Ribosomal Protein Complex Research Articles

Curcumin interferes with sepsis-induced cardiomyocyte apoptosis via TLR1 inhibition

ObjectiveSepsis-induced cardiomyopathy is the leading cause of death in sepsis and is characterized by reversible myocardial depression. However, the specific mechanisms responsible for myocardial injury in sepsis are not known. The present study used bioinformatic analysis to explore the possible mechanisms of sepsis-induced myocardial injury and the therapeutic potential of curcumin. MethodsThe GSE125042 microarray gene expression matrix was obtained from the Gene Expression Omnibus database, which includes 10 septic cardiomyocyte samples from cecum ligation perforation constructs and 10 sham-operated groups cardiomyocyte samples. Background correction and matrix data normalization were performed using the robust multiarray average algorithm. Differentially expressed genes (DEGs) screening was performed using the Limma R package expression matrix, and whole gene analysis was performed using the weighted gene co-expression network analysis R package to construct gene networks and identify modules. Enrichment analysis and gene set enrichment analysis was performed on the genes to be selected. Construct cellular and animal models of myocardial injury in sepsis were assessed and the effects of curcumin on a rat or cardiac myocytes were observed. ResultsA total of 2876 DEGs were screened based on the GSE125042 chip, of which 1424 genes were upregulated and 1452 genes were down regulated. WGCNA analysis of the whole genes was also performed and a total of 20 gene modules were generated. Among them, the selected TLR1 gene was present in the most strongly correlated Brown module. Enrichment analysis of the upregulated DEGs with the Brown module showed that they were significantly enriched in biological processes related to ribosomal protein complex generation, cellular components related to phagocytic vesicles and molecular functions related to Toll-like receptor binding, affecting cardiomyocyte survival as a target for molecular intervention in septic cardiomyopathy. Animal experiments showed that curcumin reduced inflammation levels, improved cardiac function and increased survival in rats with septic myocardial injury. Cellular experiments showed that curcumin increased the survival rate of lipopolysaccharide-treated cardiomyocytes and down regulated TLR1 expression and inhibited NF-κB phosphorylation in cells in a dose-dependent manner. Molecular docking analysis revealed that curcumin interacted with TLR1 by hydrogen bonding and could be stably bound to inhibit the biological function of TLR1. ConclusionOur study shows that curcumin attenuates myocardial injury in sepsis by inhibiting TLR1 expression, which provides a molecular theoretical basis for clinical treatment.

Mimicked synthetic ribosomal protein complex for benchmarking crosslinking mass spectrometry workflows

Cross-linking mass spectrometry has matured to a frequently used tool for the investigation of protein structures as well as interactome studies up to a system-wide level. The growing community generated a broad spectrum of applications, linker types, acquisition strategies and specialized data analysis tools, which makes it challenging to decide for an appropriate analysis workflow. Here, we report a large and flexible synthetic peptide library as reliable instrument to benchmark crosslink workflows. Additionally, we provide a tool, IMP-X-FDR, that calculates the real, experimentally validated, FDR, compares results across search engine platforms and analyses crosslink properties in an automated manner. We apply the library with 6 commonly used linker reagents and analyse the data with 6 established search engines. We thereby show that the correct algorithm and search setting choice is highly important to improve identification rate and reliability. We reach identification rates of up to ~70 % of the theoretical maximum (i.e. 700 unique lysine-lysine cross-links) while maintaining a real false-discovery-rate of <3 % at cross-link level with high reproducibility, representatively showing that our test system delivers valuable and statistically solid results.

Using the Knowledge of Post-transcriptional Regulations to Guide Gene Selections for Molecular Breeding in Soybean.

The omics approaches allow the scientific community to successfully identify genomic regions associated with traits of interest for marker-assisted breeding. Agronomic traits such as seed color, yield, growth habit, and stress tolerance have been the targets for soybean molecular breeding. Genes governing these traits often undergo post-transcriptional modifications, which should be taken into consideration when choosing elite genes for molecular breeding. Post-transcriptional regulations of genes include transcript regulations, protein modifications, and even the regulation of the translational machinery. Transcript regulations involve elements such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) for the maintenance of transcript stability or regulation of translation efficiency. Protein modifications involve molecular modifications of target proteins and the alterations of their interacting partners. Regulations of the translational machinery include those on translation factors and the ribosomal protein complex. Post-transcriptional regulations usually involve a set of genes instead of a single gene. Such a property may facilitate molecular breeding. In this review, we will discuss the post-transcriptional modifications of genes related to favorable agronomic traits such as stress tolerance, growth, and nutrient uptake, using examples from soybean as well as other crops. The examples from other crops may guide the selection of genes for marker-assisted breeding in soybean.

