Alexandra Popa

Evidence for Widespread GC-biased Gene Conversion in Eukaryotes

Abstract GC-biased gene conversion (gBGC) is a process that tends to increase the GC content of recombining DNA over evolutionary time and is thought to explain the evolution of GC content in mammals and yeasts. Evidence for gBGC outside these two groups is growing but is still limited. Here, we analyzed 36 completely sequenced genomes representing four of the five major groups in eukaryotes (Unikonts, Excavates, Chromalveolates and Plantae). gBGC was investigated by directly comparing GC content and recombination rates in species where recombination data are available, that is, half of them. To study all species of our dataset, we used chromosome size as a proxy for recombination rate and compared it with GC content. Among the 17 species showing a significant relationship between GC content and chromosome size, 15 are consistent with the predictions of the gBGC model. Importantly, the species showing a pattern consistent with gBGC are found in all the four major groups of eukaryotes studied, which suggests that gBGC may be widespread in eukaryotes.

miR-193b/365a cluster controls progression of epidermal squamous cell carcinoma

Incidence of cutaneous squamous cell carcinomas (cSCCs) constantly increases in the Caucasian population. Developing preferentially on precancerous lesions such as actinic keratoses due to chronic sunlight exposure, cSCCs result from the malignant transformation of keratinocytes. Although a resection of the primary tumor is usually curative, a subset of aggressive cSCCs shows a high risk of recurrence and metastases. The characterization of the molecular dysfunctions involved in cSCC development should help to identify new relevant targets against these aggressive cSCCs. In that context, we have used small RNA sequencing to identify 100 microRNAs (miRNAs) whose expression was altered during chemically induced mouse skin tumorigenesis. The decreased expression of the miR-193b/365a cluster during tumor progression suggests a tumor suppressor role. Ectopic expression of these miRNAs in tumor cells indeed inhibited their proliferation, clonogenic potential and migration, which were stimulated in normal keratinocytes when these miRNAs were blocked with antisense oligonucleotides. A combination of in silico predictions and transcriptome analyses identified several target genes of interest. We validated KRAS and MAX as direct targets of miR-193b and miR-365a. Repression of these targets using siRNAs mimicked the effects of miR-193b and miR-365a, suggesting that these genes might mediate, at least in part, the tumor-suppressive action of these miRNAs.

Meiotic recombination favors the spreading of deleterious mutations in human populations

Although mutations that are detrimental to the fitness of organisms are expected to be rapidly purged from populations by natural selection, some disease‐causing mutations are present at high frequencies in human populations. Several nonexclusive hypotheses have been proposed to account for this apparent paradox (high new mutation rate, genetic drift, overdominance, or recent changes in selective pressure). However, the factors ultimately responsible for the presence at high frequency of disease‐causing mutations are still contentious. Here we establish the existence of an additional process that contributes to the spreading of deleterious mutations: GC‐biased gene conversion (gBGC), a process associated with recombination that tends to favor the transmission of GC‐alleles over AT‐alleles. We show that the spectrum of amino acid‐altering polymorphisms in human populations exhibits the footprints of gBGC. This pattern cannot be explained in terms of selection and is evident with all nonsynonymous mutations, including those predicted to be detrimental to protein structure and function, and those implicated in human genetic disease. We present simulations to illustrate the conditions under which gBGC can extend the persistence time of deleterious mutations in a finite population. These results indicate that gBGC meiotic drive contributes to the spreading of deleterious mutations in human populations.Hum Mutat 32: 1‐9, 2011.

MicroRNA-375/SEC23A as biomarkers of the in vitro efficacy of vandetanib.

In this study, we performed microRNA (miRNA) expression profiling on a large series of sporadic and hereditary forms of medullary thyroid carcinomas (MTC). More than 60 miRNAs were significantly deregulated in tumor vs adjacent non-tumor tissues, partially overlapping with results of previous studies. We focused our attention on the strongest up-regulated miRNA in MTC samples, miR-375, the deregulation of which has been previously observed in a variety of human malignancies including MTC. We identified miR-375 targets by combining gene expression signatures from human MTC (TT) and normal follicular (Nthy-ori 3-1) cell lines transfected with an antagomiR-375 inhibitor or a miR-375 mimic, respectively, and from an in silico analysis of thyroid cell lines of Cancer Cell Line Encyclopedia datasets. This approach identified SEC23A as a bona fide miR-375 target, which we validated by immunoblotting and immunohistochemistry of non-tumor and pathological thyroid tissue. Furthermore, we observed that miR-375 overexpression was associated with decreased cell proliferation and synergistically increased sensitivity to vandetanib, the clinically relevant treatment of metastatic MTC. We found that miR-375 increased PARP cleavage and decreased AKT phosphorylation, affecting both cell proliferation and viability. We confirmed these results through SEC23A direct silencing in combination with vandetanib, highlighting the importance of SEC23A in the miR-375-associated increased sensitivity to vandetanib. Since the combination of increased expression of miR-375 and decreased expression of SEC23A point to sensitivity to vandetanib, we question if the expression levels of miR-375 and SEC23A should be evaluated as an indicator of eligibility for treatment of MTC patients with vandetanib.

