K mutations in which the surface properties of the protein had been changed. We assessed their ability to support the formation of the three distinct RNP complexes and found that the formation of each RNP requires a distinct set of regions on the surface of 15.5K. This implies that protein-protein contacts are essential for RNP formation in each complex. Further supporting this idea, direct protein-protein interaction could be observed between hU3-55K and 15.5K. In conclusion, our data suggest that the formation of each RNP involves the direct recognition of specific elements in both 15.5K protein and the specific RNA.

More than 100 mammalian H/ACA RNAs form an equal number of ribonucleoproteins (RNPs) by associating with the same four core proteins. The function of these H/ACA RNPs is essential for biogenesis of the ribosome, splicing of precursor mRNAs (pre-mRNAs), maintenance of telomeres and probably for additional cellular processes. Recent crystal structures of archaeal H/ACA protein complexes show how the same four proteins accommodate >100 distinct but related H/ACA RNAs and reveal that a spatial mutation cluster underlies dyskeratosis congenita, a syndrome of bone marrow failure.

Each of the many different box H/ACA ribonucleoprotein particles (RNPs) present in eukaryotes and archaea consists of four common core proteins and one specific H/ACA small RNA, which bears the sequence elements H (ANANNA) and ACA. Most of the H/ACA RNPs are small nucleolar RNPs (snoRNPs), which are localized in nucleoli, and are one of the two major classes of snoRNPs. Most H/ACA RNPs direct pseudouridine synthesis in pre-rRNA and other RNAs. One H/ACA small nucleolar RNA (snoRNA), vertebrate E1/U17 (snR30 in yeast), is required for pre-rRNA cleavage processing that generates mature 18S rRNA. E1 snoRNA is encoded in introns of protein-coding genes, and the evidence suggests that human E1 RNA undergoes uridine insertional RNA editing. The vertebrate E1 RNA consensus secondary structure shows several features that are absent in other box H/ACA snoRNAs. The available UV-induced RNA-protein crosslinking results suggest that the E1 snoRNP is asymmetrical in vertebrate cells, in contrast to other H/ACA snoRNPs. The vertebrate E1 snoRNP in cells is surprisingly complex: (i) E1 RNA contacts directly and specifically several proteins which do not appear to be any of the H/ACA RNP four core proteins; and (ii) multiple E1 RNA sites are needed for E1 snoRNP formation, E1 RNA stability, and E1 RNA-protein direct interactions., ((c) 2006 Wiley-Liss, Inc.)

In archaeal rRNAs, the isomerization of uridine into pseudouridine (Psi) is achieved by the H/ACA sRNPs and the minimal set of proteins required for RNA:Psi-synthase activity is the aCBF5-aNOP10 protein pair. The crystal structure of the aCBF5-aNOP10 heterodimer from Pyrococcus abyssi was solved at 2.1 A resolution. In this structure, protein aNOP10 has an extended shape, with a zinc-binding motif at the N-terminus and an alpha-helix at the C-terminus. Both motifs contact the aCBF5 catalytic domain. Although less efficiently as does the full-length aNOP10, the aNOP10 C-terminal domain binds aCBF5 and stimulates the RNA-guided activity. We show that the C-terminal domain of aCBF5 (the PUA domain), which is wrapped by an N-terminal extension of aCBF5, plays a crucial role for aCBF5 binding to the guide sRNA. Addition of this domain in trans partially complement particles assembled with an aCBF5DeltaPUA truncated protein. In the crystal structure, the aCBF5-aNOP10 complex forms two kinds of heterotetramers with parallel and perpendicular orientations of the aNOP10 terminal alpha-helices, respectively. By gel filtration assay, we showed that aNOP10 can dimerize in solution. As both residues Y41 and L48 were needed for dimerization, the dimerization likely takes place by interaction of parallel alpha-helices.

H/ACA RNA-protein complexes, comprised of four proteins and an H/ACA guide RNA, modify ribosomal and small nuclear RNAs. The H/ACA proteins are also essential components of telomerase in mammals. Cbf5 is the H/ACA protein that catalyzes isomerization of uridine to pseudouridine in target RNAs. Mutations in human Cbf5 (dyskerin) lead to dyskeratosis congenita. Here, we describe the 2.1 A crystal structure of a specific complex of three archaeal H/ACA proteins, Cbf5, Nop10, and Gar1. Cbf5 displays structural properties that are unique among known pseudouridine synthases and are consistent with its distinct function in RNA-guided pseudouridylation. We also describe the previously unknown structures of both Nop10 and Gar1 and the structural basis for their essential roles in pseudouridylation. By using information from related structures, we have modeled the entire ribonucleoprotein complex including both guide and substrate RNAs. We have also identified a dyskeratosis congenita mutation cluster site within a modeled dyskerin structure.

Box C/D and H/ACA snoRNAs represent two abundant groups of small noncoding RNAs. The majority of box C/D and H/ACA snoRNAs function as guide RNAs in the site-specific 2'-O-methylation and pseudouridylation of rRNAs, respectively. The box C/D snoRNAs associate with fibrillarin, Nop56, Nop58, and 15.5K/NHPX proteins to form functional snoRNP particles, whereas all box H/ACA snoRNAs form complexes with the dyskerin, Nop10, Nhp2, and Gar1 snoRNP proteins. Recent studies demonstrate that the biogenesis of mammalian snoRNPs is a complex process that requires numerous trans-acting factors. Most vertebrate snoRNAs are posttranscriptionally processed from pre-mRNA introns, and the early steps of snoRNP assembly are physically and functionally coupled with the synthesis or splicing of the host pre-mRNA. The maturing snoRNPs follow a complicated intranuclear trafficking process that is directed by transport factors also involved in nucleocytoplasmic RNA transport. The human telomerase RNA (hTR) carries a box H/ACA RNA domain that shares a common Cajal-body-specific localization element with a subclass of box H/ACA RNAs, which direct pseudouridylation of spliceosomal snRNAs in the Cajal body. However, besides concentrating in Cajal bodies, hTR also accumulates at a small, structurally distinct subset of telomeres during S phase. This suggests that a cell-cycle-dependent, dynamic localization of hTR to telomeres may play an important regulatory role in human telomere synthesis.