Box C/D snoRNP Autoregulation by a cis-Acting snoRNA in the NOP56 Pre-mRNA.

Box C/D snoRNAs constitute a class of abundant noncoding RNAs that associate with common core proteins to form catalytic snoRNPs. Most of these operate in trans to assist the maturation of rRNAs by guiding and catalyzing the 2'-O-methylation of specific nucleotides. Here, we report that the human intron-hosted box C/D snoRNA snoRD86 acts in cis as a sensor and master switch controlling levels of the limiting snoRNP core protein NOP56, which is important for proper ribosome biogenesis. Our results support a model in which snoRD86 adopts different RNP conformations that dictate the usage of nearby alternative splice donors in the NOP56 pre-mRNA. Excess snoRNP core proteins prevent further production of NOP56 and instead trigger the generation of a cytoplasmic snoRD86-containing NOP56-derived lncRNA via the nonsense-mediated decay pathway. Our findings reveal a feedback mechanism based on RNA structure that controls the precise coordination between box C/D snoRNP core proteins and global snoRNA levels.

Cytokine responses of CD4+ T cells and NKT cells to periodontitis-associated bacteria in individuals with or without periodontitis.

AIM: Periodontitis is an inflammatory disease driven by opportunistic bacteria including Porphyromonas gingivalis and Fusobacterium nucleatum, where T-cell and NKT-cell responses to these bacteria in patients with periodontitis grade B or C are not fully elucidated. The objective is to determine if exaggerated proinflammatory Th-cell responses to periodontitis-associated bacteria, but not commensal bacteria, is a characteristic of increased periodontitis grade. METHODS: Mononuclear cells from patients with periodontitis grade C (n = 26) or grade B (n = 33) and healthy controls (HCs; n = 26) were stimulated with P. gingivalis, F. nucleatum or the commensal bacteria, Staphylococcus epidermidis and Cutibacterium acnes. Cytokine production by different T-cell populations and FOXP3-expression by regulatory T cells were assessed by flow cytometry. RESULTS: Compared to HCs, grade C patients had decreased frequencies of interleukin (IL)-10-producing CD4+ T cells before stimulation (p = .02) and increased frequencies of IFN-y-producing CD4+ T cells after stimulation with P. gingivalis (p = .0019). Grade B patients had decreased frequencies of FOXP3+ CD4+ T cells before (p = .030) before and after stimulation with anti-CD2/anti-CD3/anti-CD28-loaded beads (p = .047), P. gingivalis (p = .013) and S. epidermidis (p = .018). Clinical attachment loss correlated with the frequencies of IFN-y-producing Th1 cells in P. gingivalis- and F. nucleatum-stimulated cultures in grade B patients (p = .023 and p = .048, respectively) and with the frequencies of Th17 cells in P. gingivalis-stimulated cultures (p = .0062) in grade C patients. Patients with periodontitis grade C or grade B showed lower frequencies of IL-10-producing NKT cells than HCs in unstimulated cultures (p = .0043 and p = .027 respectively). CONCLUSIONS: Both periodontitis groups showed decreased frequencies of immunoregulatory T-cell and NKT cell subsets at baseline. Clinical attachment loss correlated with P. gingivalis-induced Th17-responses in grade C patients and with Th1-responses in grade B patients when cells were stimulated with P. gingivalis, supporting that dysregulated pro-inflammatory T-cell responses to periodontitis-associated bacteria contribute to the pathogenesis of periodontitis.

Influence of Soil Moisture on Bioaccumulation, Growth, and Recruitment of Folsomia candida Exposed to Phenanthrene-Polluted Soil.

Climate change has resulted in an increased occurrence of summer droughts in large parts of the world. Low soil moisture has marked impacts on the physiology of soil invertebrates and lowers degradation rates of organic contaminants in soil. Polycyclic aromatic hydrocarbons (PAHs) are hydrophobic contaminants that readily accumulate in the lipids of soil organisms. Here, we exposed springtails (Collembola, small soil living arthropods) to phenanthrene (a common PAH) in combination with a range of soil water contents to investigate the combined effects of these factors on the bioaccumulation, survival, recruitment, and body growth in a full factorial experiment. The results showed that phenanthrene up to 60 mg/kg dry soil had moderate effects on survival (<20%), whereas dry soil (4% soil water content) caused approximately 60% mortality. The bioaccumulation of phenanthrene increased almost 3-fold when soil water content decreased from 22 to 4%. We observed a joint effect of low soil water content and phenanthrene on recruitment, suggesting a synergistic interaction. The recruitment EC50 values of phenanthrene decreased from approximately 40 mg/kg dry soil at 22% soil water content to approximately 10 mg/kg dry soil at 12% soil water content. Our results show that the effects of phenanthrene are more pronounced in dry soil partly because bioaccumulation is enhanced when soils become dry.