Direct visualization of translational GTPase factor pool formed around the archaeal ribosomal P-stalk by high-speed AFM

In translation elongation, two translational guanosine triphosphatase (trGTPase) factors EF1A and EF2 alternately bind to the ribosome and promote polypeptide elongation. The ribosomal stalk is a multimeric ribosomal protein complex which plays an essential role in the recruitment of EF1A and EF2 to the ribosome and their GTP hydrolysis for efficient and accurate translation elongation. However, due to the flexible nature of the ribosomal stalk, its structural dynamics and mechanism of action remain unclear. Here, we applied high-speed atomic force microscopy (HS-AFM) to directly visualize the action of the archaeal ribosomal heptameric stalk complex, aP0•(aP1•aP1)3 (P-stalk). HS-AFM movies clearly demonstrated the wobbling motion of the P-stalk on the large ribosomal subunit where the stalk base adopted two conformational states, a predicted canonical state, and a newly identified flipped state. Moreover, we showed that up to seven molecules of archaeal EF1A (aEF1A) and archaeal EF2 (aEF2) assembled around the ribosomal P-stalk, corresponding to the copy number of the common C-terminal factor-binding site of the P-stalk. These results provide visual evidence for the factor-pooling mechanism by the P-stalk within the ribosome and reveal that the ribosomal P-stalk promotes translation elongation by increasing the local concentration of translational GTPase factors.

Uniting Native Capillary Electrophoresis and Multistage Ultraviolet Photodissociation Mass Spectrometry for Online Separation and Characterization of Escherichia coli Ribosomal Proteins and Protein Complexes.

With an overarching goal of characterizing the structure of every protein within a cell, identifying its interacting partners, and quantifying the dynamics of the states in which it exists, key developments are still necessary to achieve comprehensive native proteomics by mass spectrometry (MS). In practice, much work remains to optimize reliable online separation methods that are compatible with native MS and improve tandem MS (MS/MS) approaches with respect to when and how energy is deposited into proteins of interest. Herein, we utilize native capillary zone electrophoresis coupled with MS to characterize the proteoforms in the Escherichia coli 70S ribosome. The capabilities of 193 nm ultraviolet photodissociation (UVPD) to yield informative backbone sequence ions are compared to those of higher-energy collisional dissociation (HCD). To further improve sequence coverage values, a multistage MS/MS approach is implemented involving front-end collisional activation to disassemble protein complexes into constituent subunits that are subsequently individually isolated and activated by HCD or UVPD. In total, 48 of the 55 known E. coli ribosomal proteins are identified as 84 unique proteoforms, including 22 protein-metal complexes and 10 protein-protein complexes. Additionally, mapping metal-bound holo fragment ions resulting from UVPD of protein-metal complexes offers insight into the metal-binding sites.

Quantitative proteomic analysis of trypsin-treated extracellular vesicles to identify the real-vesicular proteins

ABSTRACT Extracellular vesicles (EVs) are nano-sized vesicles surrounded by a lipid bilayer and released into the extracellular milieu by most of cells. Although various EV isolation methods have been established, most of the current methods isolate EVs with contaminated non-vesicular proteins. By applying the label-free quantitative proteomic analyses of human colon cancer cell SW480-derived EVs, we identified trypsin-sensitive and trypsin-resistant vesicular proteins. Further systems biology and protein–protein interaction network analyses based on their cellular localization, we classified the trypsin-sensitive and trypsin-resistant vesicular proteins into two subgroups: 363 candidate real-vesicular proteins and 151 contaminated non-vesicular proteins. Moreover, the protein interaction network analyses showed that candidate real-vesicular proteins are mainly derived from plasma membrane (46.8%), cytosol (36.6%), cytoskeleton (8.0%) and extracellular region (2.5%). On the other hand, most of the contaminated non-vesicular proteins derived from nucleus, Golgi apparatus, endoplasmic reticulum and mitochondria. In addition, ribosomal protein complexes and T-complex proteins were classified as the contaminated non-vesicular proteins. Taken together, our trypsin-digested proteomic approach on EVs is an important advance to identify the real-vesicular proteins that could help to understand EV biogenesis and protein cargo-sorting mechanism during EV release, to identify more reliable EV diagnostic marker proteins, and to decode pathophysiological roles of EVs.

