The production of multiple small peptaibol families by single 14-module Peptide synthetases in Trichoderma/Hypocrea

The most common sequences of peptaibiotics are 11-residue peptaibols found widely distributed in the genus Trichoderma/Hypocrea. Frequently associated are 14-residue peptaibols sharing partial sequence identity. Genome sequencing projects of three Trichoderma strains of the major clades reveal the presence of up to three types of nonribosomal peptide synthetases with 7, 14, or 18-20 amino acid-adding modules. Here, we provide evidence that the 14-module NRPS type found in T. virens, T. reesei (teleomorph Hypocrea jecorina), and T. atroviride produces both 11- and 14-residue peptaibols based on the disruption of the respective NRPS gene of T. reesei, and bioinformatic analysis of their amino acid-activating domains and modules. The sequences of these peptides may be predicted from the gene sequences and have been confirmed by analysis of families of 11- and 14-residue peptaibols from the strain 618, termed hypojecorins A (23 sequences determined, 4 new) and B (3 sequences determined, 2 new), and the recently established trichovirins A from T. virens. The distribution of 11- and 14-residue products is strain-specific and depends on growth conditions as well. Possible mechanisms of module skipping are discussed.     

The Production of Multiple Small Peptaibol Families by Single 14‐Module Peptide Synthetases in Trichoderma/Hypocrea

The most common sequences of peptaibiotics are 11‐residue peptaibols found widely distributed in the genus Trichoderma/Hypocrea. Frequently associated are 14‐residue peptaibols sharing partial sequence identity. Genome sequencing projects of three Trichoderma strains of the major clades reveal the presence of up to three types of nonribosomal peptide synthetases with 7, 14, or 18–20 amino acid‐adding modules. Here, we provide evidence that the 14‐module NRPS type found in T. virens, T. reesei (teleomorph Hypocrea jecorina), and T. atroviride produces both 11‐ and 14‐residue peptaibols based on the disruption of the respective NRPS gene of T. reesei, and bioinformatic analysis of their amino acid‐activating domains and modules. The sequences of these peptides may be predicted from the gene sequences and have been confirmed by analysis of families of 11‐ and 14‐residue peptaibols from the strain 618, termed hypojecorins A (23 sequences determined, 4 new) and B (3 sequences determined, 2 new), and the recently established trichovirins A from T. virens. The distribution of 11‐ and 14‐residue products is strain‐specific and depends on growth conditions as well. Possible mechanisms of module skipping are discussed.     

Two classes of new peptaibols are synthesized by a single non-ribosomal peptide synthetase of Trichoderma virens

Peptaibols are a group of small peptides having a high α-aminoisobutyric acid (Aib) content and produced by filamentous fungi, especially by the members of the genus Trichoderma (anamorph Hypocrea). These antibiotics are economically important for their anti-microbial and anti-cancer properties as well as ability to induce systemic resistance in plants against microbial invasion. In this study we present sequences of two classes (11-residue and 14-residue) of peptaibols produced by the biocontrol fungus Trichoderma virens. Of the 35 11-residue peptaibols sequenced, 18 are hitherto not described, and all the 53 14-residue sequences described by us here are new. We have also identified a peptaibol synthetase (non-ribosomal peptide synthetase, NRPS) with 14 complete modules in the genome of this fungus and disruption of this single gene (designated as tex2) resulted in the loss of both the classes of peptaibols. We, thus present here an unprecedented case where a single NRPS encodes for two classes of peptaibols. The new peptaibols identified here could have applications as therapeutic agents for the management of human and plant health.     

Identification of peptaibols from Trichoderma virens and cloning of a peptaibol synthetase

The fungus Trichoderma virens is a ubiquitous soil saprophyte that has been applied as a biological control agent to protect plants from fungal pathogens. One mechanism of biocontrol is mycoparasitism, and T. virens produces antifungal compounds to assist in killing its fungal targets. Peptide synthetases produce a wide variety of peptide secondary metabolites in bacteria and fungi. Many of these are known to possess antibiotic activities. Peptaibols form a class of antibiotics known for their high alpha-aminoisobutyric acid content and their synthesis as a mixture of isoforms ranging from 7 to 20 amino acids in length. Here we report preliminary characterization of a 62.8-kb continuous open reading frame encoding a peptaibol synthetase from T. virens. The predicted protein structure consists of 18 peptide synthetase modules with additional modifying domains at the N- and C-termini. T. virens was shown to produce a mixture of peptaibols, with the largest peptides being 18 residues. Mutation of the gene eliminated production of all peptaibol isoforms. Identification of the gene responsible for peptaibol production will facilitate studies of the structure and function of peptaibol antibiotics and their contribution to biocontrol activity.     

