Various species of non‐coding RNAs (ncRNAs) are enriched in specific subcellular compartments, but the mechanisms orchestrating their localization and their local functions remain largely unknown. We ...investigated both aspects using the elongating retinal ganglion cell axon and its tip, the growth cone, as models. We reveal that specific endogenous precursor microRNAs (pre‐miRNAs) are actively trafficked to distal axons by hitchhiking primarily on late endosomes/lysosomes. Upon exposure to the axon guidance cue semaphorin 3A (Sema3A), pre‐miRNAs are processed specifically within axons into newly generated miRNAs, one of which, in turn, silences the basal translation of tubulin beta 3 class III (TUBB3), but not amyloid beta precursor protein (APP). At the organismal level, these mature miRNAs are required for growth cone steering and a fully functional visual system. Overall, our results uncover a novel mode of ncRNA transport from one cytosolic compartment to another within polarized cells. They also reveal that newly generated miRNAs are critical components of a ncRNA‐based signaling pathway that transduces environmental signals into the structural remodeling of subcellular compartments.
Synopsis
Mechanisms regulating subcellular localization and local function of non‐coding RNA are not well understood. Here, neuronal pre‐miRNAs are found to traffic along axons docked to endosomes, with axonal processing of pre‐miRNAs leading to the inhibition of local protein synthesis.
Precursor miRNAs hitchhike onto late endosomes/lysosomes to reach the growth cone.
Sema3A, but not Slit2, axon guidance cue induces local biogenesis of specific miRNAs within axons.
Newly generated miRNAs inhibit basal translation of TUBB3 but not APP upon Sema3A exposure.
miRNAs regulate growth cone steering and the establishment of functional connections.
Pre‐miRNAs transported to distal axons are locally processed upon exposure to Sema3A and are required for growth cone steering and a fully functional visual system.
Early lineage diversification is central to understand what mutational events drive species divergence. Particularly, gene misregulation in interspecific hybrids can inform about what genes and ...pathways underlie hybrid dysfunction. In Drosophila hybrids, how regulatory evolution impacts different reproductive tissues remains understudied. Here, we generate a new genome assembly and annotation in Drosophila willistoni and analyse the patterns of transcriptome divergence between two allopatrically evolved D. willistoni subspecies, their male sterile and female fertile hybrid progeny across testis, male accessory gland, and ovary. Patterns of transcriptome divergence and modes of regulatory evolution were tissue‐specific. Despite no indication for cell‐type differences in hybrid testis, this tissue exhibited the largest magnitude of expression differentiation between subspecies and between parentals and hybrids. No evidence for anomalous dosage compensation in hybrid male tissues was detected nor was a differential role for the neo‐ and the ancestral arms of the D. willistoni X chromosome. Compared to the autosomes, the X chromosome appeared enriched for transgressively expressed genes in testis despite being the least differentiated in expression between subspecies. Evidence for fine genome clustering of transgressively expressed genes suggests a role of chromatin structure on hybrid gene misregulation. Lastly, transgressively expressed genes in the testis of the sterile male progeny were enriched for GO terms not typically associated with sperm function, instead hinting at anomalous development of the reproductive tissue. Our thorough tissue‐level portrait of transcriptome differentiation between recently diverged D. willistoni subspecies and their hybrids provides a more nuanced view of early regulatory changes during speciation.
