Identification of novel proteins binding the AU-rich element of \u3b1-prothymosin mRNA through the selection of open reading frames (RIDome)

We describe here a platform for high-throughput protein expression and interaction analysis aimed at identifying the RNA-interacting domainome. This approach combines the selection of a phage library displaying "filtered" open reading frames with next-generation DNA sequencing. The method was validated using an RNA bait corresponding to the AU-rich element of \u3b1-prothymosin, an RNA motif that promotes mRNA stability and translation through its interaction with the RNA-binding protein ELAVL1. With this strategy, we not only confirmed known RNA-binding proteins that specifically interact with the target RNA (such as ELAVL1/HuR and RBM38) but also identified proteins not previously known to be ARE-binding (R3HDM2 and RALY). We propose this technology as a novel approach for studying the RNA-binding proteome

Identification of tumor-associated cassette exons in human cancer through EST-based computational prediction and experimental validation

Background: Many evidences report that alternative splicing, the mechanism which produces mRNAs and proteins with different structures and functions from the same gene, is altered in cancer cells. Thus, the identification and characterization of cancer-specific splice variants may give large impulse to the discovery of novel diagnostic and prognostic tumour biomarkers, as well as of new targets for more selective and effective therapies. Results: We present here a genome-wide analysis of the alternative splicing pattern of human genes through a computational analysis of normal and cancer-specific ESTs from seventeen anatomical groups, using data available in AspicDB, a database resource for the analysis of alternative splicing in human. By using a statistical methodology, normal and cancer-specific genes, splice sites and cassette exons were predicted in silico. The condition association of some of the novel normal/tumoral cassette exons was experimentally verified by RT-qPCR assays in the same anatomical system where they were predicted. Remarkably, the presence in vivo of the predicted alternative transcripts, specific for the nervous system, was confirmed in patients affected by glioblastoma. Conclusion: This study presents a novel computational methodology for the identification of tumor-associated transcript variants to be used as cancer molecular biomarkers, provides its experimental validation, and reports specific biomarkers for glioblastoma

Dissecting the transcriptional phenotype of ribosomal protein deficiency: implications for Diamond-Blackfan Anemia

Defects in genes encoding ribosomal proteins cause Diamond Blackfan Anemia (DBA), a red cell aplasia often associated with physical abnormalities. Other bone marrow failure syndromes have been attributed to defects in ribosomal components but the link between erythropoiesis and the ribosome remains to be fully defined. Several lines of evidence suggest that defects in ribosome synthesis lead to "ribosomal stress" with p53 activation and either cell cycle arrest or induction of apoptosis. Pathways independent of p53 have also been proposed to play a role in DBA pathogenesis. We took an unbiased approach to identify p53-independent pathways activated by defects in ribosome synthesis by analyzing global gene expression in various cellular models of DBA. Ranking-Principal Component Analysis (Ranking-PCA) was applied to the identified datasets to determine whether there are common sets of genes whose expression is altered in these different cellular models. We observed consistent changes in the expression of genes involved in cellular amino acid metabolic process, negative regulation of cell proliferation and cell redox homeostasis. These data indicate that cells respond to defects in ribosome synthesis by changing the level of expression of a limited subset of genes involved in critical cellular processes. Moreover, our data support a role for p53-independent pathways in the pathophysiology of DBA

Primer sets for cloning the human repertoire of T cell Receptor variable regions

BACKGROUND: Amplification and cloning of na\uefve T cell Receptor (TR) repertoires or antigen-specific TR is crucial to shape immune response and to develop immuno-based therapies. TR variable (V) regions are encoded by several genes that recombine during T cell development. The cloning of expressed genes as large diverse libraries from natural sources relies upon the availability of primers able to amplify as many V genes as possible. RESULTS: Here, we present a list of primers computationally designed on all functional TR V and J genes listed in the IMGT, the ImMunoGeneTics information system. The list consists of unambiguous or degenerate primers suitable to theoretically amplify and clone the entire TR repertoire. We show that it is possible to selectively amplify and clone expressed TR V genes in one single RT-PCR step and from as little as 1000 cells. CONCLUSION: This new primer set will facilitate the creation of more diverse TR libraries than has been possible using currently available primer set

The Ribosomal Basis of Diamond-Blackfan Anemia: Mutation and Database Update

International audienceDiamond-Blackfan Anemia (DBA) is characterized by a defect in erythroid progenitor differentation and, clinically, by anemia and malformations. DBA exhibits an autosomal dominant pattern of inheritance with incomplete penetrance. Currently nine genes, all encoding ribosomal proteins (RP), have been found mutated in approximately 50% of patients. Experimental evidence supports the hypothesis that DBA is primarily the result of defective ribosome synthesis. By means of a large collaboration among six centers, we report here a mutation update that includes nine genes and 220 distinct mutations, 56 of which are new. The DBA Mutation Database now includes data from 355 patients. Of those where inheritance has been examined, 125 patients carry a de novo mutation and 72 an inherited mutation. Mutagenesis may be ascribed to slippage in 65.5% of indels, whereas CpG dinucleotides are involved in 23% of transitions. Using bioinformatic tools we show that gene conversion mechanism is not common in RP genes mutagenesis, notwithstanding the abundance of RP pseudogenes. Genotype-phenotype analysis reveals that malformations are more frequently associated with mutations in RPL5 and RPL11 than in the other genes. All currently reported DBA mutations together with their functional and clinical data are included in the DBA Mutation Database