Detection of peptaibols and partial cloning of a putative peptaibol synthetase gene fromT. harzianum CECT 2413

The characterization of 11- and 18-residue peptaibols (peptides synthesized by peptide synthetases) at Trichoderma harzianum CECT 2413 (a filamentous fungus) was performed. Using a heterologous probe from tex1, the only peptaibol synthetase cloned and characterized so far in Trichoderma species, was cloned; a region that comprised 11676 bp of a second peptide synthetase gene detected in these strain (called salps2) and sequenced. The deduced sequence of Salps2 (3891 amino acids) contained three complete and a fourth incomplete module of a peptide synthetase, in which the typical adenylation, thiolation and condensation domains were found, but also an additional dehydrogenase/reductase domain in the C-terminus of the last module. Based on sequence similarity and analysis of its modular structure, it is proposed that Salps2 is a peptaibol synthetase. Additionally, analysis of =4.4-kb sequence downstream of salps2 was done and the signature sequences of Salps2 were identified and compared with those of available sequences of the other Trichoderma peptaibol synthetases.     

炭团木霉中非核糖体多肽基因NRPS1的功能研究

[目的] 木霉属真菌是应用最为广泛和潜力最大的生防真菌,其产生的典型化合物哌珀霉素（peptaibols）类抗生素在生物防治中发挥重要作用。本研究采用基因组挖掘技术（genome mining）发现炭团木霉（Trichoderma hypoxylon）的潜在哌珀霉素生物合成基因簇及对病原菌的防治作用。[方法] 生物信息学分析预测合成哌珀霉素的基因簇,利用Quick-change技术构建基因骨架敲除盒,通过PEG介导的原生质体转化方法获得敲除突变株,通过平板对峙法和菌丝生长毒力实验验证该基因簇对炭团木霉生物活性的影响。[结果] 基因挖掘鉴定一个非核糖体多肽合成酶（nonribosomal peptide synthetases,NRPS）可能合成哌珀霉素类抗生素,命名为NRPS1,对该基因进行部分敲除,成功获得3株NRPS1缺失突变株。对峙实验表明,突变株对寄生曲霉（Aspergillus parasiticus）、尖孢镰刀菌（Fusarium oxysporum）、黑白轮枝菌（Verticillium alboatrum）等9株植物病原真菌的抑制作用与野生株相比显著下降,且突变株的粗提物的抑菌活性明显弱于野生型。[结论] NRPS1是一个潜在的哌珀霉素合成基因,该基因在宿主与病原真菌对抗过程中起关键作用,该研究为炭团木霉哌珀霉素结构解析及生物防治机理研究奠定了基础。     

Diversity Profile and Dynamics of Peptaibols Produced by Green Mould Trichoderma Species in Interactions with Their Hosts Agaricus bisporus and Pleurotus ostreatus

Certain Trichoderma species are causing serious losses in mushroom production worldwide. Trichoderma aggressivum and Trichoderma pleuroti are among the major causal agents of the green mould diseases affecting Agaricus bisporus and Pleurotus ostreatus, respectively. The genus Trichoderma is well‐known for the production of bioactive secondary metabolites, including peptaibols, which are short, linear peptides containing unusual amino acid residues and being synthesised via non‐ribosomal peptide synthetases (NRPSs). The aim of this study was to get more insight into the peptaibol production of T. aggressivum and T. pleuroti. HPLC/MS‐based methods revealed the production of peptaibols closely related to hypomurocins B by T. aggressivum, while tripleurins representing a new group of 18‐residue peptaibols were identified in T. pleuroti. Putative NRPS genes enabling the biosynthesis of the detected peptaibols could be found in the genomes of both Trichoderma species. In vitro experiments revealed that peptaibols are potential growth inhibitors of mushroom mycelia, and that the host mushrooms may have an influence on the peptaibol profiles of green mould agents.     

The peptaibol database: a sequence and structure resource.