Treated periodontitis and recurrent events after first-time myocardial infarction: A Danish nationwide cohort study.

AIM: To investigate the association between previous periodontal treatment and recurrent events after first-time myocardial infarction (MI). MATERIALS AND METHODS: From the Danish nationwide registries, patients with first-time MI between 2000 and 2015 were divided into three groups according to oral health care within 1 year prior to first-time MI. A multiple logistic regression model provided adjusted odds ratios (ORs) with 95% confidence intervals (CIs) to assess the 3-year risk of major adverse cardiovascular events (MACE). RESULTS: A total of 103,949 patients were included. Patients with treated periodontitis (PD) prior to first-time MI had an adjusted 3-year risk of MACE similar to patients presumed periodontally healthy (OR 0.97 [95% CI 0.92-1.03]). Patients with no prior dental visits were significantly older, had more comorbidities and showed significantly increased adjusted 3-year risks of MACE (OR 1.47 [95% CI 1.42-1.52]), cardiovascular death (OR 1.71 [95% CI 1.64-1.78]) and heart failure (OR 1.13 [95% CI 1.07-1.20]) compared with patients presumed periodontally healthy. CONCLUSIONS: Patients with treated PD 1 year prior to first-time MI had a similar risk of recurrent cardiovascular events as patients presumed periodontally healthy. No dental visit prior to first-time MI was an independent risk factor for recurrent events.

mTORC1 promotes TOP mRNA translation through site-specific phosphorylation of LARP1.

LARP1 is a key repressor of TOP mRNA translation. It binds the m7Gppp cap moiety and the adjacent 5'TOP motif of TOP mRNAs, thus impeding the assembly of the eIF4F complex on these transcripts. mTORC1 controls TOP mRNA translation via LARP1, but the details of the mechanism are unclear. Herein we elucidate the mechanism by which mTORC1 controls LARP1's translation repression activity. We demonstrate that mTORC1 phosphorylates LARP1 in vitro and in vivo, activities that are efficiently inhibited by rapamycin and torin1. We uncover 26 rapamycin-sensitive phospho-serine and -threonine residues on LARP1 that are distributed in 7 clusters. Our data show that phosphorylation of a cluster of residues located proximally to the m7Gppp cap-binding DM15 region is particularly sensitive to rapamycin and regulates both the RNA-binding and the translation inhibitory activities of LARP1. Our results unravel a new model of translation control in which the La module (LaMod) and DM15 region of LARP1, both of which can directly interact with TOP mRNA, are differentially regulated: the LaMod remains constitutively bound to PABP (irrespective of the activation status of mTORC1), while the C-terminal DM15 'pendular hook' engages the TOP mRNA 5'-end to repress translation, but only in conditions of mTORC1 inhibition.

Periodontal care attendance in Denmark in 2012-2016 - a nationwide register-based study.

OBJECTIVE: Planning and evaluation of oral healthcare systems rely on monitoring of care patterns. Monitoring periodontal care patterns provide information on the burden and occurrence of periodontitis in the population and on the direct financial cost. The aims of the study were to describe patterns in periodontal care among dental care attenders that might incite subsequent investigation and revised treatment guidelines. Secondly, to estimate the direct societal costs of periodontal care. MATERIAL AND METHODS: A retrospective register-based study utilising data from the Danish Public Health Insurance which includes all dental care attenders in 2012-2016, three years before and one year after a national risk-based recall maintenance program was rolled out in Denmark. RESULTS: The 2.7 million yearly dental care attenders corresponded to ∼60% of the eligible population and in the range of 20-24% received periodontal care. Total expenditure for periodontal care in Denmark increased by 13% from 2012 to 2016, from €78 to €88 million. The proportion of total healthcare funding spent on periodontal care was 0.61% in 2016. CONCLUSIONS: Patients with periodontitis have large out-of-pocket yearly expenses for periodontal care. Despite small changes in periodontal clinical practice that may indicate improved targeting of patients in need of periodontal care, challenges of reaching non-attenders and non-adherence to care are unsolved. More research into outcomes from periodontal therapy in daily practice, seen from both normative and patient perspectives, would help establish knowledge of the efficiency of existing periodontal care systems and help identify barriers and facilitators for attending care in Denmark.