Rhizopus microsporus is an early-diverging fungal species with importance in ecology, agriculture, food production, and public health. Pathogenic strains of R. microsporus harbor an intracellular ...bacterial symbiont, Mycetohabitans (formerly named Burkholderia). This vertically transmitted bacterial symbiont is responsible for the production of toxins crucial to the pathogenicity of Rhizopus and remarkably also for fungal reproduction. Here we show that R. microsporus can live not only in symbiosis with bacteria but also with two viral members of the genus Narnavirus. Our experiments revealed that both viruses replicated similarly in the growth conditions we tested. Viral copies were affected by the developmental stage of the fungus, the substrate, and the presence or absence of Mycetohabitans. Absolute quantification of narnaviruses in isolated asexual sporangiospores and sexual zygospores indicates their vertical transmission. By curing R. microsporus of its viral and bacterial symbionts and reinfecting bacteria to reestablish symbiosis, we demonstrate that these viruses affect fungal biology. Narnaviruses decrease asexual reproduction, but together with Mycetohabitans, are required for sexual reproductive success. This fungal-bacterial-viral system represents an outstanding model to investigate three-way microbial symbioses and their evolution.
New sequencing technologies pose significant challenges in terms of data complexity and magnitude. It is essential that efficient software is developed with performance that scales with this growth ...in sequence information. Here we present a comprehensive and integrated set of tools for the analysis of data from large scale sequencing experiments. It supports adapter detection and removal, demultiplexing of barcodes, paired-end data, a range of read architectures and the efficient removal of sequence redundancy. Sequences can be trimmed and filtered based on length, quality and complexity. Quality control plots track sequence length, composition and summary statistics with respect to genomic annotation. Several use cases have been integrated into a single streamlined pipeline, including both mRNA and small RNA sequencing experiments. This pipeline interfaces with existing tools for genomic mapping and differential expression analysis.
In mammalian systems RNA can move between cells via vesicles. Here we demonstrate that the gastrointestinal nematode Heligmosomoides polygyrus, which infects mice, secretes vesicles containing ...microRNAs (miRNAs) and Y RNAs as well as a nematode Argonaute protein. These vesicles are of intestinal origin and are enriched for homologues of mammalian exosome proteins. Administration of the nematode exosomes to mice suppresses Type 2 innate responses and eosinophilia induced by the allergen Alternaria. Microarray analysis of mouse cells incubated with nematode exosomes in vitro identifies Il33r and Dusp1 as suppressed genes, and Dusp1 can be repressed by nematode miRNAs based on a reporter assay. We further identify miRNAs from the filarial nematode Litomosoides sigmodontis in the serum of infected mice, suggesting that miRNA secretion into host tissues is conserved among parasitic nematodes. These results reveal exosomes as another mechanism by which helminths manipulate their hosts and provide a mechanistic framework for RNA transfer between animal species.
Sylamer is a method for detecting microRNA target and small interfering RNA off-target signals in 3′ untranslated regions from a ranked gene list, sorted from upregulated to downregulated, after a ...microRNA perturbation or RNA interference experiment. The output is a landscape plot that tracks occurrence biases using hypergeometric P-values for all words across the gene ranking. We demonstrated the utility, speed and accuracy of this approach on several datasets.
miRNAs are essential regulators of cell identity, yet their role in early embryo development in plants remains largely unexplored. To determine the earliest stage at which miRNAs act to promote ...pattern formation in embryogenesis, we examined a series of mutant alleles in the Arabidopsis thaliana miRNA biogenesis enzymes DICER-LIKE 1 (DCL1), SERRATE (SE), and HYPONASTIC LEAVES 1 (HYL1). Cellular and patterning defects were observed in dcl1, se and hyl1 embryos from the zygote through the globular stage of embryogenesis. To identify miRNAs that are expressed in early embryogenesis, we sequenced mRNAs from globular stage Columbia wild type (wt) and se-1 embryos, and identified transcripts potentially corresponding to 100 miRNA precursors. Considering genome location and transcript increase between wt and se-1, 39 of these MIRNAs are predicted to be bona fide early embryo miRNAs. Among these are conserved miRNAs such as miR156, miR159, miR160, miR161, miR164, miR165, miR166, miR167, miR168, miR171, miR319, miR390 and miR394, as well as miRNAs whose function has never been characterized. Our analysis demonstrates that miRNAs promote pattern formation beginning in the zygote, and provides a comprehensive dataset for functional studies of individual miRNAs in Arabidopsis embryogenesis.
