免疫沉淀法检测RNPs的RNA组分及其应用

本文用免疫沉淀法检测各种RNPs的RNA组分,发现各种抗RNPs抗体所沉淀的RNAs是具有特征性的,抗U_1RNP抗体沉淀出U_1RNA,抗Sm抗体沉淀出U_2RNA、U_1RNA、U_4RNA、U_5RNA和U_6RNA,抗SSA抗体沉淀出了Y_1-Y_5RNAs,抗SSB抗体沉淀出Y_1-Y_5RNAs、La4.5RNA和7-2RNA,抗JO-1抗体沉淀出tRNA~(His)。因而根据待测血清所沉淀的RNAs可判断血清中各种抗BNPs抗体,结果显示,免疫沉淀法较免疫双扩散法敏感。     

RNA-guided RNA modification: functional organization of the archaeal H/ACA RNP

In eukaryotes and archaea, uridines in various RNAs are converted to pseudouridines by RNA-guided RNA modification complexes termed H/ACA RNPs. Guide RNAs within the complexes base-pair with target RNAs to direct modification of specific ribonucleotides. Cbf5, a protein component of the complex, likely catalyzes the modification. However, little is known about the organization of H/ACA RNPs and the roles of the multiple proteins thought to comprise the complexes. We have reconstituted functional archaeal H/ACA RNPs from recombinant components, defined the components necessary and sufficient for function, and determined the direct RNA-protein and protein-protein interactions that occur between the components. The results provide substantial insight into the functional organization of this RNP. The functional complex requires a guide RNA and each of four proteins: Cbf5, Gar1, L7Ae, and Nop10. Two proteins interact directly with the guide RNA: L7Ae and Cbf5. L7Ae does not interact with other H/ACA RNP proteins in the absence of the RNA. We have defined two novel functions for Cbf5. Cbf5 is the protein that specifically recognizes and binds H/ACA guide RNAs. In addition, Cbf5 recruits the two other essential proteins, Gar1 and Nop10, to the pseudouridylation guide complex.     

Multimeric forms of satellite of tobacco ringspot virus RNA

An approximately 350-nucleotide residue RNA replicates in association with tobacco ringspot virus (TobRV) and becomes encapsidated in TobRV coat protein. Here we show by electrophoretic analyses that this small satellite RNA, RNA S, is the most abundant and most rapidly migrating of a series of at least ten encapsidated RNAs with RNA S sequences. A largely double-stranded RNA fraction from infected tissue, when denatured, gave a similar series of up to 12 zones that contained both RNA S sequences and sequences that hybridized to RNA S. Analysis of the mobilities suggests a weight increment between each zone corresponding approximately to the size of RNA S. Thus the more slowly migrating zones appear to contain covalent multimers of RNA S or, for tissue RNA, both multimers of RNA S and multimers of the complement of RNA S sequences. Neither terminal structure of TobRV genomic RNAs was found in the satellite RNA. RNA S lacks detectable polyadenylate or oligoadenylate. Covalently linked protein was not detected in RNA S or its more slowly migrating forms, and satellite RNA biological activity, unlike that of the TobRV RNAs, was not protease sensitive. Polynucleotide kinase catalyzed the phosphorylation of satellite RNAs, indicating free 5'-hydroxyl groups.     

Isolation and characterization of RNA of Entamoeba histolytica

RNAs were isolated from Entamoeba histolytica with a high salt sodium dodecylsulfate-diethylpyrocarbonate technique. Majority species of 25 S, 17 S and 4 S RNAs were detected after sucrose gradient centrifugation. An additional 5 S RNA was detected by polyacrylamide gel electrophoresis. The molecular weights of these RNAs as determined by completely denaturing polyacrylamide gel electrophoresis were 1.31 X 10(6) (25 S), 0.803 X 10(6) (17 S), 4.0 X 10(4) (5 S) and 2.5 X 10(4) (4 S). The 25 S RNA was labile and dissociated under mild denaturing conditions (between 37 degrees C and 55 degrees C) into 17 S and 16 S RNAs with molecular weights of 0.700 X 10(6) and 0.614 X 10(6), respectively; under completely denaturing conditions an additional 5.8 S RNA with a molecular weight of 4.8 X 10(4) was detected. Evidence is presented which suggests that the lability of the 25 S RNA is the result of an in vivo cleavage rather than one which is generated during RNA isolation.     