Ongoing decline in insect-pollinated plants across Danish grasslands.

Loss of habitat, eutrophication and reduced grazing intensity are known drivers of landscape-level changes in plant species composition; however, consequences of the massive decline in insect abundance are still to be understood. Pollinator decline can reduce seed set in plants relying on insects for successful reproduction. This may result in a reduced recruitment of insect-pollinated plant species with associated changes in species composition. So far, large-scale studies addressing this issue have relied on few data points-typically consisting of 'historic' records of numbers of insect-pollinated plants compared to present-day records. Such comparisons can provide information as to whether the diversity of insect-pollinated plants has changed, but not whether the process is still ongoing. Here, we use nationwide monitoring data of plant species richness in Danish grasslands from the period 2004-2014, covering 244 grassland sites and encompassing more than 790 flowering plant species. We show an ongoing decrease in insect-pollinated, but not wind-pollinated, plant species across different habitat types. In both dry calcareous and Nardus grasslands, loss of insect-pollinated plants was greatest at sites with low grazing intensity. However, insect-pollinated plants also declined from sites with higher grazing intensity, and plants requiring more specialized insect pollination tended to decline most. In addition to changes in plant diversity driven by land-use intensification, loss of pollinators may also play a role in reducing the richness of insect-pollinated plants. Ongoing reduction in floral richness could further increase the threat to insects relying on these plants as a food source.

Complement split product C3c in saliva as biomarker for periodontitis and response to periodontal treatment.

BACKGROUND AND OBJECTIVE: The complement system is engaged in inflammatory reactions both in the periodontal pockets and in the periodontium itself, where it can mediate tissue destruction. The aim of this study was, first, to compare salivary levels of the total complement system protein C3 and its split product, fluid-phase C3c in patients with periodontitis and periodontally healthy controls. Next, to determine if C3 and C3c levels had biomarker potential in diagnosing and monitoring periodontitis and its treatment. We hypothesized that salivary levels of total C3 and the split product C3c associated with the severity of periodontitis and reflected decreased inflammatory activity after periodontal treatment. METHODS: At baseline, stimulated saliva samples were collected from patients with periodontitis (n = 18) and periodontally healthy controls (n = 15). Subsequently, non-surgical periodontal treatment was performed in the patients, and saliva sampling from patients was repeated two-, six-, and twelve weeks post-treatment (NCT02913248 at clinicaltrials.gov). The patients were grouped as good and poor responders to treatment according to the achieved reduction in bleeding on probing (BOP). Salivary levels of C3 and C3c were quantified using sandwich ELISA. RESULTS: Patients with periodontitis had higher baseline levels of both total C3 and the split product C3c in saliva than did periodontally healthy controls (P < .0001). Receiver operating curve (ROC) analyses discriminated patients with periodontitis from controls based on both C3 (AUC (area under curve) = 0.91, P < .001) and C3c levels (AUC = 0.84, P < .001) in saliva. Periodontal treatment improved all clinical parameters (P < .01). Good responders (n = 10) had lower baseline levels of C3c than poor responders (n = 8), (P < .05), and baseline levels of C3c discriminated between good and poor responders (AUC = 0.80, P < .05). CONCLUSION: In conclusion, patients with periodontitis had higher salivary levels of C3c, and the C3c levels were predictive of reductions in BOP, that is, the poor responders. This suggests that salivary C3c levels possess potential to serve as a biomarker predicting the clinical response to non-surgical periodontal treatment.

The effect of Bt crops on soil invertebrates: a systematic review and quantitative meta-analysis.