The peptaibols are a large family of membrane-active peptides with considerable sequence homology, but with different biological properties and three-dimensional structures. They constitute a rich resource of naturally occurring 'mutants' which are potentially valuable for structure/function studies of ion channels. A searchable on-line database of sequences and structures of the peptaibols has been created at http://www.cryst.bbk.ac.uk/peptaibol, as a resource for the biological and structural community. In this paper, the contents and organization of the website are discussed as well as procedures for submission of new entries to the database. At present, more than 300 peptaibol sequences are stored in the database. Each sequence entry contains its full literature reference and information about its biological source. Tools are provided for searching for specific peptaibol sequences or groupings of sequences, and for locating peptaibols containing specified sequence motifs. In addition the website acts as a database for structural information. The coordinates of all currently available peptaibol x-ray and NMR structures are included and complemented, where appropriate. with molecular graphics illustrations. These include figures of model channel structures and comparisons between different peptaibol structures. The peptaibol database thus provides a tool for ready access to information and a means of investigating the sequences and structures of this class of polypeptides.     

Deuteriation of sugar protons simplify NMR assignments and structure determination of large oligonucleotide by the 1H-NMR window approach.

The concept of the 1H-NMR window has been developed and examined through a comparative study of NOESY spectra of a self-complementary Dickerson's dodecamer (I) [5'd(5C6G7C8G9A10A11T12T13C-14G15C16G)2(3')], a self-complementary 20-mer (II) [(5'd(1C2G3C4G5C6G7C8G9A10A11T12T13C14G15C16G17C18G19C20G)2(3')] in which the core part consists of the same Dickerson's dodecamer sequence with the flanking CGCG residues at both 3' and 5'-ends, and the partly-deuteriated (shown by underlined CGCG residues at both 3' and 5'-ends) analogous duplex (III) [5'd(1C2G3C4G5C6G7C8G9A10A11T12T13C14G15C16G17C18G19C20G)2(3')] in which the core 5C to 16G part (i.e. 1H-NMR window) consists of the natural Dickerson's dodecamer sequence. A comparison of their NOESY spectra clearly demonstrates that the severe overlap of proton resonances in the larger DNA duplex (II) has been successfully reduced in the partly-deuterated duplex (III) as a result of specific incorporations of the sugar-deuteriated nucleotide residues in the latter [stereospecific > 97 atom % 2H enrichment at H2', H2' and H3' sites, approximately 85 atom % 2H enrichment at H4' and approximately 20 atom % 2H enrichment at H1' (see refs. 10 and 11) in the 20-mer duplex (III)]. These simplifications of the resonance overlap by the deuteriation approach have enabled unequivocal chemical shift assignments and extraction of the quantitative NOE data in the 1H-NMR window part of duplex (III). A comparison of the 12-nucleotide long 1H-NMR window in (III) with that of the 12-mer duplex (I) also shows the scope of studying the changes in conformation and dynamics of the core 12-mer region in (III) which result from the increase of molecular weight due to the DNA chain extension. It is noteworthy that such a study is clearly impossible for the natural 20-mer (II) because of the inherent problem of the overlap of resonances.     

A nonribosomal peptide synthetase involved in the biosynthesis of ampullosporins in Sepedonium ampullosporum.

Recently, the saprophytic ascomycete Sepedonium ampullosporum strain HKI-0053 was isolated from a basidiomycete on account of its premature induction of pigment formation in Phoma destructiva, a process often related to the neuroleptic activity of the inducing compound. The active substance was identified as the 15-membered peptaibol type peptide Ampullosporin. Although to date more than 300 peptaibols have been discovered, their biosynthetic machinery has not been characterized yet. By improving the culture conditions it was possible to grow S. ampullosporum in a submerged culture and to increase Ampullosporin production by more than three times to 33 mg/l at reduced fermentation times. The appearance of two high molecular weight proteins, HMWP1 (1.5 MDa) and HMWP2 (350 kDa) was closely related to the production of Ampullosporin during the course of fermentation. Both proteins showed a cross-reaction with antibodies against a core fragment of nonribosomal peptide synthetases (NRPSs). Biochemical characterization of the partially purified enzymes exhibited selectivity for the substrate amino acid alpha-aminoisobutyric acid (Aib). substantiating their involvement in Ampullosporin biosynthesis. Our data suggest that Ampullosporin synthetase has been isolated, and provides the basis for the characterization of the entire biosynthetic gene cluster. Furthermore, this knowledge will enable the manipulation of its NRPS template, in order to engineer mutant strains of Sepedonium ampullosporum which could produce more potent analogues of Ampullosporin.     

