Assembly Of Box Research Articles

Human NOP2/NSUN1 regulates ribosome biogenesis through non-catalytic complex formation with box C/D snoRNPs.

5-Methylcytosine (m5C) is a base modification broadly found on various RNAs in the human transcriptome. In eukaryotes, m5C is catalyzed by enzymes of the NSUN family composed of seven human members (NSUN1-7). NOP2/NSUN1 has been primarily characterized in budding yeast as an essential ribosome biogenesis factor required for the deposition of m5C on the 25S ribosomal RNA (rRNA). Although human NOP2/NSUN1 has been known to be an oncogene overexpressed in several types of cancer, its functions and substrates remain poorly characterized. Here, we used a miCLIP-seq approach to identify human NOP2/NSUN1 RNA substrates. Our analysis revealed that NOP2/NSUN1 catalyzes the deposition of m5C at position 4447 on the 28S rRNA. We also find that NOP2/NSUN1 binds to the 5′ETS region of the pre-rRNA transcript and regulates pre-rRNA processing through non-catalytic complex formation with box C/D snoRNAs. We provide evidence that NOP2/NSUN1 facilitates the recruitment of U3 and U8 snoRNAs to pre-90S ribosomal particles and their stable assembly into snoRNP complexes. Remarkably, expression of both WT and catalytically inactive NOP2/NSUN1 in knockdown background rescues the rRNA processing defects and the stable assembly of box C/D snoRNP complexes, suggesting that NOP2/NSUN1-mediated deposition of m5C on rRNA is not required for ribosome synthesis.

A PRC2-independent function for EZH2 in regulating rRNA 2'-O methylation and IRES-dependent translation.

Dysregulated translation is a common feature of cancer. Uncovering its governing factors and underlying mechanism are important for cancer therapy. Here, we report that enhancer of zeste homologue 2 (EZH2), previously known as a transcription repressor and lysine methyltransferase, can directly interact with fibrillarin (FBL) to exert its role in translational regulation. We demonstrate that EZH2 enhances rRNA 2'-O methylation via its direct interaction with FBL. Mechanistically, EZH2 strengthens the FBL-NOP56 interaction and facilitates the assembly of box C/D small nucleolar ribonucleoprotein. Strikingly, EZH2 deficiency impairs the translation process globally and reduces internal ribosome entry site (IRES)-dependent translation initiation in cancer cells. Our findings reveal a previously unrecognized role of EZH2 in cancer-related translational regulation.

Ozone Gas: Scientific Justification and Practical Guidelines for Improvised Disinfection using Consumer-Grade Ozone Generators and Plastic Storage Boxes.

We describe direct measurements of ozone concentration achievable in small enclosed containers (plastic storage boxes) for use as improvised decontamination systems for small articles such as disposable PPE (N95 masks, nitrile gloves, etc.), clothing, mail and small packages, food, and other miscellaneous articles. The emphasis is on the reliable and sustained generation of ozone gas concentrations of sufficient concentration and duration to create an effective virucidal environment to achieve more than 95% to 99% viral inactivation, based upon the data already published in the peer-review literature on this topic.
 The suggestion that ozone be used to inactivate virus is certainly not a new idea. Our objective in this report is to make clear that the necessary levels of ozone can be improvised using simple, easy-to-use, inexpensive, and widely available supplies, and that there is every theoretical and experimental reason to believe that this approach is as highly effective in viral inactivation by ozone as are the far more expensive, complex, cumbersome, and less available equivalent ozone (and other) disinfectant systems that have themselves become unavailable during times of pandemic crisis.
 Using multiple types of readily available commercial ozone generators, concentration in the tested improvised enclosure is tracked over time to assess ozone charging and decay rates, and the ozone quenching effects of items placed in the box. Generator performance is compared against published ozone dosage values for virucidal and antimicrobial activity. Bubbler and box-fan-type ozone generators were found to be effective at achieving and maintaining target concentrations of 10ppm ozone or higher, whereas automotive cigarette lighter and universal serial bus type plug in “air freshener” ozone generators could not achieve the target concentrations in these experiments. Calculations and practical guidelines for assembly and effective use of an ozone box for improvised decontamination are offered.
 The majority of this report is directed toward the scientific justification and rationale for this approach. The end of the document summarizes the findings and offers simplified designs for the construction and use of ozone boxes as an improvised method of disinfection.

