从基因组分析贾第虫的核糖体合成系统

为探讨贾第虫细胞核内核糖体合成系统,及与典型的真核生物有何差异,首先,确定在典型真核生物中参与核糖体合成的129条共有的保守蛋白,然后用这些蛋白搜索贾第虫基因组以调查它们在贾第虫中的直系同源蛋白的情况,以对贾第虫的核糖体合成系统作一了解。结果表明：贾第虫具有89条这些蛋白的直系同源蛋白,包括参与rRNA甲基化和假尿嘧啶化的蛋白复合体成员,以及存在于90S、40S和60S复合体中的蛋白。贾第虫的核糖体合成系统与典型的真核生物相似,但还有40条蛋白在贾第虫基因组中找不到同源蛋白。这意味着贾第虫的核糖体合成系统较典型的真核生物简单。贾第虫虽然没有核仁结构,但其核糖体亚基合成的途径和机制可能与真核细胞相似,参与的成分不同于无核仁结构的原核生物,可能相对简单。     

A U3 snoRNP protein with homology to splicing factor PRP4 and G beta domains is required for ribosomal RNA processing.

Yeast fibrillarin (NOP1) is an evolutionarily conserved, nucleolar protein necessary for multiple steps in ribosome biogenesis. Yeast mutants lacking a functional NOP1 gene can be complemented by human fibrillarin but are temperature sensitive for growth and impaired in pre-rRNA processing. In order to identify components which interact functionally with human fibrillarin in yeast, we isolated extragenic suppressors of this phenotype. One dominant suppressor, sof1-56, which is allele-specific for human fibrillarin and restores growth and pre-RNA processing at 35 degrees C, was cloned by in vivo complementation. The wild-type allele of SOF1 is essential for cell growth and encodes a novel 56 kDa protein. In its central domain, SOF1 contains a repeated sequence also found in beta-subunits of trimeric G-proteins and the splicing factor PRP4. A single amino acid exchange in the G beta-like repeat domain is responsible for the suppressing activity of sof1-56. Indirect immunofluorescence shows that SOF1 is located within the yeast nucleolus. Co-immunoprecipitation demonstrates the physical association of SOF1 with U3 small nucleolar RNA and NOP1. In vivo depletion of SOF1 leads to impaired pre-rRNA processing and inhibition of 18S rRNA production. Thus, SOF1 is a new component of the nucleolar rRNA processing machinery.     

An unusual fibrillarin gene and protein: structure and functional implications.

The diploid germinal nucleus of the ciliated protozoan Tetrahymena thermophila is unusual among eukaryotes in that it encodes a single copy of the gene for rRNA allowing identification of cis-acting mutations in rDNA affecting rRNA structure, function, and processing. The generally conserved nucleolar protein fibrillarin has been characterized from a number of systems and is involved in pre-rRNA processing. We have demonstrated that Tetrahymena has fibrillarin and have analyzed the cDNA and the genomic DNA encoding this protein. The derived amino acid sequence of the N-terminal region of Tetrahymena fibrillarin shows little similarity with the generally highly conserved glycine/arginine-rich N-terminal domain of other eukaryotic fibrillarins. The remainder of the amino acid sequence of the molecule is more conserved. Polyclonal antibodies generated against the full-length Tetrahymena fibrillarin expressed in bacteria recognize a protein of M(r) approximately 32,000 in whole-cell or nucleolar preparations. Immunocytochemistry localizes fibrillarin to nucleoli in the somatic macronuclei of vegetative cells. Transformation experiments demonstrate that fibrillarin is an essential protein in Tetrahymena. The Tetrahymena fibrillarin is expressed but does not complement a NOP1 null mutation when transformed into the yeast Saccharomyces cerevisiae, indicating less functional conservation among fibrillarins than previously suggested.     

Colocalization and interaction of the porcine arterivirus nucleocapsid protein with the small nucleolar RNA-associated protein fibrillarin

Porcine reproductive and respiratory syndrome virus (PRRSV) replicates in the cytoplasm of infected cells, but its nucleocapsid (N) protein localizes specifically to the nucleus and nucleolus. The mechanism of nuclear translocation and whether N associates with particular nucleolar components are unknown. In the present study, we show by confocal microscopy that the PRRSV N protein colocalizes with the small nucleolar RNA (snoRNA)-associated protein fibrillarin. Direct and specific interaction of N with fibrillarin was demonstrated in vivo by the mammalian two-hybrid assay in cells cotransfected with the N and fibrillarin genes and in vitro by the glutathione S-transferase pull-down assay using the expressed fibrillarin protein. Using a series of deletion mutants, the interactive domain of N with fibrillarin was mapped to a region of amino acids 30 to 37. For fibrillarin, the first 80 amino acids, which contain the glycine-arginine-rich region (the GAR domain), was determined to be the domain interactive with N. The N protein was able to bind to the full-length genomic RNA of PRRSV, and the RNA binding domain was identified as the region overlapping with the nuclear localization signal situated at positions 41 to 47. These results suggest that the N protein nuclear transport may be controlled by the binding of RNA to N. The PRRSV N protein was also able to bind to both 28S and 18S ribosomal RNAs. The protein-protein interaction between N and fibrillarin was RNA dependent but independent of N protein phosphorylation. Taken together, our studies demonstrate a specific interaction of the PRRSV nucleocapsid protein with the host cell protein fibrillarin in the nucleolus, and they imply a potential linkage of viral strategies for the modulation of host cell functions, possibly through rRNA precursor processing and ribosome biogenesis.     