Gustavo Silva: Translating the ubiquitin code.

Silva investigates how ribosomal protein complexes are regulated by K63 ubiquitination.

Molecular basis for disassembly of an importin:ribosomal protein complex by the escortin Tsr2

Disordered extensions at the termini and short internal insertions distinguish eukaryotic ribosomal proteins (r-proteins) from their anucleated archaeal counterparts. Here, we report an NMR structure of such a eukaryotic-specific segment (ESS) in the r-protein eS26 in complex with the escortin Tsr2. The structure reveals how ESS attracts Tsr2 specifically to importin:eS26 complexes entering the nucleus in order to trigger non-canonical RanGTP-independent disassembly. Tsr2 then sequesters the released eS26 and prevents rebinding to the importin, providing an alternative allosteric mechanism to terminate the process of nuclear import. Notably, a Diamond–Blackfan anemia-associated Tsr2 mutant protein is impaired in binding to ESS, unveiling a critical role for this interaction in human hematopoiesis. We propose that eS26-ESS and Tsr2 are components of a nuclear sorting system that co-evolved with the emergence of the nucleocytoplasmic barrier and transport carriers.

Expression of ribosomal proteins in normal and cancerous human prostate tissue.

Few quantifiable tissue biomarkers for the diagnosis and prognosis of prostate cancer exist. Using an unbiased, quantitative approach, this study evaluates the potential of three proteins of the 40S ribosomal protein complex as putative biomarkers of malignancy in prostate cancer. Prostate tissue arrays, constructed from 82 patient samples (245 tissue cores, stage pT3a or pT3b), were stained for antibodies against three ribosomal proteins, RPS19, RPS21 and RPS24. Semi-automated Ox-DAB signal quantification using ImageJ software revealed a significant change in expression of RPS19, RPS21 and RPS24 in malignant vs non-malignant tissue (p<0.0001). Receiver operating characteristics curves were calculated to evaluate the potential of each protein as a biomarker of malignancy in prostate cancer. Positive likelihood ratios for RPS19, RPS21 and RPS24 were calculated as 2.99, 4.21, and 2.56 respectively, indicating that the overexpression of the protein is correlated with the presence of disease. Triple-labelled, quantitative, immunofluorescence (with RPS19, RPS21 and RPS24) showed significant changes (p<0.01) in the global intersection coefficient, a measure of how often two fluorophore signals intersect, for RPS19 and RPS24 only. No change was observed in the co-localization of any other permutations of the three proteins. Our results show that RPS19, RPS21 or RPS24 are upregulated in malignant tissue and may serve as putative biomarkers for prostate cancer.

Regulatory sequences of the Arabidopsis thaliana Rps19, a nuclear gene encoding mitochondrial ribosomal protein subunit, extend into the upstream gene

Genes encoding subunits of the mitochondrial ribosomal protein complex are distributed between the mitochondrial and nuclear genomes. In Arabidopsis thaliana only seven out of the nearly 70 genes coding for mitochondrial ribosomal subunit proteins are present in the mitochondrial genome. Nevertheless, these genes are co-ordinately expressed. Here, we present the first report of characterization of promoter of a plant mitochondrial ribosomal protein gene AtRps19 (At5g47320), a single copy nuclear gene in A. thaliana. Analysis of transgenic A. thaliana plants carrying seven different AtRps19 upstream fragments linked to the uidA reporter gene revealed that the 879 bp fragment containing the 5′ UTR and the intergenic region is capable of driving gene expression in most of the vegetative tissues. However, inclusion of 447 bp of the upstream gene sequences was essential for obtaining full expression in all tissues including anthers and pollen. Thus, we provide the first experimental proof of overlapping genes in plants. qRT-PCR analysis showed that uidA and native AtRps19 transcript levels were comparable in plants carrying the D4 (−676/+650) construct, indicating that the 1326 bp D4 fragment represents the native promoter of the AtRps19 gene. Comparison of AtRps19 promoter with the widely used CaMV35S promoter showed that AtRps19 promoter is capable of driving gene expression in all tissues including anthers and pollen but was less efficient than the CaMV35S promoter.

The ubiquitin hybrid gene UBA52 regulates ubiquitination of ribosome and sustains embryonic development.

