Impact of testicular cancer on sperm small non-coding RNA signature: a pilot study.

Testicular germ cell tumours (TGCTs) are the most common tumours in young adults of European ancestry. The high heritability and the constantly increased incidence, which has doubled over the last 20 years, strongly suggest that both genetic and environmental factors are likely to shape the TGCT susceptibility. While genome-wide association studies have identified loci associated with TGCT susceptibility, the role played by environmental molecular vectors in TGCT susceptibility remains unclear. Evidence shows that sperm non-coding RNAs provide a good vision of the environmental stresses experienced by men. Here, to determine whether TGCT impacts the abundance of specific non-coding RNAs in sperm, small RNA deep sequencing analysis of sperm of 25 men aged between 19 and 42 years, diagnosed with (n = 16) or without (n = 9) TGCT was performed. The primary analysis showed no statistical significance in the sncRNA population between the TGCT and non-TGCT groups. However, when sperm physiological parameters were considered to look for differentially expressed sncRNA, we evidenced 11 differentially expressed sncRNA between patients and control which allow a clear discrimination between control and TGCT samples after Hierarchical Clustering analysis. Together, these findings indicate that sperm small non-coding RNAs abundance may have the potential for diagnosing men with TGCT. However, specific care should be taken regarding sperm physiological parameters of the TGCT patients. Hence, larger studies are needed to confirm our findings and to determine whether such a signature associates with the risks to develop TGCT.

Systemic CLIP-seq analysis and game theory approach to model microRNA mode of binding.

microRNAs (miRNAs) associate with Ago proteins to post-transcriptionally silence gene expression by targeting mRNAs. To characterize the modes of miRNA-binding, we developed a novel computational framework, called optiCLIP, which considers the reproducibility of the identified peaks among replicates based on the peak overlap. We identified 98 999 binding sites for mouse and human miRNAs, from eleven Ago2 CLIP-seq datasets. Clustering the binding preferences, we found heterogeneity of the mode of binding for different miRNAs. Finally, we set up a quantitative model, named miRgame, based on an adaptation of the game theory. We have developed a new algorithm to translate the miRgame into a score that corresponds to a miRNA degree of occupancy for each Ago2 peak. The degree of occupancy summarizes the number of miRNA-binding sites and miRNAs targeting each binding site, and binding energy of each miRNA::RNA heteroduplex in each peak. Ago peaks were stratified accordingly to the degree of occupancy. Target repression correlates with higher score of degree of occupancy and number of miRNA-binding sites within each Ago peak. We validated the biological performance of our new method on miR-155-5p. In conclusion, our data demonstrate that miRNA-binding sites within each Ago2 CLIP-seq peak synergistically interplay to enhance target repression.

Paternal multigenerational exposure to an obesogenic diet drives epigenetic predisposition to metabolic diseases in mice.

Obesity is a growing societal scourge. Recent studies have uncovered that paternal excessive weight induced by an unbalanced diet affects the metabolic health of offspring. These reports mainly employed single-generation male exposure. However, the consequences of multigenerational unbalanced diet feeding on the metabolic health of progeny remain largely unknown. Here, we show that maintaining paternal Western diet feeding for five consecutive generations in mice induces an enhancement in fat mass and related metabolic diseases over generations. Strikingly, chow-diet-fed progenies from these multigenerational Western-diet-fed males develop a 'healthy' overweight phenotype characterized by normal glucose metabolism and without fatty liver that persists for four subsequent generations. Mechanistically, sperm RNA microinjection experiments into zygotes suggest that sperm RNAs are sufficient for establishment but not for long-term maintenance of epigenetic inheritance of metabolic pathologies. Progressive and permanent metabolic deregulation induced by successive paternal Western-diet-fed generations may contribute to the worldwide epidemic of metabolic diseases.

Prediction of coronary heart disease incidence in a general male population by circulating non-coding small RNA sRNY1-5p in a nested case-control study.