Expression and purification of recombinant Giardia fibrillarin and its interaction with small nuclear RNAs

Giardia lamblia, the ancient eukaryote does not have nucleolus but produces the fibrillarin protein that may be used for pre-rRNA processing. The nucleoli of eukaryotes contain complex population of small nucleolar RNAs, known as snoRNAs, several of which are required for rRNA processing. This report describes the full-length cloning of fibrillarin gene from Giardia lamblia, using RTPCR and the production of recombinant fibrillarin protein in Escherichia coli strain BL21 (DE3) as N-terminal His-tag protein. The condition for production of soluble protein was standardized. The expressed protein was purified by using Ni-chelation chromatography and used for functional studies. The small nuclear RNAs (snRNAs), RNA D, RNA J, and RNA H, containing box C, box D, and box C/D, respectively, of Giardia were also cloned by RTPCR. Antibody raised against the recombinant protein was used to identify the fibrillarin in giardial nuclear extract. The interaction of snRNAs with recombinant fibrillarin was followed using North-Western hybridization. Gel electrophoresis mobility shift assay demonstrated that bacterially expressed protein may participate in the in vitro interaction with RNA J, RNA H, and RNA D. Our results indicate that the recombinant fibrillarin by itself is able to bind and does not require the involvement of any other protein for this binding to the three snRNAs.     

Structure determination of fibrillarin from the hyperthermophilic archaeon Pyrococcus furiosus

The methyltransferase fibrillarin is the catalytic component of ribonucleoprotein complexes that direct site-specific methylation of precursor ribosomal RNA and are critical for ribosome biogenesis in eukaryotes and archaea. Here we report the crystal structure of a fibrillarin ortholog from the hyperthermophilic archaeon Pyrococcus furiosus at 1.97A resolution. Comparisons of the X-ray structures of fibrillarin orthologs from Methanococcus jannashii and Archaeoglobus fulgidus reveal nearly identical backbone configurations for the catalytic C-terminal domain with the exception of a unique loop conformation at the S-adenosyl-l-methionine (AdoMet) binding pocket in P. furiosus. In contrast, the N-terminal domains are divergent which may explain why some forms of fibrillarin apparently homodimerize (M. jannashii) while others are monomeric (P. furiosus and A. fulgidus). Three positively charged amino acids surround the AdoMet-binding site and sequence analysis indicates that this is a conserved feature of both eukaryotic and archaeal fibrillarins. We discuss the possibility that these basic residues of fibrillarin are important for RNA-guided rRNA methylation.     

Distribution of rDNA in the nucleus of Giardia lamblia: detection by Ag-I silver stain.

Previous investigations have proved that diplomonads have primitive cell nuclei and lack a nucleolus. We determined the distribution of ribosomal DNA (rDNA) in diplomonad nuclei that lacked a nucleolus. Giardia lamblia was used as the experimental organism with Euglena gracilis as the control. The distribution of rDNA was demonstrated indirectly by the modified Ag-I silver technique that can indicate specifically the nucleolus organizing region (NOR) by both light and electron microscopy. In ultrathin sections of silver stained Euglena cells, all silver grains were concentrated in the fibrosa of the nucleolus, while no silver grains were found in the cytoplasm, nucleoplasm, condensed chromosomes or pars granulosa of the nucleus. In the silver stained Giardia cells, no nucleolus was found, but a few silver grains were scattered in the nucleus. This suggests that the rDNA of Giardia does not form an NOR-like structure and that its nucleus is in a primitive state.     