Ubiquitination is a crucial post-translational modification; however, the functions of ubiquitin-coding genes remain unclear. UBA52 encodes a fusion protein comprising ubiquitin at the N-terminus and ribosomal protein L40 (RPL40) at the C-terminus. Here we showed that Uba52-deficient mice die during embryogenesis. UBA52-deficient cells exhibited normal levels of total ubiquitin. However, UBA52-deficient cells displayed decreased protein synthesis and cell-cycle arrest. The overexpression of UBA52 ameliorated the cell-cycle arrest caused by UBA52 deficiency. Surprisingly, RPL40 expression itself is insufficient to regulate cyclin D expression. The cleavage of RPL40 from UBA52 was required for maintaining protein synthesis. Furthermore, we found that RPL40 formed a ribosomal complex with ubiquitin cleaved from UBA52. UBA52 supplies RPL40 and ubiquitin simultaneously to the ribosome. Our study demonstrated that the ubiquitin-coding gene UBA52 is not just an ubiquitin supplier to the ubiquitin pool but is also a regulator of the ribosomal protein complex. These findings provide novel insights into the regulation of ubiquitin-dependent translation and embryonic development.

Postmortem interval determination using 18S-rRNA and microRNA.

The importance of determining postmortem interval (PMI) is crucial to criminal, civil and forensic cases. The precise estimation of PMI is a critical step in many death investigations. A technique exploiting the level of RNA, 18S rRNA and microRNA to estimate PMI was investigated. 18S-rRNA is a main ribosomal RNA presented as part of the ribosomal protein complex, while microRNA is a class of small non-coding single-stranded RNA, only 21-25 nucleotides, which has a strong conservation between different species. In this study, heart tissues were removed from adult rats at various postmortem intervals. An efficient extraction and detection protocol to analyze the level of 18S-rRNA and microRNA in postmortem tissue was carried out. The process consists of total RNA extraction, transcription and visualization by quantitative real time PCR. The result indicates a characteristic parabola relationship between postmortem period and Ct values for 18S-rRNA in dead rat hearts. The result indicates that the degradation pattern of tissue 18S-rRNA and microRNA is useful in the determination of the postmortem interval within seven days.

Abstract B230: Targeting mitochondria for metastatic lung adenocarcinoma specific lethality.

Abstract Introduction: Lung cancer is the leading cause of cancer deaths in the United States. Its ability to leave the primary tumor and establish inoperable metastasis impairs successful therapy and is a major contributor to its lethality. A genetically engineered mouse model recapitulates the human disease very well, incorporating conditional alleles of the Kras oncogene and p53 tumor suppressor. Recently, cell lines generated from non-metastatic primary lung tumors (TnonMet) and metastases (Met) in this model were shown to maintain their metastatic state and exhibit dramatic differences in gene expression programs. This unique set of cell lines from early stage non-metastatic tumors and late-stage metastases provides an opportunity to understand whether cancer progression also creates unique vulnerabilities. Experimental design: A pooled lentiviral-shRNA library screen which targeted ∼20,000 genes with &amp;gt;92,000 shRNA in two TnonMet and two Met cell lines was used to systematically uncover genes that are specifically required to sustain metastasis survival or growth. High-throughput sequencing of the shRNA in the cancer cell populations before and after 20 population doublings in culture identified shRNAs that disadvantaged each cell line. Gene-set enrichment algorithm was utilized to analyse the data. qRT-PCR, cell competition assays and mitochondria-specific dyes were used to verify the results and unravel the underlying mechanism. Results and Conclusion: The analysis of our pooled shRNA screen revealed mitochondrial ribosomal protein (Mrp) complexes as the top two gene-sets; in fact individual shRNA targeting more than 15 Mrp genes selectively disadvantaging Met cells with minimal impact on TnonMet cells. These results were individually verified with qRT-PCR. Interestingly, Met cells showed increased expression of mitochondria encoded genes, pointing to a strong imbalance in mitochondrial homeostasis. Furthermore, mitochondria-targeted therapy blocking mitochondrial translation or replication significantly inhibited Met cell growth more than TnonMet cell growth. Taken together, these results point to mitochondria as a metastasis-specific therapeutic target in lung adenocarcinoma and need to be further investigated. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B230. Citation Format: Barbara M. Gruener, Chen-Hua Chuang, Ian Linde, Shin-Heng Chiou, Ben Readhead, Joel Dudley, John Doench, David Root, Monte M. Winslow. Targeting mitochondria for metastatic lung adenocarcinoma specific lethality. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B230.

