Human Fibrillarin Research Articles

A cell-based assay for detection of anti-fibrillarin autoantibodies with performance equivalent to immunoprecipitation.

Anti-fibrillarin autoantibodies are useful for the diagnosis and prognosis of systemic sclerosis (SSc). Anti-fibrillarin produces a clumpy nucleolar pattern in indirect immunofluorescence assay on HEp-2 cells (HEp-2 IFA). Here we develop and validate a reliable cell-based anti-fibrillarin assay (Fibrillarin/CBA) for use in clinical diagnostic laboratories. A TransMembrane Signal was fused to the human fibrillarin gene (TMS-fibrillarin). HEp-2 cells overexpressing transgenic TMS-fibrillarin at the cytoplasmic membrane were used as IFA substrate in the Fibrillarin/CBA. Sixty-two serum samples with nucleolar pattern in the HEp-2 IFA (41 clumpy; 21 homogeneous/punctate) were tested for anti-fibrillarin using Fibrillarin/CBA, immunoprecipitation (IP), line-blot and ELISA. In addition, samples from 106 SSc-patients were evaluated with Fibrillarin/CBA and the results were correlated with disease phenotypes. Thirty-eight of 41 samples with the clumpy nucleolar pattern (92.7%) were positive in the Fibrillarin/CBA, while all 21 samples with other nucleolar patterns were negative. Fibrillarin/CBA results agreed 100% with IP results. Among the 38 Fibrillarin/CBA-positive samples, only 15 (39.5%) and 11 (29%) were positive for anti-fibrillarin in line-blot and ELISA, respectively. Higher frequency of diffuse cutaneous SSc (dcSSc) phenotype (72.7% vs 36.8%; p=0.022), cardiac involvement (36.4% vs 6.5%; p=0.001) and scleroderma renal crisis (18.2% vs 3.3% p = 0.028) was observed in SSc patients with positive compared to negative Fibrillarin/CBA result. Performance of Fibrillarin/CBA in the detection of anti-fibrillarin autoantibodies was comparable to the gold standard IP. Positive Fibrillarin/CBA results correlated with disease phenotypes known to be associated with anti-fibrillarin autoantibodies, underscoring the clinical validation of this novel assay.

Discovery of Cofactor Competitive Inhibitors against the Human Methyltransferase Fibrillarin

Fibrillarin (FBL) is an essential and evolutionarily highly conserved S-adenosyl methionine (SAM) dependent methyltransferase. It is the catalytic component of a multiprotein complex that facilitates 2′-O-methylation of ribosomal RNAs (rRNAs), a modification essential for accurate and efficient protein synthesis in eukaryotic cells. It was recently established that human FBL (hFBL) is critical for Nipah, Hendra, and respiratory syncytial virus infections. In addition, overexpression of hFBL contributes towards tumorgenesis and is associated with poor survival in patients with breast cancer, suggesting that hFBL is a potential target for the development of both antiviral and anticancer drugs. An attractive strategy to target cofactor-dependent enzymes is the selective inhibition of cofactor binding, which has been successful for the development of inhibitors against several protein methyltransferases including PRMT5, DOT1L, and EZH2. In this work, we solved crystal structures of the methyltransferase domain of hFBL in apo form and in complex with the cofactor SAM. Screening of a fluorinated fragment library, via X-ray crystallography and 19F NMR spectroscopy, yielded seven hit compounds that competed with cofactor binding, two of which resulted in co-crystal structures. One of these structures revealed unexpected conformational variability in the cofactor binding site, which allows it to accommodate a compound significantly different from SAM. Our structural data provide critical information for the design of selective cofactor competitive inhibitors targeting hFBL, and preliminary elaboration of hit compounds has led to additional cofactor site binders.