The ongoing debate about the ecological effects of Bt-crops calls for thorough reviews about the impact on soil biodiversity and their ecosystem services. Transgenic Bt-crops have been genetically modified by inserting a Bacillus thuriengensis gene so the plant expresses a Cry toxin aimed for insect crop pests. Non-target soil invertebrates are particularly recognized for their contribution to plant nutrient availability and turnover of organic matter and it is therefore relevant to protect these invertebrate taxa. A number of studies have compared the population abundance and biomass of soil invertebrates in agricultural fields planted with genetically modified Bt crops and their conventional counterparts. Here, were review and analyze a selection of studies on Protista, nematodes, Collembola, mites, enchytraeids, and earthworms systematically to empower the evidence for asking the question whether population abundances and biomasses of soil invertebrates are changed by Bt crops compared to conventional crops. 6110 titles were captured, of which 38 studies passed our inclusion criteria, and a final number of 22 publications were subject to data extraction. A database with 2046 records was compiled covering 36 locations and the Bt types Cry1Ab, Cry1Ac, Cry3Bb1 and Cry3Aa. Comparative effect sizes in terms of Hedges' g were calculated irrespectively of statistical significance of effects of the source studies. Cry effects on populations were compared across the studies in a meta-analysis employing a hierarchical Bayesian approach of weighted data according to the level of replication. The temporal development of effect sizes was modelled, thereby taking into account the variable duration of the field experiments. There was considerable variation among soil invertebrate orders, but the sample size was insufficient and the sample heterogeneity too large to draw any credible conclusions on the effect of Cry at the order level. However, across orders there was no significant effect of Cry on soil invertebrates.

Improved microRNA suppression by WPRE-linked tough decoy microRNA sponges.

Our genes are post-transcriptionally regulated by microRNAs (miRNAs) inducing translational suppression and degradation of targeted mRNAs. Strategies to inhibit miRNAs in a spatiotemporal manner in a desired cell type or tissue, or at a desired developmental stage, can be crucial for understanding miRNA function and for pushing forward miRNA suppression as a feasible rationale for genetic treatment of disease. For such purposes, RNA polymerase II (RNA Pol II)-transcribed tough decoy (TuD) miRNA inhibitors are particularly attractive. Here, we demonstrate augmented miRNA suppression capacity of TuD RNA hairpins linked to the Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). This effect is position-dependent and evident only when the WPRE is positioned upstream of the TuD. In accordance, inclusion of the WPRE does not change nuclear export, translation, total levels of TuD-containing RNA transcripts, or cytoplasmic P-body localization, suggesting that previously reported WPRE functions are negligible for improved TuD function. Notably, deletion analysis of TuD-fused WPRE unveils truncated WPRE variants resulting in optimized miRNA suppression. Together, our findings add to the guidelines for production of WPRE-supported anti-miRNA TuDs.

Natural RNA circles function as efficient microRNA sponges.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so-called competing endogenous RNA in humans and target mimicry in plants. We previously identified a highly expressed circular RNA (circRNA) in human and mouse brain. Here we show that this circRNA acts as a miR-7 sponge; we term this circular transcript ciRS-7 (circular RNA sponge for miR-7). ciRS-7 contains more than 70 selectively conserved miRNA target sites, and it is highly and widely associated with Argonaute (AGO) proteins in a miR-7-dependent manner. Although the circRNA is completely resistant to miRNA-mediated target destabilization, it strongly suppresses miR-7 activity, resulting in increased levels of miR-7 targets. In the mouse brain, we observe overlapping co-expression of ciRS-7 and miR-7, particularly in neocortical and hippocampal neurons, suggesting a high degree of endogenous interaction. We further show that the testis-specific circRNA, sex-determining region Y (Sry), serves as a miR-138 sponge, suggesting that miRNA sponge effects achieved by circRNA formation are a general phenomenon. This study serves as the first, to our knowledge, functional analysis of a naturally expressed circRNA.

Translational coregulation of 5'TOP mRNAs by TIA-1 and TIAR.

The response of cells to changes in their environment often requires coregulation of gene networks, but little is known about how this can occur at the post-transcriptional level. An important example of post-transcriptional coregulation is the selective translational regulation in response to growth conditions of mammalian mRNAs that encode protein biosynthesis factors and contain hallmark 5'-terminal oligopyrimidine tracts (5'TOP). However, the responsible trans-factors and the mechanism by which they coregulate 5'TOP mRNAs have remained elusive. Here we identify stress granule-associated TIA-1 and TIAR proteins as key factors in human 5'TOP mRNA regulation, which upon amino acid starvation assemble onto the 5' end of 5'TOP mRNAs and arrest translation at the initiation step, as evidenced by TIA-1/TIAR-dependent 5'TOP mRNA translation repression, polysome release, and accumulation in stress granules. This requires starvation-mediated activation of the GCN2 (general control nonderepressible 2) kinase and inactivation of the mTOR (mammalian target of rapamycin) signaling pathway. Our findings provide a mechanistic explanation to the long-standing question of how the network of 5'TOP mRNAs are coregulated according to amino acid availability, thereby allowing redirection of limited resources to mount a nutrient deprivation response. This presents a fundamental example of how a group of mRNAs can be translationally coregulated in response to changes in the cellular environment.

