Noncoder : a web interface for exon array-based detection of long non-coding RNAs

Due to recent technical developments, a high number of long non-coding RNAs (lncRNAs) have been discovered in mammals. Although it has been shown that lncRNAs are regulated differently among tissues and disease statuses, functions of these transcripts are still unknown in most cases. GeneChip Exon 1.0 ST Arrays (exon arrays) from Affymetrix, Inc. have been used widely to profile genome-wide expression changes and alternative splicing of protein-coding genes. Here, we demonstrate that re-annotation of exon array probes can be used to profile expressions of tens of thousands of lncRNAs. With this annotation, a detailed inspection of lncRNAs and their isoforms is possible. To allow for a general usage to the research community, we developed a user-friendly web interface called 'noncoder'. By uploading CEL files from exon arrays and with a few mouse clicks and parameter settings, exon array data will be normalized and analysed to identify differentially expressed lncRNAs. Noncoder provides the detailed annotation information of lncRNAs and is equipped with unique features to allow for an efficient search for interesting lncRNAs to be studied further. The web interface is available at http://noncoder.mpi-bn.mpg.de

Functionally Conserved Noncoding Regulators of Cardiomyocyte Proliferation and Regeneration in Mouse and Human

BACKGROUND: The adult mammalian heart has little regenerative capacity after myocardial infarction (MI), whereas neonatal mouse heart regenerates without scarring or dysfunction. However, the underlying pathways are poorly defined. We sought to derive insights into the pathways regulating neonatal development of the mouse heart and cardiac regeneration post-MI. METHODS AND RESULTS: Total RNA-seq of mouse heart through the first 10 days of postnatal life (referred to as P3, P5, P10) revealed a previously unobserved transition in microRNA (miRNA) expression between P3 and P5 associated specifically with altered expression of protein-coding genes on the focal adhesion pathway and cessation of cardiomyocyte cell division. We found profound changes in the coding and noncoding transcriptome after neonatal MI, with evidence of essentially complete healing by P10. Over two-thirds of each of the messenger RNAs, long noncoding RNAs, and miRNAs that were differentially expressed in the post-MI heart were differentially expressed during normal postnatal development, suggesting a common regulatory pathway for normal cardiac development and post-MI cardiac regeneration. We selected exemplars of miRNAs implicated in our data set as regulators of cardiomyocyte proliferation. Several of these showed evidence of a functional influence on mouse cardiomyocyte cell division. In addition, a subset of these miRNAs, miR-144-3p, miR-195a-5p, miR- 451a, and miR-6240 showed evidence of functional conservation in human cardiomyocytes. CONCLUSIONS: The sets of messenger RNAs, miRNAs, and long noncoding RNAs that we report here merit further investigation as gatekeepers of cell division in the postnatal heart and as targets for extension of the period of cardiac regeneration beyond the neonatal period.Leducq Foundation funding via the Transatlantic Network of Excellence (Grant 11CVD01), the British Heart Foundation funding via the Imperial College Centre of Research Excellence and the Imperial Cardiovascular Regenerative Medicine Centre RM/13/1/30157

Discovery of naturally occurring ESR1 mutations in breast cancer cell lines modelling endocrine resistance.

Resistance to endocrine therapy remains a major clinical problem in breast cancer. Genetic studies highlight the potential role of estrogen receptor-α (ESR1) mutations, which show increased prevalence in the metastatic, endocrine-resistant setting. No naturally occurring ESR1 mutations have been reported in in vitro models of BC either before or after the acquisition of endocrine resistance making functional consequences difficult to study. We report the first discovery of naturally occurring ESR1 Y537C and ESR1 Y537S mutations in MCF7 and SUM44 ESR1-positive cell lines after acquisition of resistance to long-term-estrogen-deprivation (LTED) and subsequent resistance to fulvestrant (ICIR). Mutations were enriched with time, impacted on ESR1 binding to the genome and altered the ESR1 interactome. The results highlight the importance and functional consequence of these mutations and provide an important resource for studying endocrine resistance.Cancer Research U

Molecular characterisation of aromatase inhibitor-resistant advanced breast cancer: the phenotypic effect of ESR1 mutations.