Experiences during Design, Fabrication, Assembly and Factory Acceptance Test of the ITER Cryoplant Termination Cold Box

The ITER Cryodistribution (CD) system distributes cryogenic power from the cryoplant to the applications, namely superconducting magnets, cryopumps, etc. The ITER CD system comprises seven cold boxes of which the Cryoplant Termination Cold Box (CTCB) is the largest one. The CTCB plays a pivotal role in distributing the cold helium fluid with the highest mass flow rates of 4 kg/s coming from one of the 80 K plants to the applications. The physical connection between the CTCB and different applications is through nine cryolines (CLs) having diameters ranging from 0.45 m to 1.0 m. The CTCB has been manufactured and assembled with various large size components such as electrical heater system of 600 kW capacity, cryogenic control valves of size DN200, bubble panel thermal shield (TS) made of stainless steel, etc. The CTCB, which withstands interface loads of ∼1.4 times of its own weight, has been analysed for its functional and thermo-structural requirements under various load conditions. Components & subsystems were manufactured and factory tested individually in various locations in Europe and India to be finally assembled in one place. The CTCB has also been integrally tested, of which major activities were the pressure and helium leak tightness tests, followed by functionality checks of all instruments. The present paper describes the challenges involved and how they were resolved as well as lesson learnt during the design, fabrication, assembly, and factory acceptance test (FAT) phase of the CTCB project.

Implication of the box C/D snoRNP assembly factor Rsa1p in U3 snoRNP assembly.

The U3 box C/D snoRNA is one key element of 90S pre-ribosome. It contains a 5΄ domain pairing with pre-rRNA and the U3B/C and U3C΄/D motifs for U3 packaging into a unique small nucleolar ribonucleoprotein particle (snoRNP). The RNA-binding protein Snu13/SNU13 nucleates on U3B/C the assembly of box C/D proteins Nop1p/FBL and Nop56p/NOP56, and the U3-specific protein Rrp9p/U3-55K. Snu13p/SNU13 has a much lower affinity for U3C΄/D but nevertheless forms on this motif an RNP with box C/D proteins Nop1p/FBL and Nop58p/NOP58. In this study, we characterized the influence of the RNP assembly protein Rsa1 in the early steps of U3 snoRNP biogenesis in yeast and we propose a refined model of U3 snoRNP biogenesis. While recombinant Snu13p enhances the binding of Rrp9p to U3B/C, we observed that Rsa1p has no effect on this activity but forms with Snu13p and Rrp9p a U3B/C pre-RNP. In contrast, we found that Rsa1p enhances Snu13p binding on U3C΄/D. RNA footprinting experiments indicate that this positive effect most likely occurs by direct contacts of Rsa1p with the U3 snoRNA 5΄ domain. In light of the recent U3 snoRNP cryo-EM structures, our data suggest that Rsa1p has a dual role by also preventing formation of a pre-mature functional U3 RNP.

Cost efficiency, integration and assembly of a generic composite aeronautical wing box

This paper presents a cost-efficiency study of part integration with respect to reduced assembly effort within aeronautical composite structures. The study is performed through the use, and continuous improvement upon, a previously developed cost model. Focus are on the assembly and basic inspection a wing box, part of a section of a full wing, where involved parts are all considered to be manufactured from carbon fibre reinforced plastic (CFRP). Treated cases range from traditional, mechanical joining, to high integration either through co-curing or co-bonding of composite structures. The outcome of presented cost study shows that increased integration decreases the overall production cost of said considered wing box. In general it is shown that co-curing or co-bonding reduces a number of cost-expensive assembly steps in comparison to mechanical joining.

Nop17 is a key R2TP factor for the assembly and maturation of box C/D snoRNP complex.

BackgroundBox 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 R2TP and Hsp90 chaperone. Nop17, also known as Pih1, has been shown to be a constituent of the R2TP (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.ResultsTo 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.ConclusionsBased on the results shown here, we propose a model for the assembly of box C/D snoRNP, according to which R2TP complex is important for reducing the affinity of Nop58 for snoRNA, and for the binding of the other snoRNP subunits.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-015-0037-5) contains supplementary material, which is available to authorized users.

