H/ACA guide RNAs, proteins and complexes

H/ACA guide RNAs direct site-specific pseudouridylation of substrate RNAs by forming ribonucleoprotein (RNP) complexes with pseudouridine synthase Cbf5 and three accessory proteins. Recently determined crystal structures of H/ACA protein complexes and a fully assembled H/ACA RNP complex have provided significant insights into the architecture, assembly and mechanism of action of RNA-guided pseudouridine synthase. The binding of guide RNA is directed by its conserved secondary structure and sequence motifs, which enables guide RNA with different sequences to be incorporated into the same protein complex. Accessory proteins and peripheral domains crucially coordinate the position of guide RNA, and possibly regulate the reaction process.     

Functionality and substrate specificity of human box H/ACA guide RNAs

A large number of box H/ACA RNAs have been identified in human cells, and have been predicted to account for nearly all pseudouridylation sites in human rRNAs. However, the function of these mammalian H/ACA RNAs in directing pseudouridylation has been verified experimentally in only two cases. In this study, we used three in vitro reconstitution systems, including yeast and mammalian systems, to test the function of seven H/ACA RNAs guiding16 pseudouridylation sites. Our results verified 12 of these sites; four predictions were incorrect. Further analyses indicated that three components, including the stability of the hairpin structure harboring the pseudouridylation pocket, the stability of guide sequence–target RNA base-pairing interaction, and the distance between the target uridine and the box H or ACA, were critical for the guide function, as changes in these components were sufficient to alter the functionality and specificity of the pseudouridylation pocket. The dynamic functional changes in response to changes in these three important components were further tested in vivo, and the results were completely consistent with the in vitro results. Finally, we compared our results with predictions made by two computer programs, as well as predictions made by human experts using visual inspection. We found that the predictions of one program (snoGPS) agreed with our experimental results with 100% sensitivity (12/12) and 75% specificity (3/4).     

Crystal structure determination and site-directed mutagenesis of the Pyrococcus abyssi aCBF5-aNOP10 complex reveal crucial roles of the C-terminal domains of both proteins in H/ACA sRNP activity

In archaeal rRNAs, the isomerization of uridine into pseudouridine (Ψ) is achieved by the H/ACA sRNPs and the minimal set of proteins required for RNA:Ψ-synthase activity is the aCBF5–aNOP10 protein pair. The crystal structure of the aCBF5–aNOP10 heterodimer from Pyrococcus abyssi was solved at 2.1 Å resolution. In this structure, protein aNOP10 has an extended shape, with a zinc-binding motif at the N-terminus and an α-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 aCBF5ΔPUA truncated protein. In the crystal structure, the aCBF5–aNOP10 complex forms two kinds of heterotetramers with parallel and perpendicular orientations of the aNOP10 terminal α-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 α-helices.     

A novel experimental approach for systematic identification of box H/ACA snoRNAs from eukaryotes

Box H/ACA snoRNAs represent an abundant group of small non-coding RNAs mainly involved in the pseudouridylation of rRNAs and/or snRNAs in eukaryotes and Archaea. In this study, we describe a novel experimental method for systematic identification of box H/ACA snoRNAs from eukaryotes. In the specialized cDNA libraries constructed by this method with total cellular RNAs from human blood cells, the high efficiency of cloning for diverse box H/ACA snoRNAs was achieved and seven novel species of this snoRNA family were identified from human for the first time. Furthermore, the novel method has been successfully applied for the identification of the box H/ACA snoRNAs from Drosophila and the fission yeast, demonstrating a powerful ability for systematic analysis of box H/ACA snoRNAs in a broad spectrum of eukaryotes.     

Nucleolar localization signals of box H/ACA small nucleolar RNAs.