Evolutionary conservation of the human nucleolar protein fibrillarin and its functional expression in yeast

NOP1 is an essential nucleolar protein in yeast that is associated with small nucleolar RNA and required for ribosome biogenesis. We have cloned the human nucleolar protein, fibrillarin, from a HeLa cDNA library. Human fibrillarin is 70% identical to yeast NOP1 and is also the functional homologue since either human or Xenopus fibrillarin can complement a yeast nop1- mutant. Human fibrillarin is localized in the yeast nucleolus and associates with yeast small nucleolar RNAs. This shows that the signals within eucaryotic fibrillarin required for nucleolar association and nucleolar function are conserved from yeast to man. However, human fibrillarin only partially complements in yeast resulting in a temperature-sensitive growth, concomitantly altered rRNA processing and aberrant nuclear morphology. A suppressor of the human fibrillarin ts-mutant was isolated and found to map intragenically at a single amino acid position of the human nucleolar protein. The growth rate of yeast nop1- strains expressing Xenopus or human fibrillarin or the human fibrillarin suppressor correlates closely with their ability to efficiently and correctly process pre-rRNA. These findings demonstrate for the first time that vertebrate fibrillarin functions in ribosomal RNA processing in vivo.     

Two RNA-binding sites in plant fibrillarin provide interactions with various RNA substrates

Fibrillarin, one of the major proteins of the nucleolus, plays several essential roles in ribosome biogenesis including pre-rRNA processing and 2'-O-ribose methylation of rRNA and snRNAs. Recently, it has been shown that fibrillarin plays a role in virus infections and is associated with viral RNPs. Here, we demonstrate the ability of recombinant fibrillarin 2 from Arabidopsis thaliana (AtFib2) to interact with RNAs of different lengths and types including rRNA, snoRNA, snRNA, siRNA and viral RNAs in vitro. Our data also indicate that AtFib2 possesses two RNA-binding sites in the central (138-179 amino acids) and C-terminal (225-281 amino acids) parts of the protein, respectively. The conserved GCVYAVEF octamer does not bind RNA directly as suggested earlier, but may assist with the proper folding of the central RNA-binding site.     

Interaction of the plant glycine-rich RNA-binding protein MA16 with a novel nucleolar DEAD box RNA helicase protein from Zea mays

The maize RNA-binding MA16 protein is a developmentally and environmentally regulated nucleolar protein that interacts with RNAs through complex association with several proteins. By using yeast two-hybrid screening, we identified a DEAD box RNA helicase protein from Zea mays that interacted with MA16, which we named Z. maysDEAD box RNA helicase 1 (ZmDRH1). The sequence of ZmDRH1 includes the eight RNA helicase motifs and two glycine-rich regions with arginine-glycine-rich (RGG) boxes at the amino (N)- and carboxy (C)-termini of the protein. Both MA16 and ZmDRH1 were located in the nucleus and nucleolus, and analysis of the sequence determinants for their cellular localization revealed that the region containing the RGG motifs in both proteins was necessary for nuclear/nucleolar localization The two domains of MA16, the RNA recognition motif (RRM) and the RGG, were tested for molecular interaction with ZmDRH1. MA16 specifically interacted with ZmDRH1 through the RRM domain. A number of plant proteins and vertebrate p68/p72 RNA helicases showed evolutionary proximity to ZmDRH1. In addition, like p68, ZmDRH1 was able to interact with fibrillarin. Our data suggest that MA16, fibrillarin, and ZmDRH1 may be part of a ribonucleoprotein complex involved in ribosomal RNA (rRNA) metabolism.     

Mechanism of ribosome assisted protein folding: A new insight into rRNA functions

The peptidyl transferase center (PTC), present in the domain V of 23S rRNA of bacteria can act as a general protein folding modulator. Any general function of a nucleic acid polymer (DNA or RNA) is always related to specific sequence/sequences. The ribosome mediated protein folding also involves a specific interaction between the nucleotides of peptidyl transferase center and the amino acids of an unfolded protein. In this article the mechanism of rRNA assisted protein folding and its significance in the light of high resolution crystal structure of ribosome are discussed.     

Identification of a Giardia krr1 homolog gene and the secondarily anucleolate condition of Giaridia lamblia

Giaridia lamblia was long considered to be one of the most primitive eukaryotes and to lie close to the transition between prokaryotes and eukaryotes, but several supporting features, such as lack of mitochondrion and Golgi, have been challenged recently. It was also reported previously that G. lamblia lacked nucleolus, which is the site of pre-rRNA processing and ribosomal assembling in the other eukaryotic cells. Here, we report the identification of the yeast homolog gene, krr1, in the anucleolate eukaryote, G. lamblia. The krr1 gene, encoding one of the pre-rRNA processing proteins in yeast, is actively transcribed in G. lamblia. The deduced protein sequence of G. lamblia krr1 is highly similar to yeast KRR1p that contains a single-KH domain. Our database searches indicated that krr1 genes actually present in diverse eukaryotes and also seem to present in Archaea. However, only the eukaryotic homologs, including that of G. lamblia, have the single-KH domain, which contains the conserved motif KR(K)R. Fibrillarin, another important pre-rRNA processing protein has also been identified previously in G. lamblia. Moreover, our database search shows that nearly half of the other nucleolus-localized protein genes of eukaryotic cells also have their homologs in Giardia. Therefore, we suggest that a common mechanism of pre-RNA processing may operate in the anucleolate eukaryote G. lamblia and in the other eukaryotes and that like the case of "lack of mitochondrion," "lack of nucleolus" may not be a primitive feature, but a secondarily evolutionary condition of the parasite.     