During the development of atherosclerotic lesion, s-RNYs (small RNAs of about 24/34 nucleotides) are derived by the processing of long Ro-associated non-coding RNAs (RNYs) in macrophages. The levels of serum s-RNYs have been found significantly upregulated in patients with coronary heart disease (CHD) compared to age-matched CHD-free individuals. The present study aimed to examine the predictive value of serum s-RNYs for CHD events in the general male population. Within the frame of nested-case-control study, the GENES study, we measured the absolute expression of a RNY-derived small RNA, the s-RNY1-5p, in the serum of individuals (without CHD at baseline) who encountered a CHD event within 12 years of follow-up (n = 30) (Cases) and compared them to individuals who remained event-free (Controls) (n = 30). The expression of s-RNY1-5p in serum was significantly upregulated in Cases compared to Controls (p = 0.027). The proportion of CHD event-free was significantly higher among individuals with serum s-RNY1-5p below the median value (631 molecules/mL). In a multivariable model adjusted for age, smoking, hypertension, diabetes and dyslipidemia, the risk of CHD events increased more than fourfold in individuals with serum s-RNY1-5p above the median value (HR, 4.36; 95% CI 1.22-15.60). A positive association with CHD events was also observed when considering s-RNY1-5p as a continuous variable (p = 0.022). Based on our results, we conclude that serum s-RNY1-5p is an independent predictor of CHD events in a general male population and might be a relevant biomarker for early detection of cardiovascular diseases.

Subcellular Heterogeneity of the microRNA Machinery.

Different methods have recently been developed to understand the subcellular localization and role of microRNAs (miRNAs) as well as small RNAs associated with Argonaute (AGO) proteins. The heterogeneity of the protein complexes associated with miRNAs, along with their subcellular localization, provides clues into their biochemical mechanism of function. Subcellular diversity indicates that miRNAs localized to different cellular regions could have different functions, including transcriptional regulation on chromatin or post-transcriptional control, providing global regulation of gene expression by miRNAs. Herein, I review the current knowledge and most recent discoveries relating to the subcellular function of miRNAs and other AGO-associated small RNAs, revealing the emergence of a multitude of functions of the miRNA pathway to control different steps of the gene expression program(s).

Recent computational developments on CLIP-seq data analysis and microRNA targeting implications.

Cross-Linking Immunoprecipitation associated to high-throughput sequencing (CLIP-seq) is a technique used to identify RNA directly bound to RNA-binding proteins across the entire transcriptome in cell or tissue samples. Recent technological and computational advances permit the analysis of many CLIP-seq samples simultaneously, allowing us to reveal the comprehensive network of RNA-protein interaction and to integrate it to other genome-wide analyses. Therefore, the design and quality management of the CLIP-seq analyses are of critical importance to extract clean and biological meaningful information from CLIP-seq experiments. The application of CLIP-seq technique to Argonaute 2 (Ago2) protein, the main component of the microRNA (miRNA)-induced silencing complex, reveals the direct binding sites of miRNAs, thus providing insightful information about the role played by miRNA(s). In this review, we summarize and discuss the most recent computational methods for CLIP-seq analysis, and discuss their impact on Ago2/miRNA-binding site identification and prediction with a regard toward human pathologies.

Paternal obesity: how bad is it for sperm quality and progeny health?

There is substantial evidence that paternal obesity is associated not only with an increased incidence of infertility, but also with an increased risk of metabolic disturbance in adult offspring. Apparently, several mechanisms may contribute to the sperm quality alterations associated with paternal obesity, such as physiological/hormonal alterations, oxidative stress, and epigenetic alterations. Along these lines, modifications of hormonal profiles namely reduced androgen levels and elevated estrogen levels, were found associated with lower sperm concentration and seminal volume. Additionally, oxidative stress in testis may induce an increase of the percentage of sperm with DNA fragmentation. The latter, relate to other peculiarities such as alteration of the embryonic development, increased risk of miscarriage, and development of chronic morbidity in the offspring, including childhood cancers. Undoubtedly, epigenetic alterations (ie, DNA methylation, chromatin modifications, and small RNA deregulation) of sperm related to paternal obesity and their consequences on the progeny are poorly understood determinants of paternal obesity-induced transmission. In this review, we summarize and discuss the data available in the literature regarding the biological, physiological, and molecular consequences of paternal obesity on male fertility potential and ultimately progeny health.