A stepwise assembly of a molecular box from 16-electron half-sandwich precursors [Cp*M(pdt)] (M = Rh, Ir).

The coordinatively-unsaturated 16-electron half-sandwich precursors [Cp*M(pdt)] (M = Rh, Ir; pdt = pyrazine-2,3-dithiol) have been synthesized. X-ray crystallography in combination with (1)H NMR analysis was used to elucidate the nature of the precursors. The Rh(iii) precursor displays a dimeric form in the trans arrangement in the solid state, formulated as [(Cp*Rh)2(μ(S)-pdt)2] (), in which covalent Rh-S bonds bridge the metal centers. In solution, however, dimers and monomers coexist in equilibrium. The dissociation equilibrium of in DMSO-d6 was evaluated by (1)H NMR at several temperatures between 20 and 80 °C. The Ir(iii) precursor [Cp*Ir(pdt)] () is the monomeric form, and stable in the solid state and solution. Due to their unsaturation and bridging properties, these precursors were further used in stepwise assembly reactions with the binuclear building blocks to give open macrocycles and a closed molecular box.

Proteomic and 3D structure analyses highlight the C/D box snoRNP assembly mechanism and its control.

In vitro, assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) involves the sequential recruitment of core proteins to snoRNAs. In vivo, however, assembly factors are required (NUFIP, BCD1, and the HSP90-R2TP complex), and it is unknown whether a similar sequential scheme applies. In this paper, we describe systematic quantitative stable isotope labeling by amino acids in cell culture proteomic experiments and the crystal structure of the core protein Snu13p/15.5K bound to a fragment of the assembly factor Rsa1p/NUFIP. This revealed several unexpected features: (a) the existence of a protein-only pre-snoRNP complex containing five assembly factors and two core proteins, 15.5K and Nop58; (b) the characterization of ZNHIT3, which is present in the protein-only complex but gets released upon binding to C/D snoRNAs; (c) the dynamics of the R2TP complex, which appears to load/unload RuvBL AAA(+) adenosine triphosphatase from pre-snoRNPs; and (d) a potential mechanism for preventing premature activation of snoRNP catalytic activity. These data provide a framework for understanding the assembly of box C/D snoRNPs.

Co-expression and co-purification of archaeal and eukaryal box C/D RNPs.

Box C/D ribonucleoprotein particles (RNPs) are 2′-O-methylation enzymes required for maturation of ribosomal and small nuclear RNA. Previous biochemical and structural studies of the box C/D RNPs were limited by the unavailability of purified intact RNPs. We developed a bacterial co-expression strategy based on the combined use of a multi-gene expression system and a tRNA-scaffold construct that allowed the expression and purification of homogeneous archaeal and human box C/D RNPs. While the co-expressed and co-purified archaeal box C/D RNP was found to be fully active in a 2′-O-methylation assay, the intact human U14 box C/D RNP showed no detectable catalytic activity, consistent with the earlier findings that assembly of eukaryotic box C/D RNPs is nonspontaneous and requires additional protein factors. Our systems provide a means for further biochemical and structural characterization of box C/D RNPs and their assembly factors.

Effect of post welding heat treatment of the HCPB TBM on Eurofer and lithium orthosilicate pebbles

In the breeder units (BUs) of the HCPB TBM, the lithium orthosilicate (OSi) pebbles are contained by cooling plates which are made of Eurofer. The assembly of the TBM box and BUs is based on welding processes which require post welding heat treatment (PWHT). According to the proposed TBM manufacture strategy, the BUs will be packed with the OSi pebbles during the TBM assembly; consequently the pebbles and the Eurofer plates will undergo the PWHT. Therefore the objective of this study is to investigate the effect of the PWHT on both Eurofer and OSi and their interaction. The test unit is a Eurofer box packed with OSi pebbles and Eurofer specimens. This test unit and all Eurofer specimens were heat treated inside a furnace which is capable of providing the required heating and cooling rates under a controlled atmosphere of vacuum or argon. Charpy impact and tensile tests of the Eurofer specimens and crush load tests of the OSi pebbles were performed after the heat treatment to investigate any changes in their mechanical properties. The results of these mechanical tests revealed that both materials did not suffer any significant degradation in their mechanical properties or chemical compatibility problems. The metallurgical analyses of both materials showed minimum chemical interaction.