New Trichobrachins, 11-residue peptaibols from a marine strain of Trichoderma longibrachiatum

A marine strain of Trichoderma longibrachiatum isolated from blue mussels (Mytilus edulis) was investigated for short peptaibol production. Various 11-residue peptaibols, obtained as microheterogenous mixtures after a chromatographic fractionation, were identified by positive mass spectrometry fragmentation (ESI-IT-MS(n), CID-MS(n) and GC/EI-MS). Thirty sequences were identified, which is the largest number of analogous sequences so far observed at once. Twenty-one sequences were new, and nine others corresponded to peptaibols already described. These peptaibols belonged to the same peptidic family based on the model Ac-Aib-xxx-xxx-xxx-Aib-Pro-xxx-xxx-Aib-Pro-xxol. They were named trichobrachin A when the residue in position 2 was an Asn, and trichobrachin C when it was a Gln. Major chromatographic sub-fractions, corresponding to purified peptaibols, were assayed for their cytotoxic activity. Trichobrachin A-IX and trichobrachin C exhibited the highest activities. There was an exponential relation between their relative hydrophobicity and their cytotoxicity on KB cells.     

Total synthesis of the natural,medium-length,peptaibol pentadecaibin and study of the chemical features responsible for its membrane activity

Peptaibols are naturally occurring, antimicrobial peptides endowed with well-defined helical conformations and resistance to proteolysis. Both features stem from the presence in their sequence of several, C^α-tetrasubstituted, α-aminoisobutyric acid (Aib) residues. Peptaibols interact with biological membranes, usually causing their leakage. All of the peptaibol–membrane interaction mechanisms proposed so far begin with peptide aggregation or accumulation. The long-length alamethicin, the most studied peptaibol, acts by forming pores in the membranes. Conversely, the carpet mechanism has been claimed for short-length peptaibols, such as trichogin. The mechanism of medium-length peptaibols is far less studied, and this is partly due to the difficulties of their synthesis. They are believed to perturb membrane permeability in different ways, depending on the membrane properties. The present work focuses on pentadecaibin, a recently discovered, medium-length peptaibol. In contrast to the majority of its family members, its sequence does not comprise hydroxyprolines or prolines, and its helix is not kinked. A reliable and effective synthesis procedure is described that allowed us to produce also two shorter analogs. By a combination of techniques, we were able to establish a 3D-structure–activity relationship. In particular, the membrane activity of pentadecaibin heavily depends on the presence of three consecutive Aib residues that are responsible for the clear, albeit modest, amphiphilic character of its helix. The shortest analog, devoid of two of these three Aib residues, preserves a well-defined helical conformation, but not its amphipathicity, and loses almost completely the ability to cause membrane leakage. We conclude that pentadecaibin amphiphilicity is probably needed for the peptide ability to perturb model membranes.     

木霉菌抗菌活肽peptaibols合成调控机制研究进展

木霉菌Trichoderma spp.是广泛存在于土壤环境的丝状真菌,能够产生丰富的次生代谢物,具有抑制病原菌和促植物生长等功效,在农业和医药领域有广泛应用。Peptaibols是一类由非核糖体肽合成酶（non-ribosomal peptide synthetase,NRPS）合成的富含α-氨基异丁酸（Aib）的线性、具抗菌活性及长度不等的多肽。本文基于国内外关于木霉菌产生peptaibols的研究发展现状,重点介绍了peptaibols生物合成酶NRPSs基因簇,合成途径和调控模式,提出未来peptaibols类抗菌肽研究关注的焦点,有利于深入挖掘更具生物医药价值的抗菌肽产物。     

Peptaibol production by sepedonium strains parasitizing boletales

Fungi of the genus Sepedonium (anamorphic ascomycetes) are known to infect fruiting bodies of Basidiomycetes of the order Boletales. We have characterized twelve Sepedonium isolates by intact-cell mass spectrometry (IC-MS) with the help of respective biomarkers and their metabolite spectra focusing on peptaibol production. A strain of mycoparasitic S. chalcipori was grown in solid-state fermentation, and tylopeptin production was found, suggesting an ascomycete origin of these peptaibols, which were first described in the basidiomycete Tylopilus neofelleus. In addition, the structures of two new peptaibols, chalciporin A (=Ac-Trp-Val-Aib-Val-Ala-Gln-Ala-Aib-Ser-Leu-Ala-Leu-Aib-Gln-Leuol) and chalciporin B (=Ac-Trp-Val-Aib-Val-Ala-Gln-Ala-Aib-Gln-Aib-Ala-Leu-Aib-Gln-Leuol) are presented. The IC-MS technique was applied for in situ peptaibol analysis of Sepedonium strains growing on Boletales, in particular S. chrysospermum infecting Xerocomus cf. badius. We found chrysospermins at the surface and within basidiomycete tissue, as well as in the cultivated parasite.     

