Distinct hypoxia-induced translational profiles of embryonic and adult-derived macrophages.

Tissue resident macrophages are largely of embryonic (fetal liver) origin and long-lived, while bone marrow-derived macrophages (BMDM) are recruited following an acute perturbation, such as hypoxia in the setting of myocardial ischemia. Prior transcriptome analyses identified BMDM and fetal liver-derived macrophage (FLDM) differences at the RNA expression level. Posttranscriptional regulation determining mRNA stability and translation rate may override transcriptional signals in response to hypoxia. We profiled differentially regulated BMDM and FLDM transcripts in response to hypoxia at the level of mRNA translation. Using a translating ribosome affinity purification (TRAP) assay and RNA-seq, we identified non-overlapping transcripts with increased translation rate in BMDM (Ly6e, vimentin, PF4) and FLDM (Ccl7, Ccl2) after hypoxia. We further identified hypoxia-induced transcripts within these subsets that are regulated by the RNA-binding protein HuR. These findings define translational differences in macrophage subset gene expression programs, highlighting potential therapeutic targets in ischemic myocardium.

A double-negative gene regulatory circuit underlies the virgin behavioral state.

Virgin females of many species conduct distinctive behaviors, compared with post-mated and/or pregnant individuals. In Drosophila, this post-mating switch is initiated by seminal factors, implying that the default female state is virgin. However, we recently showed that loss of miR-iab-4/8-mediated repression of the transcription factor Homothorax (Hth) within the abdominal ventral nerve cord (VNC) causes virgins to execute mated behaviors. Here, we use genomic analysis of mir-iab-4/8 deletion and hth-microRNA (miRNA) binding site mutants (hth[BSmut]) to elucidate doublesex (dsx) as a critical downstream factor. Dsx and Hth proteins are highly complementary in CNS, and Dsx is downregulated in miRNA/hth[BSmut] mutants. Moreover, virgin behavior is highly dose sensitive to developmental dsx function. Strikingly, depletion of Dsx from very restricted abdominal neurons (SAG-1 cells) abrogates female virgin conducts, in favor of mated behaviors. Thus, a double-negative regulatory pathway in the VNC (miR-iab-4/8 ˧ Hth ˧ Dsx) specifies the virgin behavioral state.

MicroRNA-dependent regulation of biomechanical genes establishes tissue stiffness homeostasis.

Vertebrate tissues exhibit mechanical homeostasis, showing stable stiffness and tension over time and recovery after changes in mechanical stress. However, the regulatory pathways that mediate these effects are unknown. A comprehensive identification of Argonaute 2-associated microRNAs and mRNAs in endothelial cells identified a network of 122 microRNA families that target 73 mRNAs encoding cytoskeletal, contractile, adhesive and extracellular matrix (CAM) proteins. The level of these microRNAs increased in cells plated on stiff versus soft substrates, consistent with homeostasis, and suppressed targets via microRNA recognition elements within the 3' untranslated regions of CAM mRNAs. Inhibition of DROSHA or Argonaute 2, or disruption of microRNA recognition elements within individual target mRNAs, such as connective tissue growth factor, induced hyper-adhesive, hyper-contractile phenotypes in endothelial and fibroblast cells in vitro, and increased tissue stiffness, contractility and extracellular matrix deposition in the zebrafish fin fold in vivo. Thus, a network of microRNAs buffers CAM expression to mediate tissue mechanical homeostasis.

Identification and dynamic changes of RNAs isolated from RALY-containing ribonucleoprotein complexes.

