We present here miRTrace, the first algorithm to trace microRNA sequencing data back to their taxonomic origins. This is a challenge with profound implications for forensics, parasitology, food ...control, and research settings where cross-contamination can compromise results. miRTrace accurately (> 99%) assigns real and simulated data to 14 important animal and plant groups, sensitively detects parasitic infection in mammals, and discovers the primate origin of single cells. Applying our algorithm to over 700 public datasets, we find evidence that over 7% are cross-contaminated and present a novel solution to clean these computationally, even after sequencing has occurred. miRTrace is freely available at https://github.com/friedlanderlab/mirtrace .
Abstract
Single-cell RNA sequencing studies on gene co-expression patterns could yield important regulatory and functional insights, but have so far been limited by the confounding effects of ...differentiation and cell cycle. We apply a tailored experimental design that eliminates these confounders, and report thousands of intrinsically covarying gene pairs in mouse embryonic stem cells. These covariations form a network with biological properties, outlining known and novel gene interactions. We provide the first evidence that miRNAs naturally induce transcriptome-wide covariations and compare the relative importance of nuclear organization, transcriptional and post-transcriptional regulation in defining covariations. We find that nuclear organization has the greatest impact, and that genes encoding for physically interacting proteins specifically tend to covary, suggesting importance for protein complex formation. Our results lend support to the concept of post-transcriptional RNA operons, but we further present evidence that nuclear proximity of genes may provide substantial functional regulation in mammalian single cells.
Previous large-scale studies have uncovered many features that determine the processing of microRNA (miRNA) precursors; however, they have been conducted in vitro. Here, we introduce MapToCleave, ...a method to simultaneously profile processing of thousands of distinct RNA structures in living cells. We find that miRNA precursors with a stable lower basal stem are more efficiently processed and also have higher expression in vivo in tissues from 20 animal species. We systematically compare the importance of known and novel sequence and structural features and test biogenesis of miRNA precursors from 10 animal and plant species in human cells. Lastly, we provide evidence that the GHG motif better predicts processing when defined as a structure rather than sequence motif, consistent with recent cryogenic electron microscopy (cryo-EM) studies. In summary, we apply a screening assay in living cells to reveal the importance of lower basal stem stability for miRNA processing and in vivo expression.
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•MapToCleave method allows simultaneous screening of 12,472 RNA structures in cells•The biogenesis of ∼15% of human miRNAs is influenced by cell-dependent factors•We perform a systematic comparison of the importance of miRNA biogenesis features•Stability of first seven base pairs of the stem tunes processing in cells and tissues
Numerous miRNA features that facilitate biogenesis are known, but most have been identified in vitro. Kang et al. re-evaluate miRNA biogenesis in living cells and in animal tissues, and they find that stability of the first seven base pairs of the stem is particularly important for processing in cells.
Accumulating behavioural data indicate that aggregation pheromones may mediate the formation and maintenance of mosquito swarms. However, chemical cues possibly luring mosquitoes to swarms have not ...been adequately investigated, and the likely molecular incitants of these complex reproductive behaviours remain unknown. Here we show that males of the important malaria vector species Anopheles arabiensis and An. gambiae produce and release aggregation pheromones that attract individuals to the swarm and enhance mating success. We found that males of both species released significantly higher amounts of 3-hydroxy-2-butanone (acetoin), 6-methyl-5-hepten-2-one (sulcatone), octanal, nonanal and decanal during swarming in the laboratory. Feeding males with stable-isotope-labelled glucose revealed that the males produced these five compounds. A blend composed of synthetic analogues to these swarming odours proved highly attractive to virgin males and females of both species under laboratory conditions and substantially increased mating in five African malaria vectors (An. gambiae, An. coluzzii, An. arabiensis, An. merus and An. funestus) in semi-field experiments. Our results not only narrow a conspicuous gap in understanding a vital aspect of the chemical ecology of male mosquitoes but also demonstrate fundamental roles of rhythmic and metabolic genes in the physiology and behavioural regulation of these vectors. These identified aggregation pheromones have great potential for exploitation against these highly dangerous insects. Manipulating such pheromones could increase the efficacy of malaria-vector control programmes.