Experimental investigation on the possible techniques of pebbles packing for the HCPB Test Blanket Module

The Helium Cooled Pebble Bed Test Blanket Module (TBM) features a structural box that consists of the first wall, two caps and a stiffening grid. Inside the stiffening grid the breeding units (BUs), consisting of the beryllium and lithium ceramic pebble beds and cooling plates, are accommodated. The BUs are closed by the BU back plates and several structural plates of the manifold system as well as the TBM back plate consequently the BUs may not be accessed directly after the assembly of the TBM box; however, access is possible through dedicated penetrations in the TBM caps. According to the current manufacturing strategy, the assembly of the TBM structural sub-components is based on several welding processes which require post-welding heat treatments (PWHT) at temperatures which exceed the temperature limit of the beryllium pebbles. For that reason the beryllium pebble beds will be packed after the TBM box is assembled and heat treated. The packing of the BUs will be performed using a small-diameter (5 mm) tube that will be inserted into some penetrations in the TBM caps. It is expected that the lithium ceramic pebbles can withstand the high temperatures of the PWHT (this assumption needs to be verified) therefore the current strategy is to pack the ceramic pebble beds during the TBM box assembly. This study experimentally demonstrates the packing procedures for the beryllium beds using a full-scale Plexiglas mock-up as well as the optimization of the packing process by dedicated measures such as vibrating and tilting of the mock-up. In addition the impacts of the experimental results on the TBM design are summarized and the paper is concluded by proposing a packing strategy that can be used to achieve a packing factor of 63.6%.

Dynamic guide-target interactions contribute to sequential 2'-O-methylation by a unique archaeal dual guide box C/D sRNP.

Assembly and guide-target interaction of an archaeal box C/D-guide sRNP was investigated under various conditions by analyzing the lead (II)-induced cleavage of the guide RNA. Guide and target RNAs derived from Haloferax volcanii pre-tRNA(Trp) were used with recombinant Methanocaldococcus jannaschii core proteins in the reactions. Core protein L7Ae binds differentially to C/D and C'/D' motifs of the guide RNA, and interchanging the two motifs relative to the termini of the guide RNA did not affect L7Ae binding or sRNA function. L7Ae binding to the guide RNA exposes its D'-guide sequence first followed by the D guide. These exposures are reduced when aNop5p and aFib proteins are added. The exposed guide sequences did not pair with the target sequences in the presence of L7Ae alone. The D-guide sequence could pair with the target in the presence of L7Ae and aNop5p, suggesting a role of aNop5p in target recruitment and rearrangement of sRNA structure. aFib binding further stabilizes this pairing. After box C/D-guided modification, target-guide pairing at the D-guide sequence is disrupted, suggesting that each round of methylation may require some conformational change or reassembly of the RNP. Asymmetric RNPs containing only one L7Ae at either of the two box motifs can be assembled, but a functional RNP requires L7Ae at the box C/D motif. This arrangement resembles the asymmetric eukaryal snoRNP. Observations of initial D-guide-target pairing and the functional requirement for L7Ae at the box C/D motif are consistent with our previous report of the sequential 2'-O-methylations of the target RNA.

Cwc24p, a Novel Saccharomyces cerevisiae Nuclear Ring Finger Protein, Affects Pre-snoRNA U3 Splicing

U3 snoRNA is transcribed from two intron-containing genes in yeast, snR17A and snR17B. Although the assembly of the U3 snoRNP has not been precisely determined, at least some of the core box C/D proteins are known to bind pre-U3 co-transcriptionally, thereby affecting splicing and 3'-end processing of this snoRNA. We identified the interaction between the box C/D assembly factor Nop17p and Cwc24p, a novel yeast RING finger protein that had been previously isolated in a complex with the splicing factor Cef1p. Here we show that, consistent with the protein interaction data, Cwc24p localizes to the cell nucleus, and its depletion leads to the accumulation of both U3 pre-snoRNAs. U3 snoRNA is involved in the early cleavages of 35 S pre-rRNA, and the defective splicing of pre-U3 detected in cells depleted of Cwc24p causes the accumulation of the 35 S precursor rRNA. These results led us to the conclusion that Cwc24p is involved in pre-U3 snoRNA splicing, indirectly affecting pre-rRNA processing.