De plus en plus de données tendent à montrer que l'obésité paternelle a non seulement des effets néfastes sur la santé métabolique et reproductive de l'individu mais également sur celle de sa descendance. Les mécanismes mis en jeu dans ce processus incluraient des altérations physiologiques et hormonales des fonctions reproductives de l'homme obèse ainsi que des altérations épigénétiques au niveau du génome spermatique. Les modifications hormonales associées à l'obésité et qui se caractérisent principalement par une réduction du taux d'androgènes et une augmentation du niveau d'estrogène induiraient une altération des paramètres spermatiques, une diminution de la concentration ou de la numération totale en spermatozoïde et du volume séminal. Le stress oxydatif dans le testicule induirait une augmentation de la fragmentation de l'ADN spermatique et pourrait rendre compte de l'augmentation des risques de fausses-couches, des problèmes de développement embryonnaire ainsi que de l'augmentation des risques de mortalité chez la descendance, problèmes fréquemment rencontrés lorsque le père est. obèse. Les modifications épigénétiques (altérations des profils de méthylation de l'ADN, de la structure de la chromatine ou/et des profils d'expression des ARN spermatiques) induites par l'obésité sont, quant à elles, loin d'être comprises, même si elles sont, surement, les vecteurs clés de la transmission épigénétique paternelle des maladies métaboliques. L'objet de cette revue est. de résumer puis de discuter les différentes études expérimentales et épidémiologiques publiés à ce jour sur les conséquences physiologiques et moléculaire de l'obésité paternelle sur la santé de l'individu et sur celle de sa descendance.

Post-transcriptional gene silencing mediated by microRNAs is controlled by nucleoplasmic Sfpq.

There is a growing body of evidence about the presence and the activity of the miRISC in the nucleus of mammalian cells. Here, we show by quantitative proteomic analysis that Ago2 interacts with the nucleoplasmic protein Sfpq in an RNA-dependent fashion. By a combination of HITS-CLIP and transcriptomic analyses, we demonstrate that Sfpq directly controls the miRNA targeting of a subset of binding sites by local binding. Sfpq modulates miRNA targeting in both nucleoplasm and cytoplasm, indicating a nucleoplasmic commitment of Sfpq-target mRNAs that globally influences miRNA modes of action. Mechanistically, Sfpq binds to a sizeable set of long 3'UTRs forming aggregates to optimize miRNA positioning/recruitment at selected binding sites, including let-7a binding to Lin28A 3'UTR. Our results extend the miRNA-mediated post-transcriptional gene silencing into the nucleoplasm and indicate that an Sfpq-dependent strategy for controlling miRNA activity takes place in cells, contributing to the complexity of miRNA-dependent gene expression control.

Viruses and miRNAs: More Friends than Foes.

There is evidence that eukaryotic miRNAs (hereafter called host miRNAs) play a role in the replication and propagation of viruses. Expression or targeting of host miRNAs can be involved in cellular antiviral responses. Most times host miRNAs play a role in viral life-cycles and promote infection through complex regulatory pathways. miRNAs can also be encoded by a viral genome and be expressed in the host cell. Viral miRNAs can share common sequences with host miRNAs or have totally different sequences. They can regulate a variety of biological processes involved in viral infection, including apoptosis, evasion of the immune response, or modulation of viral life-cycle phases. Overall, virus/miRNA pathway interaction is defined by a plethora of complex mechanisms, though not yet fully understood. This article review summarizes recent advances and novel biological concepts related to the understanding of miRNA expression, control and function during viral infections. The article also discusses potential therapeutic applications of this particular host-pathogen interaction.