The siRNA and piRNA pathways have been shown in insects to be essential for regulation of gene expression and defence against exogenous and endogenous genetic elements (viruses and transposable ...elements). The vast majority of endogenous small RNAs produced by the siRNA and piRNA pathways originate from repetitive or transposable elements (TE). In D. melanogaster, TE-derived endogenous siRNAs and piRNAs are involved in genome surveillance and maintenance of genome integrity. In the medically relevant malaria mosquito Anopheles gambiae TEs constitute 12-16% of the genome size. Genetic variations induced by TE activities are known to shape the genome landscape and to alter the fitness in An. gambiae.
Here, using bioinformatics approaches we analyzed the small RNA data sets from 6 libraries formally reported in a previous study and examined the expression of the mixed germline/somatic siRNAs and piRNAs produced in adult An. gambiae females. We characterized a large population of TE-derived endogenous siRNAs and piRNAs, which constitutes 56-60% of the total siRNA and piRNA reads in the analysed libraries. Moreover, we identified a number of protein coding genes producing gene-specific siRNAs and piRNAs that were generally expressed at much lower levels than the TE-associated small RNAs. Detailed sequence analysis revealed that An. gambiae piRNAs were produced by both "ping-pong" dependent (TE-associated piRNAs) and independent mechanisms (genic piRNAs). Similarly to D. melanogaster, more than 90% of the detected piRNAs were produced from TE-associated clusters in An. gambiae. We also found that biotic stress as blood feeding and infection with Plasmodium parasite, the etiological agent of malaria, modulated the expression levels of the endogenous siRNAs and piRNAs in An. gambiae.
We identified a large and diverse set of the endogenously derived siRNAs and piRNAs that share common and distinct aspects of small RNA expression across insect species, and inferred their impact on TE and gene activity in An. gambiae. The TE-specific small RNAs produced by both the siRNA and piRNA pathways represent an important aspect of genome stability and genetic variation, which might have a strong impact on the evolution of the genome and vector competence in the malaria mosquitoes.
The Drosophila bHLH proneural factors Achaete (Ac) and Scute (Sc) are expressed in clusters of cells (proneural clusters), providing the cells with the potential to develop a neural fate. Mediodorsal ...proneural patterning is mediated through the GATA transcription factor Pannier (Pnr) that activates ac/sc directly through binding to the dorsocentral (DC) enhancer of ac/sc. Besides, the Gfi transcription factor Senseless (Sens), a target of Ac/Sc, synergizes with ac/sc in the presumptive sensory organ precursors (SOPs). Here we investigate, through new genetic tools, the function of dLMO, the Drosophila LIM only transcription factor that was already known to control wing development. We show that dLMO gene encodes two isoforms, dLMO-RA and dLMO-RB. dLMO null and dLMO-RA− deletions have similar phenotypes, lacking thoracic and wing margin sensory organs (SO), while dLMO-RB− deletion has normal SOs. At early stages, dLMO-RA is expressed in proneural clusters, however later it is excluded from the SOPs. We found that dLMO functions as a Pnr coactivator to promote ac/sc expression. In the late SOPs, where dLMO-PA is not expressed, Pnr participates to the Sens-dependent regulation of ac/sc. Taken together these results suggest that dLMO-PA is the major isoform that is required for early activation of ac/sc expression.