The two major families of small nucleolar RNAs (snoRNAs), Box C/D and Box H/ACA, are generated in the nucleoplasm and transported to the nucleolus where they function in rRNA processing and modification. We have investigated the sequences involved in the intranuclear transport of Box H/ACA snoRNAs by assaying the localization of injected fluorescent RNAs in Xenopus oocyte nuclear spreads. Our analysis of U17, U64 and U65 has revealed that disruption of either of the conserved sequence elements, Box H or Box ACA, eliminates nucleolar localization. In addition, the stem present at the base of the 3' hairpin is required for efficient nucleolar localization of U65. Fragments or rearrangements of U65 that consist of Box H and Box ACA flanking either the 5' or 3' hairpin are targeted to the nucleolus. The targeting is dependent on the presence of the Box sequences, but not on their orientation. Our results indicate that in each of the two major families of snoRNAs, a motif composed of the signature conserved sequences and an adjacent structural element that tethers the sequence elements directs the nucleolar localization of the RNAs. We demonstrate that telomerase RNA is also targeted to the nucleolus by a Box ACA-dependent mechanism.     

靶向宿主致病菌sRNA的发现及其靶标确定的实验技术进展

人类时常暴露于充满各种致病菌的环境中,这些致病菌与人体细胞或组织之间存在多种相互作用。在相互作用的过程中,细菌通过调节自身毒性、侵袭性等致病性,以适应宿主环境并生存下来,同样,宿主细胞也会通过调动自身的免疫系统来抵抗致病菌的入侵。然而,大多数研究者主要聚焦于致病菌sRNA (small RNA, sRNA)自身生理功能的研究,致病菌与宿主相互作用的认识仍然处于起步阶段。因此,如何使用高灵敏性、高分辨率的方法研究致病菌与宿主之间的相互作用成为当前研究面临的一大难题。本文综合国内外相关研究,概述了目前研究致病菌与宿主相互作用常用的技术方法及实验流程,提高对其机制原理的理解,为致病菌sRNA-宿主靶标的相关研究提供技术参考。     

Ribozymes which cleave arenavirus RNAs: identification of susceptible target sites and inhibition by target site secondary structure.

The development of safe and effective antiviral agents has been a slow process, largely because of the difficulty in distinguishing between virus and host functions; materials toxic to the virus are frequently harmful also to the host in which the agent resides. Recently, techniques which target nucleic acid sequences as a means of reducing gene expression have emerged. This antisense armamentarium includes ribozymes, RNA enzymes which cleave other RNA molecules in a sequence-specific manner. We wish to assess the ability of ribozymes to control animal virus infection. Reasoning that the viruses most vulnerable to ribozyme intervention will be those whose complete life cycle is based on RNA (with no DNA stage), we have begun to develop ribozymes directed toward lymphocytic choriomeningitis virus (LCMV), the prototype of the arenavirus family. Using ribozymes of the hammerhead variety, we have identified several sites on the LCMV genome which can be efficiently cleaved in trans. The efficiency of cleavage is site dependent, and we demonstrate that secondary structure at the target site can abolish ribozyme cleavage. Computer-assisted analysis indicates that much of the LCMV genome may be involved in base pairing, which may render it similarly resistant to ribozyme attack. The few remaining open regions of LCMV lack a GUC target site, on which most studies to date have relied. Here we show that AUC, CUC, and AUU are alternative sites which can be cleaved by trans-acting ribozymes. This finding is important given the aforementioned restriction of available sites, imposed by secondary structure.     

Binding sites of the antibiotics pactamycin and celesticetin on ribosomal RNAs.

The binding sites of the antibiotics pactamycin and celesticetin on the rRNAs of Escherichia coli ribosomes were investigated by a chemical footprinting procedure. Pactamycin protected residues G-693 and C-795 in 16S RNA which are located in an important functional region of the 30S subunit participating in initiation complex formation and ribosomal subunit interaction. Celesticetin altered the reactivities of 5 residues A-2058, A-2059, A-2062, A-2451 and G-2505 within the central loop of domain V of 23S RNA which has been implicated in peptidyltransferase activity. Inferences are drawn concerning the mode of action of the antibiotics.     

1H, 13C, and 15N NMR assignments of the Pyrococcus abyssi DNA polymerase II intein

Protein splicing is a precise post-translational process mediated by inteins. Inteins are intervening proteins that cleave themselves from a precursor protein while joining the flanking sequences. Here we report the ¹⁵N, ¹³C, and ¹H chemical shift assignments of the intein from DNA polymerase II of Pyrococcus abyssi (Pab PolII intein), which has been recombinantly overexpressed and isotopically labeled. The NMR assignments of Pab PolII intein are essential for solution structure determination and protein dynamics study.     