Characterization of the Activity and Biological Function of Human Protein Arginine Methyltransferase 9 (PRMT9)

Protein arginine methyltransferases (PRMTs) make up a family of enzymes that transfer methyl groups onto arginine residues and play important roles in DNA repair, splicing, transcriptional control and signaling. In mammals, a family of nine PRMTs are known. PRMT1,2,3,4,6, and 8 catalyze the formation of asymmetric dimethylarginine (ADMA) and w‐monomethylarginine (MMA), PRMT5 catalyzes symmetric dimethylarginine (SDMA) and MMA formation, and PRMT7 catalyzes only MMA formation. The last member of this family, PRMT9 (also designated PRMT10), has not previously been characterized. In humans, it is encoded on a gene on chromosome 4 in position 4q31. We have now expressed human PRMT9 both as a GST‐fusion protein in bacterial cells and as a GFP‐fusion protein in human HEK293 cells. For both expression systems, we have also prepared catalytic mutants to serve as controls for possible contamination of other types of PRMTs. Using an in vitro methylation assay, we show that both forms of PRMT9 catalyze the methylation of myelin basic protein and a GST‐fusion protein of the N‐terminal domain of human fibrillarin. Studies are in progress to characterize the type of activity and physiological substrates for PRMT9.

Characterization of the activity and biological function of human protein arginine methyltransferase 9 (555.6)

Protein arginine methyltransferases (PRMTs) make up a family of enzymes that transfer methyl groups onto arginine residues and play important roles in DNA repair, splicing, transcriptional control and signaling. In mammals, a family of nine PRMTs are known. PRMT1,2,3,4,6, and 8 catalyze the formation of asymmetric dimethylarginine (ADMA) and ω‐monomethylarginine (MMA), PRMT5 catalyzes symmetric dimethylarginine (SDMA) and MMA formation, and PRMT7 catalyzes only MMA formation. The last member of this family, PRMT9 (also designated PRMT10), has not previously been characterized. In humans, it is encoded on a gene on chromosome 4 in position 4q31. We have now expressed human PRMT9 both as a GST‐fusion protein in bacterial cells and as a GFP‐fusion protein in human HEK293 cells. For both expression systems, we have also prepared catalytic mutants to serve as controls for possible contamination of other types of PRMTs. Using an in vitro methylation assay, we show that both forms of PRMT9 catalyze the methylation of myelin basic protein and a GST‐fusion protein of the N‐terminal domain of human fibrillarin. Studies are in progress to characterize the type of activity and physiological substrates for PRMT9.Grant Funding Source: Supported by Ruth L. Kirschstein National Research Service Award GM007185

Structure ofAeropyrum pernixfibrillarin in complex with natively boundS-adenosyl-L-methionine at 1.7 Å resolution

Fibrillarin is the key methyltransferase associated with the C/D class of small nuclear ribonucleoproteins (snRNPs) and participates in the preliminary step of pre-ribosomal rRNA processing. This molecule is found in the fibrillar regions of the eukaryotic nucleolus and is involved in methylation of the 2'-O atom of ribose in rRNA. Human fibrillarin contains an N-terminal GAR domain, a central RNA-binding domain comprising an RNP-2-like superfamily consensus sequence and a catalytic C-terminal helical domain. Here, Aeropyrum pernix fibrillarin is described, which is homologous to the C-terminal domain of human fibrillarin. The protein was crystallized with an S-adenosyl-L-methionine (SAM) ligand bound in the active site. The molecular structure of this complex was solved using X-ray crystallography at a resolution of 1.7 Å using molecular replacement with fibrillarin structural homologs. The structure shows the atomic details of SAM and its active-site interactions; there are a number of conserved residues that interact directly with the cofactor. Notably, the adenine ring of SAM is stabilized by π-π interactions with the conserved residue Phe110 and by electrostatic interactions with the Asp134, Ala135 and Gln157 residues. The π-π interaction appears to play a critical role in stabilizing the association of SAM with fibrillarin. Furthermore, comparison of A. pernix fibrillarin with homologous structures revealed different orientations of Phe110 and changes in α-helix 6 of fibrillarin and suggests key differences in its interactions with the adenine ring of SAM in the active site and with the C/D RNA. These differences may play a key role in orienting the SAM ligand for catalysis as well as in the assembly of other ribonucleoproteins and in the interactions with C/D RNA.