Comparison of circular RNA prediction tools.

CircRNAs are novel members of the non-coding RNA family. For several decades circRNAs have been known to exist, however only recently the widespread abundance has become appreciated. Annotation of circRNAs depends on sequencing reads spanning the backsplice junction and therefore map as non-linear reads in the genome. Several pipelines have been developed to specifically identify these non-linear reads and consequently predict the landscape of circRNAs based on deep sequencing datasets. Here, we use common RNAseq datasets to scrutinize and compare the output from five different algorithms; circRNA_finder, find_circ, CIRCexplorer, CIRI, and MapSplice and evaluate the levels of bona fide and false positive circRNAs based on RNase R resistance. By this approach, we observe surprisingly dramatic differences between the algorithms specifically regarding the highly expressed circRNAs and the circRNAs derived from proximal splice sites. Collectively, this study emphasizes that circRNA annotation should be handled with care and that several algorithms should ideally be combined to achieve reliable predictions.

Exonuclease hDIS3L2 specifies an exosome-independent 3'-5' degradation pathway of human cytoplasmic mRNA.

Turnover of mRNA in the cytoplasm of human cells is thought to be redundantly conducted by the monomeric 5'-3' exoribonuclease hXRN1 and the 3'-5' exoribonucleolytic RNA exosome complex. However, in addition to the exosome-associated 3'-5' exonucleases hDIS3 and hDIS3L, the human genome encodes another RNase II/R domain protein-hDIS3L2. Here, we show that hDIS3L2 is an exosome-independent cytoplasmic mRNA 3'-5' exonuclease, which exhibits processive activity on structured RNA substrates in vitro. hDIS3L2 associates with hXRN1 in an RNA-dependent manner and can, like hXRN1, be found on polysomes. The impact of hDIS3L2 on cytoplasmic RNA metabolism is revealed by an increase in levels of cytoplasmic RNA processing bodies (P-bodies) upon hDIS3L2 depletion, which also increases half-lives of investigated mRNAs. Consistently, RNA sequencing (RNA-seq) analyses demonstrate that depletion of hDIS3L2, like downregulation of hXRN1 and hDIS3L, causes changed levels of multiple mRNAs. We suggest that hDIS3L2 is a key exosome-independent effector of cytoplasmic mRNA metabolism.

Plant colonization and survival along a hydrological gradient: demography and niche dynamics.

Predicting the effect of a changing environment, e.g., caused by climate change, on realized niche dynamics, and consequently, biodiversity is a challenging scientific question that needs to be addressed. One promising approach is to use estimated demographic parameters for predicting plant abundance and occurrence probabilities. Using longitudinal pinpoint cover data sampled along a hydrological gradient in the Marais poitevin grasslands, France, the effect of the gradient on the demographic probabilities of colonization and survival was estimated. The estimated probabilities and calculated elasticities of survival and colonization covaried with the observed cover of the different species along the hydrological gradient. For example, the flooding tolerant grass A. stolonifera showed a positive response in both colonization and survival to flooding, and the hydrological gradient is clearly the most likely explanation for the occurrence pattern observed for A. stolonifera. The results suggest that knowledge on the processes of colonization and survival of the individual species along the hydrological gradient is sufficient for at least a qualitative understanding of species occurrences along the gradient. The results support the hypothesis that colonization has a predominant role for determining the ecological success along the hydrological gradient compared to survival. Importantly, the study suggests that it may be possible to predict the realized niche of different species from demographic studies. This is encouraging for the important endeavor of predicting realized niche dynamics.

Molecular mechanisms in DM1 - a focus on foci.