RALY is a member of the heterogeneous nuclear ribonucleoprotein family (hnRNP), a large family of RNA-binding proteins involved in many aspects of RNA metabolism. Although RALY interactome has been recently characterized, a comprehensive global analysis of RALY-associated RNAs is lacking and the biological function of RALY remains elusive. Here, we performed RIP-seq analysis to identify RALY interacting RNAs and assessed the role of RALY in gene expression. We demonstrate that RALY binds specific coding and non-coding RNAs and associates with translating mRNAs of mammalian cells. Among the identified transcripts, we focused on ANXA1 and H1FX mRNAs, encoding for Annexin A1 and for the linker variant of the histone H1X, respectively. Both proteins are differentially expressed by proliferating cells and are considered as markers for tumorigenesis. We demonstrate that cells lacking RALY expression exhibit changes in the levels of H1FX and ANXA1 mRNAs and proteins in an opposite manner. We also provide evidence for a direct binding of RALY to the U-rich elements present within the 3΄UTR of both transcripts. Thus, our results identify RALY as a poly-U binding protein and as a regulator of H1FX and ANXA1 in mammalian cells.

MicroRNAs Establish Uniform Traits during the Architecture of Vertebrate Embryos.

Proper functioning of an organism requires cells and tissues to behave in uniform, well-organized ways. How this optimum of phenotypes is achieved during the development of vertebrates is unclear. Here, we carried out a multi-faceted and single-cell resolution screen of zebrafish embryonic blood vessels upon mutagenesis of single and multi-gene microRNA (miRNA) families. We found that embryos lacking particular miRNA-dependent signaling pathways develop a vascular trait similar to wild-type, but with a profound increase in phenotypic heterogeneity. Aberrant trait variance in miRNA mutant embryos uniquely sensitizes their vascular system to environmental perturbations. We discovered a previously unrecognized role for specific vertebrate miRNAs to protect tissue development against phenotypic variability. This discovery marks an important advance in our comprehension of how miRNAs function in the development of higher organisms.

In vivo mutagenesis of miRNA gene families using a scalable multiplexed CRISPR/Cas9 nuclease system.

A large number of microRNAs (miRNAs) are grouped into families derived from the same phylogenetic ancestors. miRNAs within a family often share the same physiological functions despite differences in their primary sequences, secondary structures, or chromosomal locations. Consequently, the generation of animal models to analyze the activity of miRNA families is extremely challenging. Using zebrafish as a model system, we successfully provide experimental evidence that a large number of miRNAs can be simultaneously mutated to abrogate the activity of an entire miRNA family. We show that injection of the Cas9 nuclease and two, four, ten, and up to twenty-four multiplexed single guide RNAs (sgRNAs) can induce mutations in 90% of the miRNA genomic sequences analyzed. We performed a survey of these 45 mutations in 10 miRNA genes, analyzing the impact of our mutagenesis strategy on the processing of each miRNA both computationally and in vivo. Our results offer an effective approach to mutate and study the activity of miRNA families and pave the way for further analysis on the function of complex miRNA families in higher multicellular organisms.

A Dicer-miR-107 Interaction Regulates Biogenesis of Specific miRNAs Crucial for Neurogenesis.

Dicer controls the biogenesis of microRNAs (miRNAs) and is essential for neurogenesis. Recent reports show that the levels and substrate selectivity of DICER result in the preferential biogenesis of specific miRNAs in vitro. However, how dicer expression levels and miRNA biogenesis are regulated in vivo and how this affects neurogenesis is incompletely understood. Here we show that during zebrafish hindbrain development dicer expression levels are controlled by miR-107 to tune the biogenesis of specific miRNAs, such as miR-9, whose levels regulate neurogenesis. Loss of miR-107 function stabilizes dicer levels and miR-9 biogenesis across the ventricular hindbrain zone, resulting in an increase of both proliferating progenitors and postmitotic neurons. miR-9 ectopic accumulation in differentiating neuronal cells recapitulated the excessive neurogenesis phenotype. We propose that miR-107 modulation of dicer levels in differentiating neuronal cells is required to maintain the homeostatic levels of specific miRNAs, whose precise accumulation is essential for neurogenesis.

Proteome-wide characterization of the RNA-binding protein RALY-interactome using the in vivo-biotinylation-pulldown-quant (iBioPQ) approach.