•dcl1, serrate and hyl1 show phenotypes beginning in the zygote.•Early dcl1, serrate and hyl1 phenotypes resemble yoda, wrky2, wox8/wox9 and auxin mutants.•At least 39 MIRNAs are expressed in globular stage embryos.•Embryo expressed MIRNAs correspond to well-known and uncharacterized miRNAs.
The process of erythropoiesis must be efficient and robust to supply the organism with red bloods cells both under condition of homeostasis and stress. The microRNA (miRNA) pathway was recently shown ...to regulate erythroid development. Here, we show that expression of the locus encoding miR-144 and miR-451 is strictly dependent on Argonaute 2 and is required for erythroid homeostasis. Mice deficient for the miR-144/451 cluster display a cell autonomous impairment of late erythroblast maturation, resulting in erythroid hyperplasia, splenomegaly, and a mild anemia. Analysis of gene expression profiles from wild-type and miR-144/451-deficient erythroblasts revealed that the miR-144/451 cluster acts as a "tuner" of gene expression, influencing the expression of many genes. MiR-451 imparts a greater impact on target gene expression than miR-144. Accordingly, mice deficient in miR-451 alone exhibited a phenotype indistinguishable from miR-144/451-deficient mice. Thus, the miR-144/451 cluster tunes gene expression to impart a robustness to erythropoiesis that is critical under conditions of stress.
Summary
The RNAi machinery is generally involved in genome protection in filamentous fungi; however, the physiological role of RNAi has been poorly studied in fungal models. Here, we report that in ...the filamentous fungus Trichoderma atroviride, the products of the dcr2 and rdr3 genes control reproductive development, because mutations in these genes affect conidiation. In addition, Dcr1 together with Dcr2 control vegetative growth since Δdcr1, Δdcr2 and Δdcr1Δdcr2 present morphological alterations. Whole‐genome transcriptional analysis of WT, Δdcr1, Δdcr2 and Δdcr1Δdcr2 show that each Dicer controls different biological processes, such as development or metabolism, which could explain the lack of conidiation in the mutants. Finally, we observed sRNAs that are differentially expressed in the WT and Δdcr2. The expression of some of these sRNAs correlates with the expression of differential transcripts, suggesting that these mRNAs may contain the corresponding targets. Together these data show that in T. atroviride, the RNAi machinery plays a central role in endogenous processes such as development and fitness, beyond controlling genome protection against invasive nucleic acids as reported for other fungi.
Root systems consist of different root types (RTs) with distinct developmental and functional characteristics. RTs may be individually reprogrammed in response to their microenvironment to maximize ...adaptive plasticity. Molecular understanding of such specific remodeling—although crucial for crop improvement—is limited. Here, RT-specific transcriptomes of adult rice crown, large and fine lateral roots were assessed, revealing molecular evidence for functional diversity among individual RTs. Of the three rice RTs, crown roots displayed a significant enrichment of transcripts associated with phytohormones and secondary cell wall (SCW) metabolism, whereas lateral RTs showed a greater accumulation of transcripts related to mineral transport. In nature, arbuscular mycorrhizal (AM) symbiosis represents the default state of most root systems and is known to modify root system architecture. Rice RTs become heterogeneously colonized by AM fungi, with large laterals preferentially entering into the association. However, RT-specific transcriptional responses to AM symbiosis were quantitatively most pronounced for crown roots despite their modest physical engagement in the interaction. Furthermore, colonized crown roots adopted an expression profile more related to mycorrhizal large lateral than to noncolonized crown roots, suggesting a fundamental reprogramming of crown root character. Among these changes, a significant reduction in SCW transcripts was observed that was correlated with an alteration of SCW composition as determined by mass spectrometry. The combined change in SCW, hormone- and transport-related transcript profiles across the RTs indicates a previously overlooked switch of functional relationships among RTs during AM symbiosis, with a potential impact on root system architecture and functioning.
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