Mutations in the nucleolar proteins Tma23 and Nop6 suppress the malfunction of the Nep1 protein

The nucleolar Saccharomyces cerevisiae protein Nep1 was previously shown to bind to a specific site of the 18S rRNA and to be involved in assembly of Rps19p into pre-40S ribosome subunits. Here we report on the identification of tma23 and nop6 mutations as recessive suppressors of a nep1(ts) mutant allele and the nep1 deletion as well. Green fluorescent protein fusions localized Tma23p and Nop6p within the nucleolus, indicating their function in ribosome biogenesis. The high lysine content of both proteins and an RNA binding motif in the Nop6p amino acid sequence suggest RNA-binding functions for both factors. Surprisingly, in contrast to Nep1p, Tma23p and Nop6p seem to be specific for fungi as no homologues could be found in higher eukaryotes. In contrast to most other ribosome biogenesis factors, Tma23p and Nop6p are nonessential in S. cerevisiae. Interestingly, the tma23 mutants showed a considerably increased resistance against the aminoglycoside G418, probably due to a structural change in the 40S ribosomal subunit, which could be the result of incorrectly folded 18S rRNA gene, missing rRNA modifications or the lack of a ribosomal protein.     

Molecular organization of dinoflagellate ribosomal DNA: evolutionary implications of the deduced 5.8 S rRNA secondary structure

The 5.8 S rRNA gene of Prorocentrum micans, a primitive dinoflagellate, has been cloned and its 159 base pairs (bp) have been sequenced along with the two flanking internal transcribed spacers (ITS 1 and 2), respectively, 212 and 195 bp long. Nucleotide sequence homologies between several previously published 5.8 S rRNA gene sequences including those from another dinoflagellate, an ascomycetous yeast, protozoans, a higher plant and a mammal have been determined by sequence alignment. Two prokaryotic 5'-ends of the 23 S rRNA gene have been compared owing to their probable common origin with eucaryotic 5.8 S rRNA genes. Several nucleotides are distinctive for dinoflagellates when compared with either typical eucaryotes or procaryotes. This is consistent with an early divergence of the dinoflagellate lineage from the typical eucaryotes. The secondary structure of dinoflagellate 5.8 S rRNA molecules fits the model of Walker et al. (1983). Conserved nucleotides which distinguish dinoflagellate 5.8 S rRNA from that of other eucaryotes are located in specific loops which are assumed to play a structural role in the ribosome. A 5.8 S rRNA phylogenetic tree which is proposed, based on sequence data, supports our initial assumption of the dinoflagellates.     

Unusual features of fibrillarin cDNA and gene structure in Euglena gracilis: evolutionary conservation of core proteins and structural predictions for methylation-guide box C/D snoRNPs throughout the domain Eucarya

Box C/D ribonucleoprotein (RNP) particles mediate O^2′-methylation of rRNA and other cellular RNA species. In higher eukaryotic taxa, these RNPs are more complex than their archaeal counterparts, containing four core protein components (Snu13p, Nop56p, Nop58p and fibrillarin) compared with three in Archaea. This increase in complexity raises questions about the evolutionary emergence of the eukaryote-specific proteins and structural conservation in these RNPs throughout the eukaryotic domain. In protists, the primarily unicellular organisms comprising the bulk of eukaryotic diversity, the protein composition of box C/D RNPs has not yet been extensively explored. This study describes the complete gene, cDNA and protein sequences of the fibrillarin homolog from the protozoon Euglena gracilis, the first such information to be obtained for a nucleolus-localized protein in this organism. The E.gracilis fibrillarin gene contains a mixture of intron types exhibiting markedly different sizes. In contrast to most other E.gracilis mRNAs characterized to date, the fibrillarin mRNA lacks a spliced leader (SL) sequence. The predicted fibrillarin protein sequence itself is unusual in that it contains a glycine-lysine (GK)-rich domain at its N-terminus rather than the glycine-arginine-rich (GAR) domain found in most other eukaryotic fibrillarins. In an evolutionarily diverse collection of protists that includes E.gracilis, we have also identified putative homologs of the other core protein components of box C/D RNPs, thereby providing evidence that the protein composition seen in the higher eukaryotic complexes was established very early in eukaryotic cell evolution.