Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor

BACKGROUND: Epidermal Growth Factor (EGF) plays an important function in the regulation of cell growth, proliferation, and differentiation by binding to its receptor (EGFR) and providing cancer cells with increased survival responsiveness. Signal transduction carried out by EGF has been extensively studied at both transcriptional and post-transcriptional levels. Little is known about the involvement of microRNAs (miRNAs) in the EGF signaling pathway. miRNAs have emerged as major players in the complex networks of gene regulation, and cancer miRNA expression studies have evidenced a direct involvement of miRNAs in cancer progression. RESULTS: In this study, we have used an integrative high content analysis approach to identify the specific miRNAs implicated in EGF signaling in HeLa cells as potential mediators of cancer mediated functions. We have used microarray and deep-sequencing technologies in order to obtain a global view of the EGF miRNA transcriptome with a robust experimental cross-validation. By applying a procedure based on Rankprod tests, we have delimited a solid set of EGF-regulated miRNAs. After validating regulated miRNAs by reverse transcription quantitative PCR, we have derived protein networks and biological functions from the predicted targets of the regulated miRNAs to gain insight into the potential role of miRNAs in EGF-treated cells. In addition, we have analyzed sequence heterogeneity due to editing relative to the reference sequence (isomiRs) among regulated miRNAs. CONCLUSIONS: We propose that the use of global genomic miRNA cross-validation derived from high throughput technologies can be used to generate more reliable datasets inferring more robust networks of co-regulated predicted miRNA target genes

Characterization of alternatively spliced products and tissue-specific isoforms of USP28 and USP25

Background: The ubiquitin-dependent protein degradation pathway is essential for the proteolysis of intracellular proteins and peptides. Deubiquitinating enzymes constitute a complex protein family involved in a multitude of cellular processes. The ubiquitin-specific proteases (UBP) are a group of enzymes whose predicted function is to reverse the ubiquitinating reaction by removing ubiquitin from a large variety of substrates. We have lately reported the characterization of human USP25, a specific-ubiquitin protease gene at 21q11.2, with a specific pattern of expression in murine fetal brains and adult testis. Results: Database homology searches at the DNA and protein levels and cDNA library screenings led to the identification of a new UBP member in the human genome, named USP28, at 11q23. This novel gene showed preferential expression in heart and muscle. Moreover, cDNA, expressed sequence tag and RT-PCR analyses provided evidence for alternatively spliced products and tissue-specific isoforms. Concerning function, USP25 overexpression in Down syndrome fetal brains was shown by real-time PCR. Conclusions: On the basis of the genomic and protein sequence as well as the functional data, USP28 and USP25 establish a new subfamily of deubiquitinating enzymes. Both genes have alternatively spliced exons that could generate protein isoforms with distinct tissue-specific activity. The overexpression of USP25 in Down syndrome fetal brains supports the gene-dosage effects suggested for other UBP members related to aneuploidy syndromes

Characterization of alternatively spliced products and tissue-specific isoforms of USP28 and USP25

Background: The ubiquitin-dependent protein degradation pathway is essential for the proteolysis of intracellular proteins and peptides. Deubiquitinating enzymes constitute a complex protein family involved in a multitude of cellular processes. The ubiquitin-specific proteases (UBP) are a group of enzymes whose predicted function is to reverse the ubiquitinating reaction by removing ubiquitin from a large variety of substrates. We have lately reported the characterization of human USP25, a specific-ubiquitin protease gene at 21q11.2, with a specific pattern of expression in murine fetal brains and adult testis. Results: Database homology searches at the DNA and protein levels and cDNA library screenings led to the identification of a new UBP member in the human genome, named USP28, at 11q23. This novel gene showed preferential expression in heart and muscle. Moreover, cDNA, expressed sequence tag and RT-PCR analyses provided evidence for alternatively spliced products and tissue-specific isoforms. Concerning function, USP25 overexpression in Down syndrome fetal brains was shown by real-time PCR. Conclusions: On the basis of the genomic and protein sequence as well as the functional data, USP28 and USP25 establish a new subfamily of deubiquitinating enzymes. Both genes have alternatively spliced exons that could generate protein isoforms with distinct tissue-specific activity. The overexpression of USP25 in Down syndrome fetal brains supports the gene-dosage effects suggested for other UBP members related to aneuploidy syndromes

Truncating variant burden in high functioning autism and pleiotropic effects of lrp1 across psychiatric phenotypes

Background: Previous research has implicated de novo and inherited truncating mutations in autism-spectrum disorder. We aim to investigate whether the load of inherited truncating mutations contributes similarly to high-functioning autism, and to characterize genes that harbour de novo variants in high-functioning autism. Methods: We performed whole-exome sequencing in 20 high-functioning autism families (average IQ = 100). Results: We observed no difference in the number of transmitted versus nontransmitted truncating alleles for high-functioning autism (117 v. 130, p = 0.78). Transmitted truncating and de novo variants in high-functioning autism were not enriched in gene ontology (GO) or Kyoto Encyclopedia of Genes and Genomes (KEGG) categories, or in autism-related gene sets. However, in a patient with high-functioning autism we identified a de novo variant in a canonical splice site of LRP1, a postsynaptic density gene that is a target for fragile X mental retardation protein (FRMP). This de novo variant leads to in-frame skipping of exon 29, removing 2 of 6 blades of the β-propeller domain 4 of LRP1, with putative functional consequences. Large data sets implicate LRP1 across a number of psychiatric disorders: de novo variants are associated with autism-spectrum disorder (p = 0.039) and schizophrenia (p = 0.008) from combined sequencing projects; common variants using genome-wide association study data sets from the Psychiatric Genomics Consortium show gene-based association in schizophrenia (p = 6.6 × E−07) and in a meta-analysis across 7 psychiatric disorders (p = 2.3 × E−03); and the burden of ultra-rare pathogenic variants has been shown to be higher in autism-spectrum disorder (p = 1.2 × E−05), using whole-exome sequencing from 6135 patients with schizophrenia, 1778 patients with autism-spectrum disorder and 7875 controls. Limitations: We had a limited sample of patients with high-functioning autism, related to difficulty in recruiting probands with high cognitive performance and no family history of psychiatric disorders. Conclusion: Previous studies and ours suggest an effect of truncating mutations restricted to severe autism-spectrum disorder phenotypes that are associated with intellectual disability. We provide evidence for pleiotropic effects of common and rare variants in the LRP1 gene across psychiatric phenotypes

Multiple platform assessmant of the EGF dependent transcritpome by microarrays and deep TAG sequencing analysis

Abstract Background: Epidermal Growth Factor (EGF) is a key regulatory growth factor activating many processes relevant to normal development and disease, affecting cell proliferation and survival. Here we use a combined approach to study the EGF dependent transcriptome of HeLa cells by using multiple long oligonucleotide based microarray platforms (from Agilent, Operon, and Illumina) in combination with digital gene expression profiling (DGE) with the Illumina Genome Analyzer. Results: By applying a procedure for cross-platform data meta-analysis based on RankProd and GlobalAncova tests, we establish a well validated gene set with transcript levels altered after EGF treatment. We use this robust gene list to build higher order networks of gene interaction by interconnecting associated networks, supporting and extending the important role of the EGF signaling pathway in cancer. In addition, we find an entirely new set of genes previously unrelated to the currently accepted EGF associated cellular functions. Conclusions: We propose that the use of global genomic cross-validation derived from high content technologies (microarrays or deep sequencing) can be used to generate more reliable datasets. This approach should help to improve the confidence of downstream in silico functional inference analyses based on high content data