A universal model for the secondary structure of 5.8S ribosomal RNA molecules, their contact sites with 28S ribosomal RNAs, and their prokaryotic equivalent

The phylogenetic approach (ref. 1) has been utilized in construction of a universal 5.8S rRNA secondary structure model, in which about 65% of the residues exist in paired structures. Conserved nucleotides primarily occupy unpaired regions. Multiple compensating base changes are demonstrated to be present in each of the five postulated helices, thereby forming a major basis for their proof. The results of chemical and enzymatic probing of 5.8S rRNAs (ref. 13, 32) are fully consistent with, and support, our model. This model differs in several ways from recently proposed 5.8S rRNA models (ref. 3, 4), which are discussed. Each of the helices in our model has been extended to the corresponding bacterial, chloroplast and mitochondrial sequences, which are demonstrated to be positionally conserved by alignment with their eukaryotic counterparts. This extension is also made for the base paired 5.8S/28S contact points, and their prokaryotic and organelle counterparts. The demonstrated identity of secondary structure in these diverse molecules strongly suggests that they perform equivalent functions in prokaryotic and eukaryotic ribosomes.     

Computational RNomics:Structure identification and functional prediction of non-coding RNAs in silico

The eukaryotic genome contains varying numbers of non-coding RNA(ncRNA) genes.Computational RNomics takes a multidisciplinary approach,like information science,to resolve the structure and function of ncRNAs.Here,we review the main issues in Computational RNomics of data storage and management,ncRNA gene identification and characterization,ncRNA target identification and functional prediction,and we summarize the main methods and current content of computational RNomics.     

Revisiting the structure/function relationships of H/ACA(-like) RNAs: a unified model for Euryarchaea and Crenarchaea

A structural and functional classification of H/ACA and H/ACA-like motifs is obtained from the analysis of the H/ACA guide RNAs which have been identified previously in the genomes of Euryarchaea (Pyrococcus) and Crenarchaea (Pyrobaculum). A unified structure/function model is proposed based on the common structural determinants shared by H/ACA and H/ACA-like motifs in both Euryarchaea and Crenarchaea. Using a computational approach, structural and energetic rules for the guide:target RNA-RNA interactions are derived from structural and functional data on the H/ACA RNP particles. H/ACA(-like) motifs found in Pyrococcus are evaluated through the classification and their biological relevance is discussed. Extra-ribosomal targets found in both Pyrococcus and Pyrobaculum might support the hypothesis of a gene regulation mediated by H/ACA(-like) guide RNAs in archaea.     

Physiological Responses to Stress Conditions and Barophilic Behavior of the Hyperthermophilic Vent Archaeon Pyrococcus abyssi

The physiology of the deep-sea hyperthermophilic, anaerobic vent archaeon Pyrococcus abyssi, originating from the Fiji Basin at a depth of 2,000 m, was studied under diverse conditions. The emphasis of these studies lay in the growth and survival of this archaeon under the different conditions present in the natural habitat. Incubation under in situ pressure (20 MPa) and at 40 MPa increased the maximal and minimal growth temperatures by 4(deg)C. In situ pressure enhanced survival at a lethal high temperature (106 to 112(deg)C) relative to that at low pressure (0.3 MPa). The whole-cell protein profile, analyzed by one-dimensional sodium dodecyl sulfate gel electrophoresis, did not change in cultures grown under low or high pressure at optimal and minimal growth temperatures, but several changes were observed at the maximal growth temperature under in situ pressure. The complex lipid pattern of P. abyssi grown under in situ and 0.1- to 0.5-MPa pressures at different temperatures was analyzed by thin-layer chromatography. The phospholipids became more complex at a low growth temperature at both pressures but their profiles were not superimposable; fewer differences were observed in the core lipids. The polar lipids were composed of only one phospholipid in cells grown under in situ pressure at high temperatures. Survival in the presence of oxygen and under starvation conditions was examined. Oxygen was toxic to P. abyssi at growth range temperature, but the strain survived for several weeks at 4(deg)C. The strain was not affected by starvation in a minimal medium for at least 1 month at 4(deg)C and only minimally affected at 95(deg)C for several days. Cells were more resistant to oxygen in starvation medium. A drastic change in protein profile, depending on incubation time, was observed in cells when starved at growth temperature.