A Spliceosomal Intron Binding Protein, IBP160, Links Position-Dependent Assembly of Intron-Encoded Box C/D snoRNP to Pre-mRNA Splicing

Pre-mRNA splicing in vertebrates is molecularly linked to other processes. We previously reported that splicing is required for efficient assembly of intron-encoded box C/D small nucleolar ribonucleoprotein (snoRNP). In the spliceosomal C1 complex, snoRNP proteins efficiently assemble onto snoRNA sequences if they are located about 50 nt upstream of the intron branchpoint. Here, we identify the splicing factor responsible for coupling snoRNP assembly to intron excision. Intron binding protein (IBP) 160, a helicase-like protein previously detected in the spliceosomal C1 complex, binds the pre-mRNA in a sequence-independent manner, contacting nucleotides 33-40 upstream of the intron branch site, regardless of whether a snoRNA is present. Depletion of IBP160 abrogates snoRNP assembly in vitro. IBP160 binding directly to a snoRNA located too close to the intron branch site interferes with snoRNP assembly. Thus, IBP160 is the key factor linking snoRNP biogenesis and perhaps other postsplicing events to pre-mRNA splicing.

Assembly of the Archaeal Box C/D sRNP Can Occur via Alternative Pathways and Requires Temperature-facilitated sRNA Remodeling

Archaeal dual-guide box C/D small nucleolar RNA-like RNAs (sRNAs) bind three core proteins in sequential order at both terminal box C/D and internal C′/D′ motifs to assemble two ribonuclear protein (RNP) complexes active in guiding nucleotide methylation. Experiments have investigated the process of box C/D sRNP assembly and the resultant changes in sRNA structure or “remodeling” as a consequence of sRNP core protein binding. Hierarchical assembly of the Methanocaldococcus jannaschii sR8 box C/D sRNP is a temperature-dependent process with binding of L7 and Nop56/58 core proteins to the sRNA requiring elevated temperature to facilitate necessary RNA structural dynamics. Circular dichroism (CD) spectroscopy and RNA thermal denaturation revealed an increased order and stability of sRNA folded structure as a result of L7 binding. Subsequent binding of the Nop56/58 and fibrillarin core proteins to the L7–sRNA complex further remodeled sRNA structure. Assessment of sR8 guide region accessibility using complementary RNA oligonucleotide probes revealed significant changes in guide region structure during sRNP assembly. A second dual-guide box C/D sRNA from M. jannaschii, sR6, also exhibited RNA remodeling during temperature-dependent sRNP assembly, although core protein binding was affected by sR6′s distinct folded structure. Interestingly, the sR6 sRNP followed an alternative assembly pathway, with both guide regions being continuously exposed during sRNP assembly. Further experiments using sR8 mutants possessing alternative guide regions demonstrated that sRNA folded structure induced by specific guide sequences impacted the sRNP assembly pathway. Nevertheless, assembled sRNPs were active for sRNA-guided methylation independent of the pathway followed. Thus, RNA remodeling appears to be a common and requisite feature of archaeal dual-guide box C/D sRNP assembly and formation of the mature sRNP can follow different assembly pathways in generating catalytically active complexes.

