Jeppe Vinther

Identification of Let-7–Regulated Oncofetal Genes

MicroRNAs (miRNA) are small RNA molecules of approximately 20 to 22 nucleotides that reduce expression of proteins through mRNA degradation and/or translational silencing. Each known miRNA has a large number of predicted targets. Members of the let-7/miR-98 family of miRNAs are up-regulated at the end of embryonic development. Let-7 is often down-regulated early during cancer development, suggesting that let-7-regulated oncofetal genes (LOG) may become reexpressed in cancer cells. Using comparative bioinformatics, we have identified 12 conserved LOGs that include HMGA2 and IMP-1/CRD-BP. IMP-1 has growth-promoting activities through stabilization of c-myc mRNA. We experimentally confirmed that IMP-1 is a direct let-7 target that promotes cell growth and motility of tumor cells, and we confirmed by proteomics analysis that IMP-1 and HMGA2 are major miRNA targets. Our data suggest that a substantial part of the growth inhibitory activities of let-7 comes from suppressing the expression of IMP-1. LOGs could be novel therapeutic targets and potential biomarkers for cancer treatment.

Identification of miRNA targets with stable isotope labeling by amino acids in cell culture

miRNAs are small noncoding RNAs that regulate gene expression. We have used stable isotope labeling by amino acids in cell culture (SILAC) to investigate the effect of miRNA-1 on the HeLa cell proteome. Expression of 12 out of 504 investigated proteins was repressed by miRNA-1 transfection. This repressed set of genes significantly overlaps with miRNA-1 regulated genes that have been identified with DNA array technology and are predicted by computational methods. Moreover, we find that the 3′-untranslated region for the repressed set are enriched in miRNA-1 complementary sites. Our findings demonstrate that SILAC can be used for miRNA target identification and that one highly expressed miRNA can regulate the levels of many different proteins.

Pharmaco-miR: linking microRNAs and drug effects

MicroRNAs (miRNAs) are short regulatory RNAs that down-regulate gene expression. They are essential for cell homeostasis and active in many disease states. A major discovery is the ability of miRNAs to determine the efficacy of drugs, which has given rise to the field of ‘miRNA pharmacogenomics’ through ‘Pharmaco-miRs’. miRNAs play a significant role in pharmacogenomics by down-regulating genes that are important for drug function. These interactions can be described as triplet sets consisting of a miRNA, a target gene and a drug associated with the gene. We have developed a web server which links miRNA expression and drug function by combining data on miRNA targeting and protein–drug interactions. miRNA targeting information derive from both experimental data and computational predictions, and protein–drug interactions are annotated by the Pharmacogenomics Knowledge base (PharmGKB). Pharmaco-miR’s input consists of miRNAs, genes and/or drug names and the output consists of miRNA pharmacogenomic sets or a list of unique associated miRNAs, genes and drugs. We have furthermore built a database, named Pharmaco-miR Verified Sets (VerSe), which contains miRNA pharmacogenomic data manually curated from the literature, can be searched and downloaded via Pharmaco-miR and informs on trends and generalities published in the field. Overall, we present examples of how Pharmaco-miR provides possible explanations for previously published observations, including how the cisplatin and 5-fluorouracil resistance induced by miR-148a may be caused by miR-148a targeting of the gene KIT. The information is available at www.Pharmaco-miR.org.

Quantitative regulation of alternative splicing in evolution and development

Alternative splicing (AS) is a widespread mechanism with an important role in increasing transcriptome and proteome diversity by generating multiple different products from the same gene. Evolutionary studies of AS have focused primarily on the conservation of alternatively spliced sequences or of the AS pattern of those sequences itself. Less is known about the evolution of the regulation of AS, but several studies, working from different perspectives, have recently made significant progress. Here, we categorize the different levels of AS evolution, and summarize the studies on evolution of AS regulation, which point to a high level of evolutionary conservation of the regulation of AS events conserved between related species. This suggests that the quantitative regulation of AS is an intrinsic part of AS function. We discuss the potential role of changes in developmental regulation of AS as an additional layer in complex gene regulatory networks and in the emergence of genetic novelties.

Massive parallel-sequencing-based hydroxyl radical probing of RNA accessibility

Hydroxyl Radical Footprinting (HRF) is a tried-and-tested method for analysis of the tertiary structure of RNA and for identification of protein footprints on RNA. The hydroxyl radical reaction breaks accessible parts of the RNA backbone, thereby allowing ribose accessibility to be determined by detection of reverse transcriptase termination sites. Current methods for HRF rely on reverse transcription of a single primer and detection by fluorescent fragments by capillary electrophoresis. Here, we describe an accurate and efficient massive parallel-sequencing-based method for probing RNA accessibility with hydroxyl radicals, called HRF-Seq. Using random priming and a novel barcoding scheme, we show that HRF-Seq dramatically increases the throughput of HRF experiments and facilitates the parallel analysis of multiple RNAs or experimental conditions. Moreover, we demonstrate that HRF-Seq data for the Escherichia coli 16S rRNA correlates well with the ribose accessible surface area as determined by X-ray crystallography and have a resolution that readily allows the difference in accessibility caused by exposure of one side of RNA helices to be observed.