Subgenomic messenger RNAs: mastering regulation of (+)-strand RNA virus life cycle

Many (+)-strand RNA viruses use subgenomic (SG) RNAs as messengers for protein expression, or to regulate their viral life cycle. Three different mechanisms have been described for the synthesis of SG RNAs. The first mechanism involves internal initiation on a (−)-strand RNA template and requires an internal SGP promoter. The second mechanism makes a prematurely terminated (−)-strand RNA which is used as template to make the SG RNA. The third mechanism uses discontinuous RNA synthesis while making the (−)-strand RNA templates. Most SG RNAs are translated into structural proteins or proteins related to pathogenesis: however other SG RNAs regulate the transition between translation and replication, function as riboregulators of replication or translation, or support RNA-RNA recombination. In this review we discuss these functions of SG RNAs and how they influence viral replication, translation and recombination.     

Reconstitution and structural analysis of the yeast box H/ACA RNA-guided pseudouridine synthase

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.     

Production of cucumber mosaic virus RNA5 and its role in recombination

Cucumber Mosaic Virus (CMV) is a plant infecting tripartite positive-strand RNA virus. In addition to three genomic and two known subgenomic RNAs, CMV strains of subgroup II (e.g. Q-CMV), but not subgroup I (e.g. Fny-CMV), produce and package a redundant RNA5 encompassing the 3′ 304-307 nucleotides of RNAs 2 and 3. The mechanism regulating RNA5 production and its role in CMV life cycle is unknown. In this study, transient expression of Q2 or Q3 by agroinfiltration into Nicotiana benthamiana plants resulted in efficient accumulation of RNA5 suggesting that its production is independent of CMV replication. Deletion and point mutations engineered into a highly conserved region (Box1) adjacent to the 5′ end of RNA5 identified sequences required for its efficient production. An experimental system, involving a chimera of Q3 (Q3B3) characterized by having a 3′ tRNA-like structure (3′TLS) from Brome mosaic virus (BMV) and RNA5 defective variants of Q1 (Q1Δ), Q2 (Q2Δ) and Q3B3 (Q3ΔB3), was used to evaluate in vivo the contribution of RNA5 in promoting RNA recombination. Generation of precise homologous recombinants was strictly dependent on sequence identity. When both parental RNAs carried the Box1, recombination occurred preferentially within the Box1. In contrast, generation of non-homologous recombinants occurred only when Q1 and Q2 were competent to produce RNA5. A mechanistic model explaining the functional role played by the RNA5 in generating CMV recombinants was presented.     

6.5 S RNA; Preliminary characterisation of unusual small RNAs in Trypanosoma brucei

In this paper we report the preliminary characterisation of some unusual small rRNAs isolated from Trypanosoma brucei. These molecules correspond closely in size and properties to the 5 S and 5.8 S rRNAs of other eukaryotes and in addition there are two further small RNAs which we designate 6.5 S RNA. These have been characterised by sucrose gradient centrifugation and electrophoresis in denaturing gels. The two 6.5 S RNAs may be distinguished from each other on the basis of their size and the different conditions required for their dissociation from the ribosomes. They are present in the same abundance as 5.8 S RNA suggesting that they occur once per ribosome.     

Autoantibodies in human anti-Ro sera specifically recognize deproteinized hY5 Ro RNA.

We report the existence of a novel autoantibody specificity linked to anti-Ro antibodies. Sera from two patients with anti-Ro ribonucleoprotein (RNP) antibodies also contained antibodies that immunoprecipitated specifically either the deproteinized RNA component of the RohY5 RNP particle, or intact in vitro transcribed hY5 RNA. No serum recognized specifically the other hY RNAs. A mutant hY5 RNA with additional nucleotides (nt) at both extremities was not immunoprecipitated, possibly because of altered secondary structure. Following digestion of hY5 RNA with ribonuclease T1, the smallest immunoprecipitable RNA fragments were 27 and 31 nt long, and respectively mapped to the 5' and 3' ends of hY5 RNA, excluding the La-binding region. Base pairing between the 27 and 31 nt long fragments was required for recognition by antibodies. Our data indicate that the epitope bound by anti-hY5 RNA antibodies is conformational. We have previously reported that most anti-Ro sera contain a population of antibodies specific for the RohY5 RNP. Since antibodies to the deproteinized hY RNAs within anti-Ro sera are also restricted to anti-hY5 RNA, a direct role for the human-specific RohY5 particles in the immunization process leading to the production of anti-Ro antibodies is suggested.     