Cotranscriptional Recognition of Human Intronic Box H/ACA snoRNAs Occurs in a Splicing-Independent Manner

Processing from pre-mRNA introns is a widespread mechanism to generate human box C/D and H/ACA snoRNAs. Recent studies revealed that an optimal position relative to the 3' splice site is important for efficient processing of most box C/D snoRNAs and that assembly of box C/D snoRNPs is stimulated by splicing factors likely bound to the branch point region. Here we have investigated the processing of another major class of human intron-encoded RNAs, the box H/ACA snoRNAs. Analysis of 80 H/ACA RNA genes revealed that human H/ACA RNAs possess no preferential localization close to the 3' or 5' splice site. In vivo processing experiments confirmed that H/ACA intronic snoRNAs are processed in a position-independent manner, indicating that there is no synergy between H/ACA RNA processing and splicing. We also showed that recognition of intronic H/ACA snoRNAs and assembly of pre-snoRNPs is an early event that occurs during transcription elongation parallel with pre-mRNA splice site selection. Finally, we found that efficient processing and correct nucleolar localization of the human U64 H/ACA snoRNA requires RNA polymerase II-mediated synthesis of the U64 precursor. This suggests that polymerase II-associated factors direct the efficient assembly and determine the correct subnuclear trafficking of human H/ACA snoRNPs.

Assembly and Maturation of the U3 snoRNP in the Nucleoplasm in a Large Dynamic Multiprotein Complex

The assembly and maturation of box C/D snoRNPs, factors essential for ribosome biogenesis, occur in the nucleoplasm. To investigate this process, we have analyzed non-snoRNP factors associated with the nucleoplasmic human U3 snoRNA. We show that both the precursor and mature length nucleoplasmic U3 snoRNAs are present in larger multiprotein complexes that contain the core box C/D proteins as well as many non-snoRNP factors linked to snoRNP assembly (TIP48, TIP49, Nopp140), RNA processing (TGS1, La, LSm4, hRrp46), and subcellular localization (CRM1, PHAX). Using RNAi, we show that most of these factors are essential for box C/D snoRNA accumulation. Furthermore, we demonstrate that the core proteins undergo a restructuring event that stabilizes their binding to the snoRNA. Importantly, restructuring, which may be mediated by the putative remodeling factor TIP49, appears to be linked to nucleolar localization. We believe that the assembly complex coordinates snoRNA processing, snoRNP assembly, restructuring, and localization.

Cbf5p, the putative pseudouridine synthase of H/ACA-type snoRNPs, can form a complex with Gar1p and Nop10p in absence of Nhp2p and box H/ACA snoRNAs.

Box C/D and box H/ACA small ribonucleoprotein particles (sRNPs) are found from archaea to humans, and some of these play key roles during the biogenesis of ribosomes or components of the splicing apparatus. The protein composition of the core of both types of particles is well established and the assembly pathway of box C/D sRNPs has been extensively investigated both in archaeal and eukaryotic systems. In contrast, knowledge concerning the mode of assembly and final structure of box H/ACA sRNPs is much more limited. In the present study, we have investigated the protein/protein interactions taking place between the four protein components of yeast box H/ACA small nucleolar RNPs (snoRNPs), Cbf5p, Gar1p, Nhp2p, and Nop10p. We provide evidence that Cbf5p, Gar1p, and Nop10p can form a complex devoid of Nhp2p and small nucleolar RNA (snoRNA) components of the particles and that Cbf5p and Nop10p can directly bind to each other. We also show that the absence of any component necessary for assembly of box H/ACA snoRNPs inhibits accumulation of Cbf5p, Gar1p, or Nop10p, whereas Nhp2p levels are little affected.

Molecular Basis of Box C/D RNA-Protein Interactions: Cocrystal Structure of Archaeal L7Ae and a Box C/D RNA

We have determined and refined a crystal structure of the initial assembly complex of archaeal box C/D sRNPs comprising the Archaeoglobus fulgidus (AF) L7Ae protein and a box C/D RNA. The box C/D RNA forms a classical kink-turn (K-turn) structure and the resulting protein-RNA complex serves as a distinct platform for recruitment of the fibrillarin-Nop5p complex. The cocrystal structure confirms previously proposed secondary structure of the box C/D RNA that includes a protruded U, a UU mismatch, and two sheared tandem GA base pairs. Detailed structural comparisons of the AF L7Ae-box C/D RNA complex with previously determined crystal structures of L7Ae homologs in complex with functionally distinct K-turn RNAs revealed a set of remarkably conserved principles in protein-RNA interactions. These analyses provide a structural basis for interpreting the functional roles of the box C/D sequences in directing specific assembly of box C/D sRNPs.