Comprehensive Cell-specific Protein Analysis in Early and Late Pollen Development from Diploid Microsporocytes to Pollen Tube Growth

Pollen development in angiosperms is one of the most important processes controlling plant reproduction and thus productivity. At the same time, pollen development is highly sensitive to environmental fluctuations, including temperature, drought, and nutrition. Therefore, pollen biology is a major focus in applied studies and breeding approaches for improving plant productivity in a globally changing climate. The most accessible developmental stages of pollen are the mature pollen and the pollen tubes, and these are thus most frequently analyzed. To reveal a complete quantitative proteome map, we additionally addressed the very early stages, analyzing eight stages of tobacco pollen development: diploid microsporocytes, meiosis, tetrads, microspores, polarized microspores, bipolar pollen, desiccated pollen, and pollen tubes. A protocol for the isolation of the early stages was established. Proteins were extracted and analyzed by means of a new gel LC-MS fractionation protocol. In total, 3817 protein groups were identified. Quantitative analysis was performed based on peptide count. Exceedingly stage-specific differential protein regulation was observed during the conversion from the sporophytic to the gametophytic proteome. A map of highly specialized functionality for the different stages could be revealed from the metabolic activity and pronounced differentiation of proteasomal and ribosomal protein complex composition up to protective mechanisms such as high levels of heat shock proteins in the very early stages of development.

S6:S18 ribosomal protein complex interacts with a structural motif present in its own mRNA

Prokaryotic ribosomal protein genes are typically grouped within highly conserved operons. In many cases, one or more of the encoded proteins not only bind to a specific site in the ribosomal RNA, but also to a motif localized within their own mRNA, and thereby regulate expression of the operon. In this study, we computationally predicted an RNA motif present in many bacterial phyla within the 5' untranslated region of operons encoding ribosomal proteins S6 and S18. We demonstrated that the S6:S18 complex binds to this motif, which we hereafter refer to as the S6:S18 complex-binding motif (S6S18CBM). This motif is a conserved CCG sequence presented in a bulge flanked by a stem and a hairpin structure. A similar structure containing a CCG trinucleotide forms the S6:S18 complex binding site in 16S ribosomal RNA. We have constructed a 3D structural model of a S6:S18 complex with S6S18CBM, which suggests that the CCG trinucleotide in a specific structural context may be specifically recognized by the S18 protein. This prediction was supported by site-directed mutagenesis of both RNA and protein components. These results provide a molecular basis for understanding protein-RNA recognition and suggest that the S6S18CBM is involved in an auto-regulatory mechanism.

Insights into the Catalytic Mechanism of 16S rRNA Methyltransferase RsmE (m3U1498) from Crystal and Solution Structures

RsmE is the founding member of a new RNA methyltransferase (MTase) family responsible for methylation of U1498 in 16S ribosomal RNA in Escherichia coli. It is well conserved across bacteria and plants and may play an important role in ribosomal intersubunit communication. The crystal structure in monomer showed that it consists of two distinct but structurally related domains: the PUA (pseudouridine synthases and archaeosine-specific transglycosylases)-like RNA recognition and binding domain and the conserved MTase domain with a deep trefoil knot. Analysis of small-angle X-ray scattering data revealed that RsmE forms a flexible dimeric conformation that may be essential for substrate binding. The S-adenosyl-l-methionine (AdoMet)-binding characteristic determined by isothermal titration calorimetry suggested that there is only one AdoMet molecule bound in the subunit of the homodimer. In vitro methylation assay of the mutants based on the RsmE-AdoMet-uridylic acid complex model showed key residues involved in substrate binding and catalysis. Comprehensive comparisons of RsmE with closely related MTases, combined with the biochemical experiments, indicated that the MTase domain of one subunit in dimeric RsmE is responsible for binding of one AdoMet molecule and catalytic process while the PUA-like domain in the other subunit is mainly responsible for recognition of one substrate molecule (the ribosomal RNA fragment and ribosomal protein complex). The methylation process is required by collaboration of both subunits, and dimerization is functionally critical for catalysis. In general, our study provides new information on the structure-function relationship of RsmE and thereby suggests a novel catalytic mechanism.