Formation of atroviridin by Hypocrea atroviridis is conidiation associated and positively regulated by blue light and the G protein GNA3

Species of the mycoparasitic fungal genus Hypocrea/Trichoderma are prominent producers of peptaibols, a class of small linear peptides of fungal origin. Some of these peptaibols have been shown to act synergistically with cell-wall-degrading enzymes in the inhibition of the growth of other fungi in vitro and in vivo. Here we present the structure of the Hypocrea atroviridis peptaibol synthetase gene (pbs1), deduced from the genome sequence of H. atroviridis. It consists of 19 typical peptide synthetase modules with the required additional modifying domains at the N and C termini. Phylogenetic and similarity analyses of the individual amino acid-activating modules is consistent with its ability to synthesize atroviridins. Matrix-assisted laser desorption ionization-time of flight mass spectrometry of surface-grown cultures of H. atroviridis showed that no peptaibols were formed during vegetative growth, but a microheterogenous mixture of atroviridins accumulated when the colonies started to sporulate. This correlation between sporulation and atroviridin formation was shown to be independent of the pathway inducing sporulation (i.e., light, mechanical injury and carbon starvation, respectively). Atroviridin formation was dependent on the function of the two blue light regulators, BLR1 and BLR2, under some but not all conditions of sporulation and was repressed in a pkr1 (regulatory subunit of protein kinase A) antisense strain with constitutively active protein kinase A. Conversely, however, loss of function of the Galpha-protein GNA3, which is a negative regulator of sporulation and leads to a hypersporulating phenotype, fully impairs atroviridin formation. Our data show that formation of atroviridin by H. atroviridis occurs in a sporulation-associated manner but is uncoupled from it at the stage of GNA3.     

Cereulide, the emetic toxin of Bacillus cereus, is putatively a product of nonribosomal peptide synthesis

AIMS: To determine if cereulide, the emetic toxin produced by Bacillus cereus, is produced by a nonribosomal peptide synthetase (NRPS). METHODS AND RESULTS: NC Y, an emetic strain of Bacillus cereus, was examined for a NRPS gene using PCR with primers recognizing a fragment of a NRPS gene from the cyanobacterium Microcystis. The amplicon was sequenced and compared with other gene sequences using BLAST analysis, which showed that the amplicon from strain NC Y was similar in sequence to peptide synthetase genes in other micro-organisms, including Bacillus subtilis and B. brevis, while no such sequence was found in the complete genome sequence of a nonemetic strain of B. cereus. Specific PCR primers were then designed and used to screen 40 B. cereus isolates previously implicated in outbreaks of foodborne illness. The isolates were also screened for toxin production using the MTT cell cytotoxicity assay. PCR and MTT assay screening of the B. cereus isolates revealed a high correlation between the presence of the NRPS gene and cereulide production. CONCLUSIONS: The results indicate that cereulide is produced by a NRPS complex. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide evidence identifying the mechanism of production of cereulide, the emetic toxin of B. cereus. The PCR primers developed in the study allow determination of the potential for cereulide production among isolates of B. cereus.     

Molecular dynamics of zervamicin II and its analogs in water and methanol

A comparative study has been made of the molecular dynamics of zervamicin II (an antimicrobial peptide of the peptaibol group, which has channel-forming activity) in water and methanol. The influence of amino acid substitutions on the dynamics and stability of the peptide structure has been investigated. The amino acid sequence responsible for the absence of swivel motions in short peptaibols has been determined.     