Analysis of nucleolar protein fibrillarin mobility and functional state in living HeLa cells

Fibrillarin is an evolutionarily-conserved and obligatory protein component of eukaryotic cell nucleoli involved in pre-rRNA processing and methylation. In vertebrates the fibrillarin molecule contains two cysteine residues (Cys99 and Cys268) whose sulfhydryl groups are able to establish intramolecular-S-S- bridges. However, the functional state of fibrillarin with reduced or oxidized thiol groups is still practically unstudied. Besides, there are no data in the literature concerning existence of the -S-S- fibrillarin form in human cells. To answer these questions, we used plasmids encoding native human fibrillarin and its mutant form devoid of cysteine residues (fibrillarinC99/268S) fused with EGFP for temporary transfection of HeLa cells. The mobile fraction localizing the enzymatically active protein molecules and the fluorescence half-recovery time characterizing the rate of enzymatic reactions were determined by the FRAP technique using a confocal laser scanning microscope. Measurements were carried out at 37 and 27°C. The results show that the fibrillarin pool in HeLa cells includes two protein forms, with reduced SH groups and with oxidized SH groups forming intramolecular -S-S- bridges between Cys99 and Cys268. However, the absence of Cys99 and Cys268 has no effect on intracellular localization of fibrillarin and its main dynamic parameters. The human fibrillarin form without disulfide bridges is included into the mobile protein fraction and is consistent with its functionally active state.

A Kinetic Study of Human Protein Arginine N-Methyltransferase 6 Reveals a Distributive Mechanism

Human protein arginine N-methyltransferase 6 (PRMT6) transfers methyl groups from the co-substrate S-adenosyl-L-methionine to arginine residues within proteins, forming S-adenosyl-L-homocysteine as well as omega-N(G)-monomethylarginine (MMA) and asymmetric dimethylarginine (aDMA) residues in the process. We have characterized the kinetic mechanism of recombinant His-tagged PRMT6 using a mass spectrometry method for monitoring the methylation of a series of peptides bearing a single arginine, MMA, or aDMA residue. We find that PRMT6 follows an ordered sequential mechanism in which S-adenosyl-L-methionine binds to the enzyme first and the methylated product is the first to dissociate. Furthermore, we find that the enzyme displays a preference for the monomethylated peptide substrate, exhibiting both lower K(m) and higher V(max) values than what are observed for the unmethylated peptide. This difference in substrate K(m) and V(max), as well as the lack of detectable aDMA-containing product from the unmethylated substrate, suggest a distributive rather than processive mechanism for multiple methylations of a single arginine residue. In addition, we speculate that the increased catalytic efficiency of PRMT6 for methylated substrates combined with lower K(m) values for native protein methyl acceptors may obscure this distributive mechanism to produce an apparently processive mechanism.

Regulation of Protein Arginine Methyltransferase 8 (PRMT8) Activity by Its N-terminal Domain

Human protein arginine methyltransferase PRMT8 has been recently described as a type I enzyme in brain that is localized to the plasma membrane by N-terminal myristoylation. The amino acid sequence of human PRMT8 is almost 80% identical to human PRMT1, the major protein arginine methyltransferase activity in mammalian cells. However, the activity of a recombinant PRMT8 GST fusion protein toward methyl-accepting substrates is much lower than that of a GST fusion of PRMT1. We show here that both His-tagged and GST fusion species lacking the initial 60 amino acid residues of PRMT8 have enhanced enzymatic activity, suggesting that the N-terminal domain may regulate PRMT8 activity. This conclusion is supported by limited proteolysis experiments showing an increase in the activity of the digested full-length protein, consistent with the loss of the N-terminal domain. In contrast, the activity of the N-terminal truncated protein was slightly diminished by limited proteolysis. Significantly, we detect automethylation at two sites in the N-terminal domain, as well as binding sites for SH3 domain-containing proteins. We suggest that the N-terminal domain may function as an autoregulator that may be displaced by interaction with one or more physiological inducers.