Pharmacogenomics genes show varying perceptibility to microRNA regulation.

Objective The aim of pharmacogenomics is to identify individual differences in genome and transcriptome composition and their effect on drug efficacy. MicroRNAs (miRNAs) are short noncoding RNAs that negatively regulate expression of the majority of animal genes, including many genes involved in drug efficacy. Consequently, differences in the miRNA expression among individuals could be an important factor contributing to differential drug response. Pharmacogenomics genes can be divided into drug target genes termed as pharmacodynamics genes (PD) and genes involved in drug transport and metabolism termed as pharmacokinetics genes (PK). To clarify the regulatory potential of miRNAs in pharmacogenomics, we have examined the potential regulation by miRNAs of PK and PD genes. Methods We identified PK and PD genes as annotated by the Pharmacogenomics Knowledge Base and examined miRNA targeting of genes in the two groups according to several miRNA target prediction databases. We furthermore studied how differences between the two groups are reflected in the gene structure and across gene families. Lastly, we studied changes in expression levels of PK versus PD genes in cells depleted for miRNAs by shRNA-mediated knockdown of the miRNA-processing enzyme Dicer. Results Our analysis identify a striking difference in the level of miRNA regulation between PK and PD genes, with the former having less than half predicted conserved miRNA binding sites compared with the latter. Importantly, this finding is reflected in a highly significant difference in the shift in expression levels of PD versus PK genes after depletion of miRNAs. Conclusion Our study emphasizes an intrinsic difference between PK and PD genes and helps clarify the role of miRNAs in pharmacogenomics.

Mutation of miRNA target sequences during human evolution

It has long-been hypothesized that changes in non-protein-coding genes and the regulatory sequences controlling expression could undergo positive selection. Here we identify 402 putative microRNA (miRNA) target sequences that have been mutated specifically in the human lineage and show that genes containing such deletions are more highly expressed than their mouse orthologs. Our findings indicate that some miRNA target mutations are fixed by positive selection and might have been involved in the evolution of human-specific traits.

Use of tiling array data and RNA secondary structure predictions to identify noncoding RNA genes

BackgroundWithin the last decade a large number of noncoding RNA genes have been identified, but this may only be the tip of the iceberg. Using comparative genomics a large number of sequences that have signals concordant with conserved RNA secondary structures have been discovered in the human genome. Moreover, genome wide transcription profiling with tiling arrays indicate that the majority of the genome is transcribed.ResultsWe have combined tiling array data with genome wide structural RNA predictions to search for novel noncoding and structural RNA genes that are expressed in the human neuroblastoma cell line SK-N-AS. Using this strategy, we identify thousands of human candidate RNA genes. To further verify the expression of these genes, we focused on candidate genes that had a stable hairpin structures or a high level of covariance. Using northern blotting, we verify the expression of 2 out of 3 of the hairpin structures and 3 out of 9 high covariance structures in SK-N-AS cells.ConclusionOur results demonstrate that many human noncoding, structured and conserved RNA genes remain to be discovered and that tissue specific tiling array data can be used in combination with computational predictions of sequences encoding structural RNAs to improve the search for such genes.

Effect of chronic uremia on the transcriptional profile of the calcified aorta analyzed by RNA sequencing

The development of vascular calcification (VC) in chronic uremia (CU) is a tightly regulated process controlled by factors promoting and inhibiting mineralization. Next-generation high-throughput RNA sequencing (RNA-seq) is a powerful and sensitive tool for quantitative gene expression profiling and the detection of differentially expressed genes. In the present study, we, for the first time, used RNA-seq to examine rat aorta transcriptomes from CU rats compared with control rats. Severe VC was induced in CU rats, which lead to extensive changes in the transcriptional profile. Among the 10,153 genes with an expression level of >1 reads/kilobase transcript/million mapped reads, 2,663 genes were differentially expressed with 47% upregulated genes and 53% downregulated genes in uremic rats. Significantly deregulated genes were enriched for ontologies related to the extracellular matrix, response to wounding, organic substance, and ossification. The individually affected genes were of relevance to osteogenic transformation, tissue calcification, and Wnt modulation. Downregulation of the Klotho gene in uremia is believed to be involved in the development of VC, but it is debated whether the effect is caused by circulating Klotho only or if Klotho is produced locally in the vasculature. We found that Klotho was neither expressed in the normal aorta nor calcified aorta by RNA-seq. In conclusion, we demonstrated extensive changes in the transcriptional profile of the uremic calcified aorta, which were consistent with a shift in phenotype from vascular tissue toward an osteochondrocytic transcriptome profile. Moreover, neither the normal vasculature nor calcified vasculature in CU expresses Klotho.