From benchmarking HITS-CLIP peak detection programs to a new method for identification of miRNA-binding sites from Ago2-CLIP data.

Experimental evidence indicates that about 60% of miRNA-binding activity does not follow the canonical rule about the seed matching between miRNA and target mRNAs, but rather a non-canonical miRNA targeting activity outside the seed or with a seed-like motifs. Here, we propose a new unbiased method to identify canonical and non-canonical miRNA-binding sites from peaks identified by Ago2 Cross-Linked ImmunoPrecipitation associated to high-throughput sequencing (CLIP-seq). Since the quality of peaks is of pivotal importance for the final output of the proposed method, we provide a comprehensive benchmarking of four peak detection programs, namely CIMS, PIPE-CLIP, Piranha and Pyicoclip, on four publicly available Ago2-HITS-CLIP datasets and one unpublished in-house Ago2-dataset in stem cells. We measured the sensitivity, the specificity and the position accuracy toward miRNA binding sites identification, and the agreement with TargetScan. Secondly, we developed a new pipeline, called miRBShunter, to identify canonical and non-canonical miRNA-binding sites based on de novo motif identification from Ago2 peaks and prediction of miRNA::RNA heteroduplexes. miRBShunter was tested and experimentally validated on the in-house Ago2-dataset and on an Ago2-PAR-CLIP dataset in human stem cells. Overall, we provide guidelines to choose a suitable peak detection program and a new method for miRNA-target identification.

RNY (YRNA)-derived small RNAs regulate cell death and inflammation in monocytes/macrophages.

The recent discovery of new classes of small RNAs has opened unknown territories to explore new regulations of physiopathological events. We have recently demonstrated that RNY (or Y RNA)-derived small RNAs (referred to as s-RNYs) are an independent class of clinical biomarkers to detect coronary artery lesions and are associated with atherosclerosis burden. Here, we have studied the role of s-RNYs in human and mouse monocytes/macrophages and have shown that in lipid-laden monocytes/macrophages s-RNY expression is timely correlated to the activation of both NF-κB and caspase 3-dependent cell death pathways. Loss- or gain-of-function experiments demonstrated that s-RNYs activate caspase 3 and NF-κB signaling pathways ultimately promoting cell death and inflammatory responses. As, in atherosclerosis, Ro60-associated s-RNYs generated by apoptotic macrophages are released in the blood of patients, we have investigated the extracellular function of the s-RNY/Ro60 complex. Our data demonstrated that s-RNY/Ro60 complex induces caspase 3-dependent cell death and NF-κB-dependent inflammation, when added to the medium of cultured monocytes/macrophages. Finally, we have shown that s-RNY function is mediated by Toll-like receptor 7 (TLR7). Indeed using chloroquine, which disrupts signaling of endosome-localized TLRs 3, 7, 8 and 9 or the more specific TLR7/9 antagonist, the phosphorothioated oligonucleotide IRS954, we blocked the effect of either intracellular or extracellular s-RNYs. These results position s-RNYs as relevant novel functional molecules that impacts on macrophage physiopathology, indicating their potential role as mediators of inflammatory diseases, such as atherosclerosis.

Developmental epigenetic programming of adult germ cell death disease: Polycomb protein EZH2-miR-101 pathway.