The pattern of the external sensory organs (SO) in Drosophila depends on the activity of the basic helix–loop–helix (bHLH) transcriptional activators Achaete/Scute (Ac/Sc) that are expressed in ...clusters of cells (proneural clusters) and provide the cells with the potential to develop a neural fate. In the mesothorax, the GATA1 transcription factor Pannier (Pnr), together with its cofactor Chip, activates ac/sc genes directly through binding to the dorsocentral enhancer (DC) of ac/sc. We identify the LIM-homeodomain (LIM-HD) transcription factor Islet (Isl) by genetic screening and investigate its role in the thoracic prepatterning. We show that isl loss-of-function mutations result in expanded Ac expression in DC and scutellar (SC) proneural clusters and formation of ectopic sensory organs. Overexpression of Isl decreases proneural expression and suppresses bristle development. Moreover, Isl is coexpressed with Pnr in the posterior region of the mesothorax. In the DC proneural cluster, Isl antagonizes Pnr activity both by dimerization with the DNA-binding domain of Pnr and via competitive inhibition of the Chip–bHLH interaction. We propose that sensory organ prepatterning relies on the antagonistic activity of individual Chip-binding factors. The differential affinities of these binding-factors and their precise stoichiometry are crucial in specifying prepatterns within the different proneural clusters.
microRNAs (miRNAs) are a highly abundant class of small noncoding regulatory RNAs that post-transcriptionally regulate gene expression in multicellular organisms. miRNAs are involved in a wide range ...of biological and physiological processes, including the regulation of host immune responses to microbial infections. Small-scale studies of miRNA expression in the malaria mosquito Anopheles gambiae have been reported, however no comprehensive analysis of miRNAs has been performed so far.
Using small RNA sequencing, we characterized de novo A. gambiae miRNA repertoire expressed in adult sugar- and blood-fed females. We provided transcriptional evidences for 123 miRNAs, including 58 newly identified miRNAs. Out of the newly described miRNAs, 19 miRNAs are homologs to known miRNAs in other insect species and 17 miRNAs share sequence similarity restricted to the seed sequence. The remaining 21 novel miRNAs displayed no obvious sequence homology with known miRNAs. Detailed bioinformatics analysis of the mature miRNAs revealed a sequence variation occurring at their 5'-end and leading to functional seed shifting in more than 5% of miRNAs. We also detected significant sequence heterogeneity at the 3'-ends of the mature miRNAs, mostly due to imprecise processing and post-transcriptional modifications. Comparative analysis of arm-switching events revealed the existence of species-specific production of dominant mature miRNAs induced by blood feeding in mosquitoes. We also identified new conserved and fragmented miRNA clusters and A. gambiae-specific miRNA gene duplication. Using miRNA expression profiling, we identified the differentially expressed miRNAs at an early time point after regular blood feeding and after infection with the rodent malaria parasite Plasmodium berghei. Significant changes were detected in the expression levels of 4 miRNAs in blood-fed mosquitoes, whereas 6 miRNAs were significantly upregulated after P. berghei infection.
In the current study, we performed the first systematic analysis of miRNAs in A. gambiae. We provided new insights on mature miRNA sequence diversity and functional shifts in the mosquito miRNA evolution. We identified a set of the differentially expressed miRNAs that respond to normal and infectious blood meals. The extended set of Anopheles miRNAs and their isoforms provides a basis for further experimental studies of miRNA expression patterns and biological functions in A. gambiae.
The best characterized chromatin insulator in Drosophila is the Suppressor of Hairy wing binding region contained within the gypsy retrotransposon. Although cellular functions have been suggested, no ...role has been found yet for the multitude of endogenous Suppressor of Hairy wing binding sites. Here we show that two Suppressor of Hairy wing binding sites in the intergenic region between the yellow gene and the Achaete-scute gene complex form a functional insulator. Genetic analysis shows that at least two proteins, Suppressor of Hairy wing and Modifier of MDG4, required for the activity of this insulator, are involved in the transcriptional regulation of Achaete-scute.
The GATA factor Pannier activates proneural achaete/scute (ac/sc) expression during development of the sensory organs of Drosophila through enhancer binding. Chip bridges Pannier with the ...(Ac/Sc)-Daughterless heterodimers bound to the promoter and facilitates the enhancer-promoter communication required for proneural development. We show here that this communication is regulated by Osa, which is recruited by Pannier and Chip. Osa belongs to Brahma chromatin remodeling complexes and we show that Osa negatively regulates ac/sc. Consequently, Pannier and Chip also play an essential role during repression of proneural gene expression. Our study suggests that altering chromatin structure is essential for regulation of enhancer-promoter communication.
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