BACKGROUND:Several thousand breast cancer patients develop resistance to aromatase inhibitors (AIs) each year in the UK. Rational treatment requires an improved molecular characterisation of resistant disease. MATERIALS AND METHODS:The mutational landscape of 198 regions in 16 key breast cancer genes and RNA expression of 209 genes covering key pathways was evaluated in paired biopsies before AI treatment and at progression on AI from 48 patients. Validity of findings was assessed in another five ESR1-mutated tumours progressing on AI. RESULTS:Eighty-nine mutations were identified in 41 matched pairs (PIK3CA in 27%; CDH1 in 20%). ESR1 (n = 5), ERBB2 (n = 1) and MAP2K4 (n = 1) had mutations in the secondary sample only. There was very high heterogeneity in gene expression between AI-resistant tumours with few patterns apparent. However, in the ESR1-mutated AI-resistant tumours, expression of four classical oestrogen-regulated genes (ERGs) was sevenfold higher than in ESR1 wild-type tumours, a finding confirmed in the second set of ESR1-mutated tumours. In ESR1 wild-type AI-resistant tumours ERG expression remained suppressed and was uncoupled from the recovery seen in proliferation. CONCLUSIONS:Major genotypic and phenotypic heterogeneity exists between AI-resistant disease. ESR1 mutations appear to drive oestrogen-regulated processes in resistant tumours

Analysis of splicing sensitive microarrays

Due to recent technical developments, it became evident that the mammalian transcriptome is much more complex than originally expected. Alternative splicing(AS) and the transcription of long non-coding RNAs (lncRNAs) are two phenomenas which have been greatly underestimated in their frequency. Nowadays it is accepted that almost every gene has at least one alternative isoform and the number of lncRNAs exceeds the one of protein-coding genes. We built user-friendly web interfaces which can process Affymetrix GeneChip Exon 1.0 ST Arrays (exon arrays) and GeneChip Gene 1.0 ST Arrays (gene arrays)for the analysis of alternative splicing events. Results are presented with detailed annotation information and graphics to identify splice events and to facilitate biological validations. Based on two studies using exon arrays, we show how our tools were used to profile genome-wide splicing changes under silencing of Jmjd6 and under hypoxic conditions. Since gene arrays are not intended for AS analysis originally, we demonstrated their applicability by profiling alternative splicing events during embryonic heart development. To measure lncRNAs expressions with exon arrays, we completely re-annotation all probes and built a lncRNA specific annotation. To demonstrate the applicability of exon arrays in combination with our annotation, we profiled the expression of tens of thousands of lncRNAs. Further, our custom annotation allows for a detailed inspection of lncRNAs and to distinguish between isoforms, as we validated by RTPCR. To allow for a general usage to the research community, we integrated the annotation in an easy-to-use web interface, which provides various helpful features for the analysis of lncRNAs

Samtykkekravet i pasient- og brukerrettighetsloven §§ 4-1 og 4-2. Med særlig fokus på lovmessigheten av å benytte hypotetisk samtykke som selvstendig kompetansegrunnlag for inngripen

Avhandlingen søker å redegjøre for hva som forstås med de lovregulerte vilkårene for å kunne avgi et gyldig samtykke etter pasient- og brukerrettighetsloven §§ 4-1 og 4-2. Avhandlingens hovedproblemstilling er lovmessigheten av hypotetisk samtykke som kompetansegrunnlag for inngripen. Denne problemstillingen ses spesielt under det forhold at et hypotetisk samtykke krenker den lovfestede retten til pasientautonomi. Spesielt i denne sammenheng er at hypotetisk samtykke som kompetansegrunnlag for inngripen stammer fra Rt. 1998 s. 1538 Caudia equina- dommen. Pasient- og brukerrettighetsloven som har lovfestet samtykkekravet er nyere enn denne dommen. I teorien eksisterer det allikevel en viss usikkerhet knyttet til hvorvidt hypotetisk samtykke er gyldig etter lovens ordlyd

Hypoxia-dependent alternative exon inclusion.

<p><b>A</b>) Scheme of alternative splicing of cassette exons. Introns are represented as black lines; constitutive exons as grey boxes and the cassette exon as white box. Primers are indicated as arrows and span the alternatively spliced exon. Cassette exons in <i>cask</i> (<b>B</b>), <i>sptan1</i> (<b>C</b>) and <i>pign</i> (<b>D</b>) are more often skipped under hypoxic then under normoxic conditions. Radioactive RT-PCR: N = normoxia, H = hypoxia, M = DNA size ladder. The precentage of exon inclusion is given below. SD = standard deviation. qRT-PCR: closed bars = normoxia, open bars = hypoxia. Two isoform specific forward primer (i1 and i2) spanning the exon/exon borders and one common reverse primer (i1/2) were used. Expression is normalized to <i>rplp0</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042697#s2" target="_blank">Results</a> from n = 3 experiments are shown. * = p-value<0.05, ** = p-value<0.01 (Student's t-test).</p

Analysis of hypoxia induced alternative splicing.

<p>Differentially expressed probe sets are given in bold. The number of the corresponding genes is given in brackets.</p