Interaction of Hantavirus Nucleocapsid Protein with Ribosomal Protein S19

Hantaviruses, members of the Bunyaviridae family, are emerging category A pathogens that initiate the translation of their capped mRNAs by a novel mechanism mediated by viral nucleocapsid protein (N). N specifically binds to the mRNA 5' m7G cap and 40S ribosomal subunit, a complex of 18S rRNA and multiple ribosomal proteins. Here, we show that N specifically interacts with the ribosomal protein S19 (RPS19), located at the head region of the 40S subunit. We suggest that this N-RPS19 interaction facilitates ribosome loading on capped mRNAs during N-mediated translation initiation.

Application of micro-reactor chip technique for millisecond quenching of deuterium incorporation into 70S ribosomal protein complex

The hydrogen/deuterium exchange (HDX) method is useful to analyze kinetics of large macromolecular complexes, although its time resolution requires further improvement. A newly developed micro-reactor chip was made of polydimethylsiloxane with a 100-μm deep and wide microchannel. The channel in the chip has two mixing points of Y-shaped flow and allowed us to shorten time durations from the start to quenching for the HDX in 70S ribosome with high temporal resolution. This device enabled us to quench the deuterium incorporation at as early as 20ms, detecting structural changes of individual ribosomal proteins in solution at the time scale comparable to a single reaction cycle for the peptide elongation. The profile of deuterium incorporation in individual proteins of the complex was superimposed on the X-ray crystal structure to depict the surface HDX map, revealing localization of protein movement in the ribosome. The current method serves as a useful method to visualize the regional movement of large macromolecules with high temporal resolution sufficient to examine protein dynamics.

Specific Interactions of the L10(L12)4 Ribosomal Protein Complex with mRNA, rRNA, and L11

Large ribosomal subunit proteins L10 and L12 form a pentameric protein complex, L10(L12) 4, that is intimately involved in the ribosome elongation cycle. Its contacts with rRNA or other ribosomal proteins have been only partially resolved by crystallography. In Escherichia coli, L10 and L12 are encoded from a single operon for which L10(L12) 4 is a translational repressor that recognizes a secondary structure in the mRNA leader. In this study, L10(L12) 4 was expressed from the moderate thermophile Bacillus stearothermophilus to quantitatively compare strategies for binding of the complex to mRNA and ribosome targets. The minimal mRNA recognition structure is widely distributed among bacteria and has the potential to form a kink-turn structure similar to one identified in the rRNA as part of the L10(L12) 4 binding site. Mutations in equivalent positions between the two sequences have similar effects on L10(L12) 4-RNA binding affinity and identify the kink-turn motif and a loop AA sequence as important recognition elements. In contrast to the larger rRNA structure, the mRNA apparently positions the kink-turn motif and loop for protein recognition without the benefit of Mg (2+)-dependent tertiary structure. The mRNA and rRNA fragments bind L10(L12) 4 with similar affinity ( approximately 10 (8) M (-1)), but fluorescence binding studies show that a nearby protein in the ribosome, L11, enhances L10(L12) 4 binding approximately 100-fold. Thus, mRNA and ribosome targets use similar RNA features, held in different structural contexts, to recognize L10(L12) 4, and the ribosome ensures the saturation of its L10(L12) 4 binding site by means of an additional protein-protein interaction.

Construction and characterization of a full-length cDNA library for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici).

BackgroundPuccinia striiformis is a plant pathogenic fungus causing stripe rust, one of the most important diseases on cereal crops and grasses worldwide. However, little is know about its genome and genes involved in the biology and pathogenicity of the pathogen. We initiated the functional genomic research of the fungus by constructing a full-length cDNA and determined functions of the first group of genes by sequence comparison of cDNA clones to genes reported in other fungi.ResultsA full-length cDNA library, consisting of 42,240 clones with an average cDNA insert of 1.9 kb, was constructed using urediniospores of race PST-78 of P. striiformis f. sp. tritici. From 196 sequenced cDNA clones, we determined functions of 73 clones (37.2%). In addition, 36 clones (18.4%) had significant homology to hypothetical proteins, 37 clones (18.9%) had some homology to genes in other fungi, and the remaining 50 clones (25.5%) did not produce any hits. From the 73 clones with functions, we identified 51 different genes encoding protein products that are involved in amino acid metabolism, cell defense, cell cycle, cell signaling, cell structure and growth, energy cycle, lipid and nucleotide metabolism, protein modification, ribosomal protein complex, sugar metabolism, transcription factor, transport metabolism, and virulence/infection.ConclusionThe full-length cDNA library is useful in identifying functional genes of P. striiformis.