海南东寨港真红树植物内生放线菌多样性及其抗菌活性

【目的】勘探海南东寨港真红树植物内生放线菌多样性,为发现放线菌新物种和新抗生素奠定基础。【方法】样品经表面消毒后粉碎,用10种不同培养基分离放线菌;通过PCR扩增、测定并比对16S r RNA基因序列,开展放线菌多样性分析;通过发酵、萃取等处理方法得到四类样品,包括发酵原液、乙酸乙酯提取液及水层和菌体的丙酮浸泡提取液;采用纸片扩散法对样品进行抗菌活性筛选;基于PCR的基因筛选技术探测活性菌株可能存在的NRPS、PKS I、PKS II抗生素生物合成基因。【结果】经形态特征排重,从14种真红树植物样品中共得到放线菌146株,16S r RNA基因序列比对表明它们分布于13个科18个属,其中链霉菌属为优势菌属,菌株S3Cf-2和S3Af-1的16S r RNA基因序列分别与有效发表菌株Couchioplanes caeruleus DSM44103T(X93202)和Microlunatus terrae BS6T(JF806519)的相似率最高,分别为97.45%和97.43%,可能为新物种。对其中46株放线菌发酵样品的抗菌活性检测表明,40株具有抗菌活性,总阳性率为86.96%;活性菌株中,38株菌存在至少一种所探测的生物合成基因簇,阳性率为95%,其中14株同时具有所探测的3种抗生素生物合成基因簇。【结论】海南东寨港真红树植物中存在多样性丰富的药用放线菌资源,具有从中发现放线菌新物种及新抗生素的潜力。     

NMR evidence for syn-anti interconversion of a trans opened (10R)-dA adduct of benzo[a]pyrene (7S,8R)-diol (9R,10S)-epoxide in a DNA duplex

2D NMR has been used to examine the structure and dynamics of a 12-mer DNA duplex, d(T(1)A(2)G(3)T(4)C(5)A(6)A(7)G(8)G(9)G(10)C(11)A(12))-d(T(13)G(14)C( 15)C(16)C(17)T(18)T(19)G(20)A(21)C(22)T(23)A(24)), containing a 10R adduct at dA(7) that corresponds to trans addition of the N(6)-amino group of dA(7) to (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-(S,R,R,S)-BP DE-2]. This DNA duplex contains the base sequence for the major dA mutational hot spot in the HPRT gene when Chinese hamster V79 cells are given low doses of the highly carcinogenic (+)-(R,S,S,R)-BP DE-2 enantiomer. NOE data indicate that the hydrocarbon is intercalated on the 5'-side of the modified base as has been seen previously for other oligonucleotides containing BP DE-2 (10R)-dA adducts. 2D chemical exchange-only experiments indicate dynamic behavior near the intercalation site especially at the 10R adducted dA, such that this base interconverts between the normal anti conformation and a less populated syn conformation. Ab initio molecular orbital chemical shift calculations of nucleotide and dinucleotide fragments in the syn and anti conformations support these conclusions. Although this DNA duplex containing a 10R dA adduct exhibits conformational flexibility as described, it is nevertheless more conformationally stable than the corresponding 10S adducted duplex corresponding to trans opening of the carcinogenic isomer (+)-(R,S,S, R)-BP DE-2, which was too dynamic to permit NMR structure determination. UV and imino proton NMR spectral observations indicated pronounced differences between these two diastereomeric 12-mer duplexes, consistent with conformational disorder at the adduct site and/or an equilibrium with a nonintercalated orientation of the hydrocarbon in the duplex containing the 10S adduct. The existence of conformational flexibility around adducts may be related to the occurrence of multiple mutagenic outcomes resulting from a single DE adduct.     

Yeast NSR1 protein that has structural similarity to mammalian nucleolin is involved in pre-rRNA processing.

We have identified a yeast gene encoding a protein structurally similar to mammalian nucleolin. The gene was previously cloned as a cold shock-inducible gene and found to be identical to yeast NSR1 gene, which encodes a protein that has been reported to bind sequences required for nuclear localization of protein. The carboxyl-terminal half of NSR1, consisting of two tandemly repeated putative RNA-binding domains and a glycine/arginine-rich domain, has 37% amino acid sequence identity with the same part of mammalian nucleolin, while no sequence similarities are found between their amino-terminal regions. Although a null mutation of the NSR1 gene was not lethal, it caused a severe defect on growth. Pulse-labeling analysis revealed that the nsr1 strain had reduced levels of 18 S rRNA and accumulated 35 S pre-rRNA compared with the wild-type strain. The level of 25 S rRNA was also slightly reduced in the nsr1 strain. Pulse-chase labeling experiments showed slow processing of 35 S pre-rRNA and impaired methylation of 18 S rRNA. The ratio of 40 S to 60 S ribosomal subunits in the nsr1 strain is significantly reduced and is consistent with impaired synthesis of 18 S rRNA. The results indicate that NSR1 is involved in pre-rRNA processing and ribosome biosynthesis in yeast.