CTdatabase: a knowledge-base of high-throughput and curated data on cancer-testis antigens.

The potency of the immune response has still to be harnessed effectively to combat human cancers. However, the discovery of T-cell targets in melanomas and other tumors has raised the possibility that cancer vaccines can be used to induce a therapeutically effective immune response against cancer. The targets, cancer-testis (CT) antigens, are immunogenic proteins preferentially expressed in normal gametogenic tissues and different histological types of tumors. Therapeutic cancer vaccines directed against CT antigens are currently in late-stage clinical trials testing whether they can delay or prevent recurrence of lung cancer and melanoma following surgical removal of primary tumors. CT antigens constitute a large, but ill-defined, family of proteins that exhibit a remarkably restricted expression. Currently, there is a considerable amount of information about these proteins, but the data are scattered through the literature and in several bioinformatic databases. The database presented here, CTdatabase (http://www.cta.lncc.br), unifies this knowledge to facilitate both the mining of the existing deluge of data, and the identification of proteins alleged to be CT antigens, but that do not have their characteristic restricted expression pattern. CTdatabase is more than a repository of CT antigen data, since all the available information was carefully curated and annotated with most data being specifically processed for CT antigens and stored locally. Starting from a compilation of known CT antigens, CTdatabase provides basic information including gene names and aliases, RefSeq accession numbers, genomic location, known splicing variants, gene duplications and additional family members. Gene expression at the mRNA level in normal and tumor tissues has been collated from publicly available data obtained by several different technologies. Manually curated data related to mRNA and protein expression, and antigen-specific immune responses in cancer patients are also available, together with links to PubMed for relevant CT antigen articles.

Hrp59, an hnRNP M protein in Chironomus and Drosophila, binds to exonic splicing enhancers and is required for expression of a subset of mRNAs.

Here, we study an insect hnRNP M protein, referred to as Hrp59. Hrp59 is relatively abundant, has a modular domain organization containing three RNA-binding domains, is dynamically recruited to transcribed genes, and binds to premRNA cotranscriptionally. Using the Balbiani ring system of Chironomus, we show that Hrp59 accompanies the mRNA from the gene to the nuclear envelope, and is released from the mRNA at the nuclear pore. The association of Hrp59 with transcribed genes is not proportional to the amount of synthesized RNA, and in vivo Hrp59 binds preferentially to a subset of mRNAs, including its own mRNA. By coimmunoprecipitation of Hrp59-RNA complexes and microarray hybridization against Drosophila whole-genome arrays, we identify the preferred mRNA targets of Hrp59 in vivo and show that Hrp59 is required for the expression of these target mRNAs. We also show that Hrp59 binds preferentially to exonic splicing enhancers and our results provide new insights into the role of hnRNP M in splicing regulation.

The Hrp65 self-interaction is mediated by an evolutionarily conserved domain and is required for nuclear import of Hrp65 isoforms that lack a nuclear localization signal.

Hrp65, an evolutionary conserved RNA-binding protein from the midge Chironomus tentans, has a conserved DBHS (Drosophila behavior, human splicing) domain that is also present in several mammalian proteins. In a yeast two-hybrid screening we found that Hrp65 can interact with itself. Here we confirm the Hrp65 self-interaction by in vitro pull-down experiments and map the sequences responsible for the interaction to a region that we refer to as the protein-binding domain located within the DBHS domain. We also show that the protein-binding domains of Drosophila NonA and human PSF, two other proteins with conserved DBHS domains, bind to Hrp65 in the yeast two-hybrid system. These observations indicate that the protein-binding domain can mediate homodimerization of Hrp65 as well as heterodimerization between different DBHS-containing proteins. Moreover, analyses of recombinant Hrp65 by gel-filtration chromatography show that Hrp65 can not only dimerize but also oligomerize into complexes of at least three to six molecules. Furthermore, we have analyzed the functional significance of the Hrp65 self-interaction in cotransfection assays, and our results suggest that the interaction between different Hrp65 isoforms is crucial for their intracellular localization.

An improved dual-expression concept, generating high-quality antibodies for proteomics research.

A novel, improved dual bacterial-expression system, designed for large-scale generation of high-quality polyclonal antibody preparations intended for proteomics research, is presented. The concept involves parallel expression of cDNA-encoded proteins, as a fusion with two different tags in two separate vector systems. Both systems enable convenient blotting procedures for expression screening on crude bacterial cell cultures and single-step affinity purification under denaturing conditions. One of the fusion proteins is used to elicit antibodies, and the second fusion protein is used in an immobilized form as an affinity ligand to enrich antibodies with selective reactivity to the cDNA-encoded part, common for the two fusion proteins. To evaluate the system, four cDNA clones from putative nuclear proteins from the non-biting midge Chironomus tentans were expressed. Antibodies to these cDNA-encoded proteins were generated, enriched and used in blotting and immunofluorescence procedures to determine expression patterns for the native proteins corresponding to the cDNAs. The four antibody preparations showed specific reactivity to the corresponding recombinant cDNA-encoded proteins, and three of the four antibodies gave specific staining in Western-blot analysis of nuclear cell extracts. Furthermore, two of the antibody preparations gave specific staining in immunofluorescence analysis of C. tentans cells. We conclude that the dual-vector concept presented offers a highly stringent strategy for the generation of monospecific polyclonal antibodies, which are useful in proteomics research.

HEL/UAP56 binds cotranscriptionally to the Balbiani ring pre-mRNA in an intron-independent manner and accompanies the BR mRNP to the nuclear pore.

The splicing factor UAP56/HEL/Sub2p is essential for mRNA export. It has been proposed that UAP56/HEL/Sub2p interacts with the pre-mRNA during splicing and recruits the export factor Aly/REF/Yra1 (reviewed in ) to the spliced mRNA. However, UAP56/HEL/Sub2p also participates in the transport of intronless mRNAs, and thus its role in export is not necessarily coupled to splicing. Here, we characterize the HEL protein of Chironomus tentans and we analyze in situ the interaction of HEL with a natural export substrate, the Balbiani ring pre-messenger ribonucleoprotein (BR pre-mRNP, reviewed in ). Using immunoelectron microscopy, we show that HEL binds to the BR pre-mRNP cotranscriptionally and that incorporation of HEL into the pre-mRNP is independent of the location of introns along the BR pre-mRNA. We also show that HEL accompanies the BR mRNP to the nuclear pore and is released from the BR mRNP during translocation to the cytoplasm. Aly/REF is also released from the BR mRNP during translocation but after dissociation of HEL. In summary, we have shown that binding of HEL to the BR pre-mRNA occurs independently of splicing, and we have established the point in the export pathway at which HEL and Aly/REF interact with the mRNP.