Yeast Hsl7 (histone synthetic lethal 7) catalyses the in vitro formation of ω-N G-monomethylarginine in calf thymus histone H2A

The HSL7 (histone synthetic lethal 7) gene in the yeast Saccharomyces cerevisiae encodes a protein with close sequence similarity to the mammalian PRMT5 protein, a member of the class of protein arginine methyltransferases that catalyses the formation of omega-N(G)-monomethylarginine and symmetric omega-N(G),N'(G)-dimethylarginine residues in a number of methyl-accepting species. A full-length HSL7 construct was expressed as a FLAG-tagged protein in Saccharomyces cerevisiae. We found that FLAG-tagged Hsl7 effectively catalyses the transfer of methyl groups from S-adenosyl-[methyl-3H]-L-methionine to calf thymus histone H2A. When the acid-hydrolysed radiolabelled protein products were separated by high-resolution cation-exchange chromatography, we were able to detect one tritiated species that co-migrated with an omega-N(G)-monomethylarginine standard. No radioactivity was observed that co-migrated with either the asymmetric or symmetric dimethylated derivatives. In control experiments, no methylation of histone H2A was found with two mutant constructs of Hsl7. Surprisingly, FLAG-Hsl7 does not appear to effectively catalyse the in vitro methylation of a GST (glutathione S-transferase)-GAR [glycine- and arginine-rich human fibrillarin-(1-148) peptide] fusion protein or bovine brain myelin basic protein, both good methyl-accepting substrates for the human homologue PRMT5. Additionally, FLAG-Hsl7 demonstrates no activity on purified calf thymus histones H1, H2B, H3 or H4. GST-Rmt1, the GST-fusion protein of the major yeast protein arginine methyltransferase, was also found to methylate calf thymus histone H2A. Although we detected Rmt1-dependent arginine methylation in vivo in purified yeast histones H2A, H2B, H3 and H4, we found no evidence for Hsl7-dependent methylation of endogenous yeast histones. The physiological substrates of the Hsl7 enzyme remain to be identified.

The AT-hook of the Chromatin Architectural Transcription Factor High Mobility Group A1a Is Arginine-methylated by Protein Arginine Methyltransferase 6

The HMGA1a protein belongs to the high mobility group A (HMGA) family of architectural nuclear factors, a group of proteins that plays an important role in chromatin dynamics. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes, such as transcriptional regulation, viral integration, DNA repair, RNA processing, and chromatin remodeling. The activity of HMGA proteins is finely modulated by a variety of post-translational modifications. Arginine methylation was recently demonstrated to occur on HMGA1a protein, and it correlates with the apoptotic process and neoplastic progression. Methyltransferases responsible for these modifications are unknown. Here we show that the protein arginine methyltransferase PRMT6 specifically methylates HMGA1a protein both in vitro and in vivo. By mass spectrometry, the sites of methylation were unambiguously mapped to Arg(57) and Arg(59), two residues which are embedded in the second AT-hook, a region critical for both protein-DNA and protein-protein interactions and whose modification may cause profound alterations in the HMGA network. The in vivo association of HMGA and PRMT6 place this yet functionally uncharacterized methyltransferase in the well established functional context of the chromatin structure organization.

A higher concentration of an antigen within the nucleolus may prevent its proper recognition by specific antibodies.

Transient transfection of HeLa cells with a plasmid encoding the full-length human fibrillarin fused to a green fluorescent protein (GFP) resulted in two major patterns of intensity of the nucleolar labeling for the chimeric protein: weak and strong. Both patterns were maintained in fibrillarin-GFP expressing cells after fixation with formaldehyde. When the fixed fibrillarin-GFP expressing cells were used for immunolabeling with antibodies to fibrillarin, only the nucleoli with a weak GFP-signal became strongly labeled, whereas those with the heavy signals were only lightly stained, if at all. A similar pattern was observed if the cells were immunolabeled with antibodies to GFP. These observations suggest that an increase in antigen accumulation within the nucleolus, which could take place under various physiological or experimental conditions, could prevent the antigen from being recognized by specific antibodies. These results have implications regarding contradictory data on localization of various nucleolar antigens obtained by conventional immunocytochemistry.