AIM: The Developmental Origin of Health and Disease refers to the concept that early exposure to toxicants or nutritional imbalances during perinatal life induces changes that enhance the risk of developing noncommunicable diseases in adulthood. Patients/materials & methods: An experimental model with an adult chronic germ cell death phenotype resulting from exposure to a xenoestrogen was used. RESULTS: A reciprocal negative feedback loop involving decreased EZH2 protein level and increased miR-101 expression was identified. In vitro and in vivo knockdown of EZH2 induced an apoptotic process in germ cells through increased levels of apoptotic factors (BIM and BAD) and DNA repair alteration via topoisomerase 2B deregulation. The increased miR-101 levels were observed in the animal blood, meaning that miR-101 may be a part of a circulating mark of germ cell death. CONCLUSION: miR-101-EZH2 pathway deregulation could represent a novel pathophysiological epigenetic basis for adult germ cell disease with environmental and developmental origins.

RNY-derived small RNAs as a signature of coronary artery disease.

BACKGROUND: Data from next generation sequencing technologies uncovered the existence of many classes of small RNAs. Recent studies reported that small RNAs are released by cells and can be detected in the blood. In this report, we aimed to discover the occurrence of novel circulating small RNAs in coronary artery disease (CAD). METHODS: We used high-throughput sequencing of small RNAs from human and mouse apoptotic primary macrophages, and analyzed the data by empirical Bayes moderated t-statistics to assess differential expression and the Benjamini and Hochberg method to control the false discovery rate. Results were then confirmed by Northern blot and RT-qPCR in foam cells and in two animal models for atherosclerosis, namely ApoE(-/-) and Ldlr(-/-) mouse lines. Quantitative RT-PCR to detect identified small RNAs, the RNY-derived small RNAs, was performed using sera of 263 patients with CAD compared to 514 matched healthy controls; the Student t-test was applied to statistically assess differences. Associations of small RNAs with clinical characteristics and biological markers were tested using Spearman's rank correlations, while multivariate logistic regressions were performed to test the statistical association of small RNA levels with CAD. RESULTS: Here, we report that, in macrophages stimulated with pro-apoptotic or pro-atherogenic stimuli, the Ro-associated non-coding RNAs, called RNYs or Y-RNAs, are processed into small RNAs (~24-34 nt) referred to as small-RNYs (s-RNYs), including s-RNY1-5p processed from RNY1. A significant upregulation of s-RNY expression was found in aortic arches and blood plasma from ApoE(-/-) and Ldlr(-/-) mice and in serum from CAD patients (P <0.001). Biostatistical analysis revealed a positive association of s-RNY1-5p with hs-CRP and ApoB levels; however, no statistical interaction was found between either of these two markers and s-RNY1-5p in relation to the CAD status. Levels of s-RNY1-5p were also independent from statin and fibrate therapies. CONCLUSION: Our results position the s-RNY1-5p as a relevant novel independent diagnostic biomarker for atherosclerosis-related diseases. Measurement of circulating s-RNY expression would be a valuable companion diagnostic to monitor foam cell apoptosis during atherosclerosis pathogenesis and to evaluate patient's responsiveness to future therapeutic strategies aiming to attenuate apoptosis in foam cells in advanced atherosclerotic lesions.

Regulation of stimulus-inducible gene expression in myeloid cells.

One of the best-characterized and biologically important gene expression programmes in myeloid cells is their response to pro-inflammatory stimuli. Macrophages and DCs in particular are key mediators of immune responses, and are widely-used as prototypes to understand and define the determinants of specific and inducible gene expression. In this review we summarize advances and concepts which have been made towards the understanding of inducible gene expression, with a particular focus on insights gained using the myeloid system as a model. We discuss the emerging concept of layered control of gene regulation and cell identity by different functional classes of transcription factors; and examine recent progress to understanding the molecular processes involved, including the involvement of nucleosome positioning, chromatin modifications, and nuclear architecture. We also address the exciting but less-well understood role of non-coding RNAs in controlling specific gene expression programmes in myeloid and other cell-types.

DICER- and AGO3-dependent generation of retinoic acid-induced DR2 Alu RNAs regulates human stem cell proliferation.