Cellular proteins mediate 5′-3′ end contacts of Norwalk virus genomic RNA

Long-range RNA-RNA interactions between the 5′ and 3′ ends are a common feature involved in the regulation of both the initiation of translation and the synthesis of the viral genomic RNAs. These interactions either take place by direct RNA-RNA contacts or can be mediated by proteins. By in silico analysis, we found three possible complementary sequences (CS) between the 5′ and the 3′ ends of the Norwalk virus genomic RNA. Co-precipitation assays demonstrated that physical contacts between the 5′ and the 3′ ends of the NV genomic RNA were stabilized by cellular proteins. Mutations and deletions within these regions, that altered the formation of the CS-1 motif disrupted the 5′-3′ end contacts, while mutations that restore complementarity of the CS-1 motif, recover the ability to form these contacts. These results suggest that the NV genomic 5′-3′ end contacts initially occur by RNA-RNA interactions but are further stabilized by cellular proteins.     

Anti-5S RNA/protein (RNP) antibody levels correlate with disease activity in a patient with systemic lupus erythematosus (SLE) nephritis.

A patient with systemic lupus erythematosus (SLE) and nephritis without antibodies to dsDNA but with antibodies to a 5S RNA/protein (RNP) complex is presented. Combined RNA precipitation and Western blotting experiments strongly suggested that these newly identified autoantibodies recognized a distinct epitope on the L5 ribosomal protein of the L5/5S RNP complex first described by Steitz et al. [1]. Quantification of the anti-5S RNP antibody levels was done by hybridizing Northern blots of immunoprecipitated RNA from serial serum samples with a 32P-labelled oligoprobe specific for the 5S ribosomal RNA. These studies revealed a strong association between anti-5S RNP autoantibody titre and severity of SLE nephritis over a 3-year prospective study. Our results indicate that the L5/5S RNP can be a target of autoimmune response, and and may serve, in some cases, as marker of SLE severity and response to therapy.     

Paramyxovirus Sendai virus C proteins are essential for maintenance of negative-sense RNA genome in virus particles

The Sendai virus (SeV) C proteins are a nested set of four accessory proteins, C', C, Y1, and Y2, encoded on the P mRNA from an alternate reading frame. The C proteins are multifunctional proteins involved in viral pathogenesis, inhibition of viral RNA synthesis, counteracting the innate immune response of the host cell, inhibition of virus-induced apoptosis, and facilitating virus-like particle (VLP)/virus budding. Among these functions, the roles for pathogenesis and counteracting host cell interferon (IFN) responses have been studied extensively, but the others are less well understood. In this paper, we found that the C proteins contributed in many ways to the efficient production of infectious virus particles by using a series of SeV recombinants without one or more C protein expression. Knockout of both C' and C protein expression resulted in reduced virus release despite higher viral protein synthesis in the cells. Interestingly, for the viruses without C' and C, or all four C protein expression, non-infectious virions containing antigenomic RNAs were produced predominantly compared to genomic RNA-containing infectious virions, due to aberrant viral RNA synthesis. Our results demonstrate for the first time that the C proteins regulate balance of viral genome and antigenome RNA synthesis for efficient production of infectious virus particles in the course of virus infection.     