Myotonic dystrophy type 1 is caused by abnormal expansion of a CTG-trinucleotide repeat in the gene encoding Dystrophia Myotonica Protein Kinase (DMPK), which in turn leads to global deregulation of gene expression in affected individuals. The transcribed mRNA contains a massive CUG-expansion in the 3' untranslated region (3'UTR) facilitating nucleation of several regulatory RNA-binding proteins, which are thus unable to perform their normal cellular function. These CUG-expanded mRNA-protein aggregates form distinct, primarily nuclear foci. In differentiated muscle cells, most of the CUG-expanded RNA remains in the nuclear compartment, while in dividing cells such as fibroblasts a considerable fraction of the mutant RNA reaches the cytoplasm, consistent with findings that both nuclear and cytoplasmic events are mis-regulated in DM1. Recent evidence suggests that the nuclear aggregates, or ribonuclear foci, are more dynamic than previously anticipated and regulated by several proteins, including RNA helicases. In this review, we focus on the homeostasis of DMPK mRNA foci and discuss how their dynamic regulation may affect disease-causing mechanisms in DM1.

La-related Protein 1 (LARP1) Represses Terminal Oligopyrimidine (TOP) mRNA Translation Downstream of mTOR Complex 1 (mTORC1).

The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of protein synthesis. The best studied targets of mTORC1 in translation are the eukaryotic initiation factor-binding protein 1 (4E-BP1) and ribosomal protein S6 kinase 1 (S6K1). In this study, we identify the La-related protein 1 (LARP1) as a key novel target of mTORC1 with a fundamental role in terminal oligopyrimidine (TOP) mRNA translation. Recent genome-wide studies indicate that TOP and TOP-like mRNAs compose a large portion of the mTORC1 translatome, but the mechanism by which mTORC1 controls TOP mRNA translation is incompletely understood. Here, we report that LARP1 functions as a key repressor of TOP mRNA translation downstream of mTORC1. Our data show the following: (i) LARP1 associates with mTORC1 via RAPTOR; (ii) LARP1 interacts with TOP mRNAs in an mTORC1-dependent manner; (iii) LARP1 binds the 5'TOP motif to repress TOP mRNA translation; and (iv) LARP1 competes with the eukaryotic initiation factor (eIF) 4G for TOP mRNA binding. Importantly, from a drug resistance standpoint, our data also show that reducing LARP1 protein levels by RNA interference attenuates the inhibitory effect of rapamycin, Torin1, and amino acid deprivation on TOP mRNA translation. Collectively, our findings demonstrate that LARP1 functions as an important repressor of TOP mRNA translation downstream of mTORC1.

Intraspecific genetic variation and species coexistence in plant communities.

Many studies report that intraspecific genetic variation in plants can affect community composition and coexistence. However, less is known about which traits are responsible and the mechanisms by which variation in these traits affect the associated community. Focusing on plant-plant interactions, we review empirical studies exemplifying how intraspecific genetic variation in functional traits impacts plant coexistence. Intraspecific variation in chemical and architectural traits promotes species coexistence, by both increasing habitat heterogeneity and altering competitive hierarchies. Decomposing species interactions into interactions between genotypes shows that genotype × genotype interactions are often intransitive. The outcome of plant-plant interactions varies with local adaptation to the environment and with dominant neighbour genotypes, and some plants can recognize the genetic identity of neighbour plants if they have a common history of coexistence. Taken together, this reveals a very dynamic nature of coexistence. We outline how more traits mediating plant-plant interactions may be identified, and how future studies could use population genetic surveys of genotype distribution in nature and methods from trait-based ecology to better quantify the impact of intraspecific genetic variation on plant coexistence.

miRdentify: high stringency miRNA predictor identifies several novel animal miRNAs.

During recent years, miRNAs have been shown to play important roles in the regulation of gene expression. Accordingly, much effort has been put into the discovery of novel uncharacterized miRNAs in various organisms. miRNAs are structurally defined by a hairpin-loop structure recognized by the two-step processing apparatus, Drosha and Dicer, necessary for the production of mature ∼ 22-nucleotide miRNA guide strands. With the emergence of high-throughput sequencing applications, tools have been developed to identify miRNAs and profile their expression based on sequencing reads. However, as the read depth increases, false-positive predictions increase using established algorithms, underscoring the need for more stringent approaches. Here we describe a transparent pipeline for confident miRNA identification in animals, termed miRdentify. We show that miRdentify confidently discloses more than 400 novel miRNAs in humans, including the first male-specific miRNA, which we successfully validate. Moreover, novel miRNAs are predicted in the mouse, the fruit fly and nematodes, suggesting that the pipeline applies to all animals. The entire software package is available at www.ncrnalab.dk/mirdentify.