RiboProfiling: a Bioconductor package for standard Ribo-seq pipeline processing.

The ribosome profiling technique (Ribo-seq) allows the selective sequencing of translated RNA regions. Recently, the analysis of genomic sequences associated to Ribo-seq reads has been widely employed to assess their coding potential. These analyses led to the identification of differentially translated transcripts under different experimental conditions, and/or ribosome pausing on codon motifs. In the context of the ever-growing need for tools analyzing Ribo-seq reads, we have developed ‘RiboProfiling’, a new Bioconductor open-source package. ‘RiboProfiling’ provides a full pipeline to cover all key steps for the analysis of ribosome footprints. This pipeline has been implemented in a single R workflow. The package takes an alignment (BAM) file as input and performs ribosome footprint quantification at a transcript level. It also identifies footprint accumulation on particular amino acids or multi amino-acids motifs. Report summary graphs and data quantification are generated automatically. The package facilitates quality assessment and quantification of Ribo-seq experiments. Its implementation in Bioconductor enables the modeling and statistical analysis of its output through the vast choice of packages available in R. This article illustrates how to identify codon-motifs accumulating ribosome footprints, based on data from Escherichia coli.

SigmaR1 regulates membrane electrical activity in response to extracellular matrix stimulation to drive cancer cell invasiveness

The sigma 1 receptor (Sig1R) is a stress-activated chaperone that regulates ion channels and is associated with pathologic conditions, such as stroke, neurodegenerative diseases, and addiction. Aberrant expression levels of ion channels and Sig1R have been detected in tumors and cancer cells, such as myeloid leukemia and colorectal cancer, but the link between ion channel regulation and Sig1R overexpression during malignancy has not been established. In this study, we found that Sig1R dynamically controls the membrane expression of the human voltage-dependent K(+) channel human ether-à-go-go-related gene (hERG) in myeloid leukemia and colorectal cancer cell lines. Sig1R promoted the formation of hERG/β1-integrin signaling complexes upon extracellular matrix stimulation, triggering the activation of the PI3K/AKT pathway. Consequently, the presence of Sig1R in cancer cells increased motility and VEGF secretion. In vivo, Sig1R expression enhanced the aggressiveness of tumor cells by potentiating invasion and angiogenesis, leading to poor survival. Collectively, our findings highlight a novel function for Sig1R in mediating cross-talk between cancer cells and their microenvironment, thus driving oncogenesis by shaping cellular electrical activity in response to extracellular signals. Given the involvement of ion channels in promoting several hallmarks of cancer, our study also offers a potential strategy to therapeutically target ion channel function through Sig1R inhibition.

Pateamine A-sensitive ribosome profiling reveals the scope of translation in mouse embryonic stem cells

BackgroundOpen reading frames are common in long noncoding RNAs (lncRNAs) and 5’UTRs of protein coding transcripts (uORFs). The question of whether those ORFs are translated was recently addressed by several groups using ribosome profiling. Most of those studies concluded that certain lncRNAs and uORFs are translated, essentially based on computational analysis of ribosome footprints. However, major discrepancies remain on the scope of translation and the translational status of individual ORFs. In consequence, further criteria are required to reliably identify translated ORFs from ribosome profiling data.ResultsWe examined the effect of the translation inhibitors pateamine A, harringtonine and puromycin on murine ES cell ribosome footprints. We found that pateamine A, a drug that targets eIF4A, allows a far more accurate identification of translated sequences than previously used drugs and computational scoring schemes. Our data show that at least one third but less than two thirds of ES cell lncRNAs are translated. We also identified translated uORFs in hundreds of annotated coding transcripts including key pluripotency transcripts, such as dicer, lin28, trim71, and ctcf.ConclusionPateamine A inhibition data clearly increase the precision of the detection of translated ORFs in ribosome profiling experiments. Our data show that translation of lncRNAs and uORFs in murine ES cells is rather common although less pervasive than previously suggested. The observation of translated uORFs in several key pluripotency transcripts suggests that translational regulation by uORFs might be part of the network that defines mammalian stem cell identity.