PRMT7, a New Protein Arginine Methyltransferase That Synthesizes Symmetric Dimethylarginine

The cDNA for PRMT7, a recently discovered human protein-arginine methyltransferase (PRMT), was cloned and expressed in Escherichia coli and mammalian cells. Immunopurified PRMT7 actively methylated histones, myelin basic protein, a fragment of human fibrillarin (GAR) and spliceosomal protein SmB. Amino acid analysis showed that the modifications produced were predominantly monomethylarginine and symmetric dimethylarginine (SDMA). Examination of PRMT7 expressed in E. coli demonstrated that peptides corresponding to sequences contained in histone H4, myelin basic protein, and SmD3 were methylated. Furthermore, analysis of the methylated proteins showed that symmetric dimethylarginine and relatively small amounts of monomethylarginine and asymmetric dimethylarginine were produced. SDMA was also formed when a GRG tripeptide was methylated by PRMT7, indicating that a GRG motif is by itself sufficient for symmetric dimethylation to occur. Symmetric dimethylation is reduced dramatically compared with monomethylation as the concentration of the substrate is increased. The data demonstrate that PRMT7 (like PRMT5) is a Type II methyltransferase capable of producing SDMA modifications in proteins.

Identification of Signal Sequences Determining the Specific Nucleolar Localization of Fibrillarin in HeLa Cells

Fibrillarin is one of the major nucleolar proteins and is involved in pre-rRNA maturation. Its three main regions are a glycine and arginine-rich (GAR) domain, an RNA-binding domain, and an alpha-helical region, which presumably has a methyltransferase activity. Yet the roles of these regions in nucleolus-specific localization of fibrillarin are still unclear. To elucidate this issue, a series of plasmids was constructed to express human fibrillarin mutants fused with the green fluorescent protein. Localization of the chimeric proteins was studied in interphase and mitotic HeLa cells after single transfection with the plasmids. Deletion or a mutation of any domain proved to alter the specific fibrillarin location coinciding with sites of pre-rRNA synthesis. The GAR domain and the first spacer together were sufficient for fibrillarin migration into the nucleolus. Fibrillarin mutants located within the interphase nucleolus did not differ in mitotic location from the wild-type fibrillarin.

Human Fibrillarin Forms a Sub-complex with Splicing Factor 2-associated p32, Protein Arginine Methyltransferases, and Tubulins α3 and β1 That Is Independent of Its Association with Preribosomal Ribonucleoprotein Complexes

Fibrillarin (FIB, Nop1p in yeast) is an RNA methyltransferase found not only in the fibrillar region of the nucleolus but also in Cajal bodies. FIB is essential for efficient processing of preribosomal RNA during ribosome biogenesis, although its precise function in this process and its role in Cajal bodies remain uncertain. Here, we demonstrate that the human FIB N-terminal glycine- and arginine-rich domain (residues 1-77) and its spacer region 1 (78-132) interact with splicing factor 2-associated p32 (SF2A-p32) and that the FIB methyltransferase-like domain (133-321) interacts with protein-arginine methyltransferase 5 (PRMT5, Janus kinase-binding protein 1). We also show that these proteins associate with several additional proteins, including PRMT1, tubulin alpha 3, and tubulin beta 1 to form a sub-complex that is principally independent of the association of FIB with preribosomal ribonucleoprotein complexes that co-immunoprecipitate with the sub-complex in human cells expressing FLAG-tagged FIB. Based on the physical association of FIB with SF2A-p32 and PRMTs, as well as the other reported results, we propose that FIB may coordinate both RNA and protein methylation during the processes of ribosome biogenesis in the nucleolus and RNA editing such as small nuclear (nucleolar) ribonucleoprotein biogenesis in Cajal bodies.

Arginine Methylation of Recombinant Murine Fibrillarin by Protein Arginine Methyltransferase

Fibrillarin is a conserved nucleolar SnoRNP with a diverse N-terminal glycine- and arginine-rich (GAR) domain in most eukaryotes. This region in human fibrillarin is known to contain modified dimethylarginines. In this report we demonstrate that recombinant murine fibrillarin is a substrate for protein arginine methyltransferase, including the purified recombinant enzyme (rat PRMT1 and yeast RMT1) and the protein methyltransferases present in lymphoblastoid cell extracts. Our results of protease digestion, methylation competition reactions, and immunoblotting with a methylarginine-specific antibody all indicate that the methylation of fibrillarin is in the N-terminal GAR domain and arginyl residues are modified. Finally, amino acid analyses revealed that the modification of recombinant murine fibrillarin forms methylarginines, mostly as dimethylarginines.