Although liganded nuclear receptors have been established to regulate RNA polymerase II (Pol II)-dependent transcription units, their role in regulating Pol III-transcribed DNA repeats remains largely unknown. Here we report that ~2-3% of the ~100,000-200,000 total human DR2 Alu repeats located in proximity to activated Pol II transcription units are activated by the retinoic acid receptor (RAR) in human embryonic stem cells to generate Pol III-dependent RNAs. These transcripts are processed, initially in a DICER-dependent fashion, into small RNAs (~28-65 nt) referred to as repeat-induced RNAs that cause the degradation of a subset of crucial stem-cell mRNAs, including Nanog mRNA, which modulate exit from the proliferative stem-cell state. This regulation requires AGO3-dependent accumulation of processed DR2 Alu transcripts and the subsequent recruitment of AGO3-associated decapping complexes to the target mRNA. In this way, the RAR-dependent and Pol III-dependent DR2 Alu transcriptional events in stem cells functionally complement the Pol II-dependent neuronal transcriptional program.

Let-7b/c enhance the stability of a tissue-specific mRNA during mammalian organogenesis as part of a feedback loop involving KSRP.

Gene silencing mediated by either microRNAs (miRNAs) or Adenylate/uridylate-rich elements Mediated mRNA Degradation (AMD) is a powerful way to post-transcriptionally modulate gene expression. We and others have reported that the RNA-binding protein KSRP favors the biogenesis of select miRNAs (including let-7 family) and activates AMD promoting the decay of inherently labile mRNAs. Different layers of interplay between miRNA- and AMD-mediated gene silencing have been proposed in cultured cells, but the relationship between the two pathways in living organisms is still elusive. We conditionally deleted Dicer in mouse pituitary from embryonic day (E) 9.5 through Cre-mediated recombination. In situ hybridization, immunohistochemistry, and quantitative reverse transcriptase-PCR revealed that Dicer is essential for pituitary morphogenesis and correct expression of hormones. Strikingly, αGSU (alpha glycoprotein subunit, common to three pituitary hormones) was absent in Dicer-deleted pituitaries. αGSU mRNA is unstable and its half-life increases during pituitary development. A transcriptome-wide analysis of microdissected E12.5 pituitaries revealed a significant increment of KSRP expression in conditional Dicer-deleted mice. We found that KSRP directly binds to αGSU mRNA, promoting its rapid decay; and, during pituitary development, αGSU expression displays an inverse temporal relationship to KSRP. Further, let-7b/c downregulated KSRP expression, promoting the degradation of its mRNA by directly binding to the 3'UTR. Therefore, we propose a model in which let-7b/c and KSRP operate within a negative feedback loop. Starting from E12.5, KSRP induces the maturation of let-7b/c that, in turn, post-transcriptionally downregulates the expression of KSRP itself. This event leads to stabilization of αGSU mRNA, which ultimately enhances the steady-state expression levels. We have identified a post-transcriptional regulatory network active during mouse pituitary development in which the expression of the hormone αGSU is increased by let7b/c through downregulation of KSRP. Our study unveils a functional crosstalk between miRNA- and AMD-dependent gene regulation during mammalian organogenesis events.

KSRP Promotes the Maturation of a Group of miRNA Precuresors.

microrNNA (mirNAs) are small noncoding RNAs that down-regulate gene expression by reducing stability and/or translation of target mRNAs. In animals, miRNAs arise from sequential processing of hairpin primary transcripts by two rNase III domain-containing enzymes, namely Drosha and Dicer, to generate a mature form of about 22 nucleotides. In this chapter we discuss our latest fndings indicating that KSRP is an integral component of both Drosha and Dicer complexes. KSRP binds to the terminal loop sequence of a subset of miRNA precursors promoting their maturation. our data indicate that the terminal loop is a pivotal structure where activators of miRNA processing as well as repressors of miRNA processing act in a coordinated way to convert cellular signals into changes in miRNA expression processing. This uncovers a new level of complexity of miRNA mechanisms for gene expression regulation.