Knockout of Vdac1 activates hypoxia-inducible factor through reactive oxygen species generation and induces tumor growth by promoting metabolic reprogramming and inflammation

BackgroundMitochondria are more than just the powerhouse of cells; they dictate if a cell dies or survives. Mitochondria are dynamic organelles that constantly undergo fusion and fission in response to environmental conditions. We showed previously that mitochondria of cells in a low oxygen environment (hypoxia) hyperfuse to form enlarged or highly interconnected networks with enhanced metabolic efficacy and resistance to apoptosis. Modifications to the appearance and metabolic capacity of mitochondria have been reported in cancer. However, the precise mechanisms regulating mitochondrial dynamics and metabolism in cancer are unknown. Since hypoxia plays a role in the generation of these abnormal mitochondria, we questioned if it modulates mitochondrial function. The mitochondrial outer-membrane voltage-dependent anion channel 1 (VDAC1) is at center stage in regulating metabolism and apoptosis. We demonstrated previously that VDAC1 was post-translationally C-terminal cleaved not only in various hypoxic cancer cells but also in tumor tissues of patients with lung adenocarcinomas. Cells with enlarged mitochondria and cleaved VDAC1 were also more resistant to chemotherapy-stimulated cell death than normoxic cancer cells.ResultsTranscriptome analysis of mouse embryonic fibroblasts (MEF) knocked out for Vdac1 highlighted alterations in not only cancer and inflammatory pathways but also in the activation of the hypoxia-inducible factor-1 (HIF-1) signaling pathway in normoxia. HIF-1α was stable in normoxia due to accumulation of reactive oxygen species (ROS), which decreased respiration and glycolysis and maintained basal apoptosis. However, in hypoxia, activation of extracellular signal-regulated kinase (ERK) in combination with maintenance of respiration and increased glycolysis counterbalanced the deleterious effects of enhanced ROS, thereby allowing Vdac1−/− MEF to proliferate better than wild-type MEF in hypoxia. Allografts of RAS-transformed Vdac1−/− MEF exhibited stabilization of both HIF-1α and HIF-2α, blood vessel destabilization, and a strong inflammatory response. Moreover, expression of Cdkn2a, a HIF-1-target and tumor suppressor gene, was markedly decreased. Consequently, RAS-transformed Vdac1−/− MEF tumors grew faster than wild-type MEF tumors.ConclusionsMetabolic reprogramming in cancer cells may be regulated by VDAC1 through vascular destabilization and inflammation. These findings provide new perspectives into the understanding of VDAC1 in the function of mitochondria not only in cancer but also in inflammatory diseases.

The sex-specific impact of meiotic recombination on nucleotide composition

Meiotic recombination is an important evolutionary force shaping the nucleotide landscape of genomes. For most vertebrates, the frequency of recombination varies slightly or considerably between the sexes (heterochiasmy). In humans, male, rather than female, recombination rate has been found to be more highly correlated with the guanine and cytosine (GC) content across the genome. In the present study, we review the results in human and extend the examination of the evolutionary impact of heterochiasmy beyond primates to include four additional eutherian mammals (mouse, dog, pig, and sheep), a metatherian mammal (opossum), and a bird (chicken). Specifically, we compared sex-specific recombination rates (RRs) with nucleotide substitution patterns evaluated in transposable elements. Our results, based on a comparative approach, reveal a great diversity in the relationship between heterochiasmy and nucleotide composition. We find that the stronger male impact on this relationship is a conserved feature of human, mouse, dog, and sheep. In contrast, variation in genomic GC content in pig and opossum is more strongly correlated with female, rather than male, RR. Moreover, we show that the sex-differential impact of recombination is mainly driven by the chromosomal localization of recombination events. Independent of sex, the higher the RR in a genomic region and the longer this recombination activity is conserved in time, the stronger the bias in nucleotide substitution pattern, through such mechanisms as biased gene conversion. Over time, this bias will increase the local GC content of the region.

Characterizing isomiR variants within the microRNA‐34/449 family

miR‐34/449 microRNAs are conserved regulators of multiciliated cell differentiation. Here, we evidence and characterize expression of two isomiR variant sequences from the miR‐34/449 family in human airway epithelial cells. These isomiRs differ from their canonical counterparts miR‐34b and miR‐449c by one supplemental uridine at their 5′‐end, leading to a one‐base shift in their seed region. Overexpression of canonical miR‐34/449 or 5′‐isomiR‐34/449 induces distinct gene expression profiles and biological effects. However, some target transcripts and functional activities are shared by both canonical microRNAs and isomiRs. Indeed, both repress important targets that result in cell cycle blockage and Notch pathway inhibition. Our findings suggest that 5′‐isomiR‐34/449 may represent additional mechanisms by which miR‐34/449 family finely controls several pathways to drive multiciliogenesis.