Clearance of cervical human papillomavirus infections

Dear Sir, It is intriguing that only a small percentage of young women infected with high-risk Human Papillomavirus (HR-HPV) in the cervix will develop a persistent infection, while the remaining young women clear the infection very rapidly.1,2 Historically, this has been attributed to differences in the immune response among individuals.3 However, whereas some studies have found that cell mediated immune responses against HPV epitopes were associated with clearance of infection,4,5 others studies have detected HPV specific T-cell responses predominantly in woman with high-grade lesions and not in women that cleared the HPV infection.6–8 Moreover, many studies have shown that HR-HPV has developed multiple strategies to efficiently evade detection and clearance by the immune system.9 Therefore, it remains possible that factors other than HPVspecific immune responses are important for the clearance of HR-HPV infections in women that possess no prior immunity. Owing to the continuous renewable nature of epithelial surfaces, cervical basal cells need to be infected in order to establish a long-term or a persistent HR-HPV infection. The basal cells consist of stem cells and transiently amplifying cells (TA-cells). Stem cells have unlimited capacity for self-renewal. In contrast, TA-cells are committed to differentiation and will divide a limited number of times before they undergo terminal differentiation and finally are pushed of the surface of the epithelium (Fig. 1). So far TA-cells have been considered irrelevant for the duration of HRHPV infection due to their transient nature and short life span. Nonetheless, the fact is that nobody knows for how long TA-cells survive in human epithelia. We believe that a subset of TA-cells in the cervical epithelium will have a large capacity for self renewal and therefore a sufficiently long life span to be host for productive HR-HPV infections. Based on this prediction, we suggest that a major contributor to the natural history of a HR-HPV infection in women without prior protective immunity is the life span of the infected cells. Thus, a stem cell infection will last until it is cleared by the immune system, whilst infections in TA-cells last for shorter periods of time that are determined by the life span of the infected cells or until cleared by the immune response (Fig. 2). This hypothesis provides a plausible molecular explanation for the observed large differences in the persistence of HR-HPV infections and clearance without any apparent activation of the immune system, although it remains a possibility that these observations are caused by inefficiency of current immunological assays. The hypothesis presented above is based on the prediction of long-lived TA-cells in the human cervical epithelium. The life span of TA-cells is coupled to the frequency of stem cell division, which is unknown for the cervical epithelium. In general, stem cells divide infrequently to ensure that harmful mutations predominantly occurring during DNA replication or mitosis will accumulate almost exclusively in TA-cells. As TA-cells have limited life spans they will be less likely to develop into cancer cells.10 In higher primates, pluripotent hematopoietic stem cells (PHSCs) have been shown to divide very infrequently (less than one division per year in adults).11,12 This is in contrast to mice PHSCs, which divide much more rapidly. A mouse lives for approximately 2 years and the mouse stem cells would therefore be expected to be able to divide much more frequently than human stem cells without causing cancer in the lifetime of the mouse.10 Epidermal stem cell research has also largely been based on experiments with mice and it is therefore possible that the frequency of stem cell division in human epidermis has been overestimated. An estimate of the frequency of human epidermal stem cell division comes from experiments with rafts of human foreskin keratinocytes transduced with a retroviral vector encoding LacZ that were grafted on nude mice. No distinctive arrangement of LacZ positive cells was found in the early postgrafting period. LacZ positive clusters of cells started to appear after 10 weeks and distinct columns representing clonal units could be seen after 40 weeks.13 These observations indicate that the life span of some TA-cells in human foreskin is between 10 and 40 weeks. The hypothesis presented here has an important implication, namely, that HR-HPV infections may frequently be cleared without immune intervention and acquisition of protective immunity. Thus, most women could experience several subclinical or transient infections during their lifetime before they develop protective immunity. This would help explain the rather high prevalence of HR-HPV infections that are observed in the general population and that reinfection with the same HR-HPV type occurs. We believe the results obtained in epidemiological studies of HR-HPV clearance are generated by a combination of immunological clearance and clearance by the mechanism described above. The HR-HPV types very efficiently evade the immune system and it is therefore possible that HR-HPV infections primarily will be detected by the immune system when the normal viral regulation somehow is abrogated. Most likely, such deregulation will in most cases require several separate events occurring in a cell. Since this is