Specific requirements for elements of the 5' and 3' terminal regions in flavivirus RNA synthesis and viral replication

We initially studied requirements for 5' and 3' terminal regions (TRs) in flavivirus negative strand synthesis in vitro. Purified West Nile (WNV) and dengue-2 (DV2) RNA polymerases were both active with all-WNV or all-DV2 subgenomic RNAs containing the 5'- and 3'TRs of the respective genomes. However, subgenomic RNAs in which the 5'-noncoding region (5'NCR) or the 5'ORF (nts 100-230) in the 5'TR were substituted by analogous sequences derived from the heterologous genome were modestly to severely defective as templates for either polymerase. We also evaluated the infectivity of substitution mutant WNV genome-length RNAs. All WNV RNAs containing the DV2 3'SL were unable to replicate. However, WNV RNAs containing substitutions of the 5'NCR, the capsid gene, and/or 3'NCR nt sequences upstream from the WNV 3'SL, by the analogous DV2 nt sequences, were infectious. Combined results suggested that replication was not dependent upon species homology between the 3'SL and NS5.     

Ro ribonucleoproteins contribute to the resistance of Deinococcus radiodurans to ultraviolet irradiation

The genome of the radiation-resistant eubacterium Deinococcus radiodurans contains an ortholog of an RNA-binding protein known as the Ro 60-kD autoantigen. This protein, which was previously identified only in higher eukaryotes, is normally bound to small RNAs known as Y RNAs. We show that the Ro protein ortholog Rsr contributes to the resistance of D. radiodurans to UV irradiation. Rsr binds several small RNAs, encoded upstream of rsr, that accumulate following UV irradiation. One of these RNAs resembles a Y RNA. These results suggest that Ro RNPs could similarly contribute to the recovery of higher cells following UV irradiation.     

Modular arrangement of viral cis-acting RNA domains in a tombusvirus satellite RNA

Satellite (sat) RNAs are parasitic sub-viral RNA replicons found associated with certain positive-strand RNA viruses. Typical sat RNAs, such as those associated with members of the genus Tombusvirus, share little or no sequence identity with their helper virus genomes. Here, we have investigated a tombusvirus sat RNA and determined that it contains two functionally-relevant higher-order RNA domains, a T-shaped domain and a downstream domain, that are similar to elements shown previously to be present in the 5' untranslated regions (UTRs) of tombusvirus genomes. Although the two sat RNA domains showed only limited sequence identity with their viral counterparts, they were able to adopt comparably-folded RNA secondary structures. Interestingly, the relative spacing between the domains in the viral and satellite contexts was notably different. In the viral 5' UTR, the two domains are adjacent and separated by a small hairpin, however, in the sat RNA they are separated by a 137-nt long segment. Despite this distal modular arrangement, the two domains were found to be united spatially in the sat RNA through the formation of an RNA-RNA bridge. This co-localization facilitated an important inter-domain interaction and was essential for efficient helper-mediated sat RNA accumulation in protoplasts. These results indicate that the tombusvirus sat RNA and helper genome contain structurally and functionally equivalent RNA domains. It is proposed that the limited sequence identity observed between these corresponding higher-order RNA structures is related to a strategy that reduces the induction of gene silencing, which presumably would be detrimental to both viral and sat RNA replicons.     

Recombination of 5' subgenomic RNA3a with genomic RNA3 of Brome mosaic bromovirus in vitro and in vivo

RNA-RNA recombination salvages viral RNAs and contributes to their genomic variability. A recombinationally-active subgenomic promoter (sgp) has been mapped in Brome mosaic bromovirus (BMV) RNA3 (Wierzchoslawski et al., 2004. J. Virol.78, 8552-8864) and mRNA-like 5′ sgRNA3a was characterized (Wierzchoslawski et al., 2006. J. Virol. 80, 12357-12366). In this paper we describe sgRNA3a-mediated recombination in both in vitro and in vivo experiments. BMV replicase-directed co-copying of (−) RNA3 with wt sgRNA3a generated RNA3 recombinants in vitro, but it failed to when 3′-truncated sgRNA3a was substituted, demonstrating a role for the 3′ polyA tail. Barley protoplast co-transfections revealed that (i) wt sgRNA3a recombines at the 3′ and the internal sites; (ii) 3′-truncated sgRNA3as recombine more upstream; and (iii) 5′-truncated sgRNA3 recombine at a low rate. In planta co-inoculations confirmed the RNA3-sgRNA3a crossovers. In summary, the non-replicating sgRNA3a recombines with replicating RNA3, most likely via primer extension and/or internal template switching.