Antifibrillarin autoantibodies present in systemic sclerosis and other connective tissue diseases interact with similar epitopes.

Autoantibodies specific against fibrillarin, a 34-kD nucleolar protein associated with U3-snRNP, are present in patients with systemic sclerosis (SSc). To understand the mechanisms involved in the induction of these autoantibodies, we prepared a series of human fibrillarin recombinant proteins covering the entire molecule and analyzed their interaction with the autoantibodies present in various connective tissue diseases. Our results showed that antifibrillarin autoantibodies are present not only in SSc, as previously reported, but also in a variety of other connective tissue diseases. Patients with SSc (58%), mixed connective tissue diseases (60%), CREST syndrome (calcinosis, Raynaud phenomenon, esophageal dismotility, sclerodactyly, and telangiectasia syndrome) (58%), systemic lupus erythematosus (39%), rheumatoid arthritis (60%), and Sjogern's syndrome (84%) showed presence of antifibrillarin autoantibodies. Results obtained from competitive inhibition radioimmunoassay and Western blot analyses with purified recombinant fusion proteins revealed that these autoantibodies react primarily with epitope(s) present in the NH2- (AA 1-80) and COOH-terminal (AA 276-321) domains of fibrillarin. Autoantibodies reacting with internal regions of fibrillarin are less frequent. Analysis of the hydrophilicity profiles of reactive peptides showed presence of three potential antigenic sites in the NH2- and two in the COOH-terminal regions. While a hexapeptide sequence NH2 terminus of fibrillarin is shared with an Epstein-Barr virus-encoded nuclear antigen, the COOH-terminal region shares sequence homology with P40, the capsid protein encoded by herpes virus type 1. Interestingly, these two regions of fibrillarin also contain the most immunodominant sequences, as predicted by surface probability and the Jameson and Wolf antigenic index. These observations suggest that molecular mimicry might play an important role in the induction of antifibrillarin autoantibodies.

Molecular cloning and sequence analysis of U3 snoRNA-associated mouse fibrillarin

We have isolated and determined the sequence of a 1.1-kb cDNA from a murine WEHI-3 macrophage library which encodes the highly conserved, nucleolar protein, fibrillarin. The murine fibrillarin protein sequence displays 94.2% identity with human fibrillarin, 82.9% identity with amphibian fibrillarin and 74.0% identity with the yeast fibrillarin homolog, NOP1. Immunoprecipitation showed that anti-fibrillarin autoantibodies from human scleroderma sera and the monoclonal autoantibody 72B9 recognize the approx. 34–36 kDa in vitro transcribed and translated protein. Mouse fibrillarin contains a N-terminal glycine- and arginine-rich (GAR) domain which although conserved among the fibrillarins is not as strongly conserved as several regions in the carboxy tail of the protein. Specific amino acid residues in yeast NOP1 thought to be associated with the synthesis and maturation of ribosomes show strong conservation between the mouse, human, amphibian and yeast protein sequences.

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.

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.

CDNA cloning and sequencing of human fibrillarin, a conserved nucleolar protein recognized by autoimmune antisera.

We have isolated a 1.1-kilobase cDNA clone that encodes human fibrillarin by screening a hepatoma library in parallel with DNA probes derived from the fibrillarin genes of Saccharomyces cerevisiae (NOP1) and Xenopus laevis. RNA blot analysis indicates that the corresponding mRNA is approximately 1300 nucleotides in length. Human fibrillarin expressed in vitro migrates on SDS gels as a 36-kDa protein that is specifically immunoprecipitated by antisera from humans with scleroderma autoimmune disease. Human fibrillarin contains an amino-terminal repetitive domain approximately 75-80 amino acids in length that is rich in glycine and arginine residues and is similar to amino-terminal domains in the yeast and Xenopus fibrillarins. The occurrence of a putative RNA-binding domain and an RNP consensus sequence within the protein is consistent with the association of fibrillarin with small nucleolar RNAs. Protein sequence alignments show that 67% of amino acids from human fibrillarin are identical to those in yeast fibrillarin and that 81% are identical to those in Xenopus fibrillarin. This identity suggests the evolutionary conservation of an important function early in the pathway for ribosome biosynthesis.