RALY is a member of the heterogeneous nuclear ribonucleoproteins, a family of RNA-binding proteins generally involved in many processes of mRNA metabolism. No quantitative proteomic analysis of RALY-containing ribonucleoparticles (RNPs) has been performed so far, and the biological role of RALY remains elusive. Here, we present a workflow for the characterization of RALY's interaction partners, termed iBioPQ, that involves in vivo biotinylation of biotin acceptor peptide (BAP)-fused protein in the presence of the prokaryotic biotin holoenzyme synthetase of BirA so that it can be purified using streptavidin-coated magnetic beads, circumventing the need for specific antibodies and providing efficient pulldowns. Protein eluates were subjected to tryptic digestion and identified using data-independent acquisition on an ion-mobility enabled high-resolution nanoUPLC-QTOF system. Using label-free quantification, we identified 143 proteins displaying at least 2-fold difference in pulldown compared to controls. Gene Ontology overrepresentation analysis revealed an enrichment of proteins involved in mRNA metabolism and translational control. Among the most abundant interacting proteins, we confirmed RNA-dependent interactions of RALY with MATR3, PABP1 and ELAVL1. Comparative analysis of pulldowns after RNase treatment revealed a protein-protein interaction of RALY with eIF4AIII, FMRP, and hnRNP-C. Our data show that RALY-containing RNPs are much more heterogeneous than previously hypothesized.

Comparison of quantitative PCR and flow cytometry as cellular viability methods to study bacterial membrane permeabilization following supercritical CO2 treatment.

Foodborne illness due to bacterial pathogens is increasing worldwide as a consequence of the higher consumption of fresh and minimally processed food products, which are more easily cross-contaminated. The efficiency of food pasteurization methods is usually measured by c.f.u. plate counts, a method discriminating viable from dead cells on the basis of the ability of cells to replicate and form colonies on standard growth media, thus ignoring viable but not cultivable cells. Supercritical CO2 (SC-CO2) has recently emerged as one of the most promising fresh food pasteurization techniques, as an alternative to traditional, heat-based methods. In the present work, using three SC-CO2-treated foodborne bacteria (Listeria monocytogenes, Salmonella enterica and Escherichia coli) we tested and compared the performance of alternative viability test methods based on membrane permeability: propidium monoazide quantitative PCR (PMA-qPCR) and flow cytometry (FCM). Results were compared based on plate counts and fluorescent microscopy measurements, which showed that the former dramatically reduced the number of cultivable cells by more than 5 log units. Conversely, FCM provided a much more detailed picture of the process, as it directly quantifies the number of total cells and distinguishes among three categories, including intact, partially permeabilized and permeabilized cells. A comparison of both PMA-qPCR and FCM with plate count data indicated that only a fraction of intact cells maintained the ability to replicate in vitro. Following SC-CO2 treatment, FCM analysis revealed a markedly higher level of bacterial membrane permeabilization of L. monocytogenes with respect to E. coli and S. enterica. Furthermore, an intermediate permeabilization state in which the cellular surface was altered and biovolume increased up to 1.5-fold was observed in L. monocytogenes, but not in E. coli or S. enterica. FCM thus compared favourably with other methods and should be considered as an accurate analytical tool for applications in which monitoring bacterial viability status is of importance, such as microbiological risk assessment in the food chain or in the environment.

Intracellular trafficking of RNASET2, a novel component of P-bodies.

BACKGROUND INFORMATION: The ribonucleases (RNases) constitute a heterogeneous group of enzymes, which exert diverse and specific biological functions. Several RNases have been shown to control gene expression and cell differentiation. RNASET2, a novel member of the Rh/T2/S family of RNases, exerts micro-environmental control of malignancy in different experimental models with a general onco-suppressor activity, since it prevents cancer proliferation. Indeed, RNASET2 was found to be downregulated at the transcript level in several primary ovarian tumours or cell lines and in melanoma cell lines. Although recent works shed light on the biological role of RNASET2 in delaying tumour growth, its trafficking within the cell is still poorly understood. RNASET2 seems to play diverse biological roles including turnover of tRNA in yeast as well as rRNA degradation in zebrafish. RESULTS: Here, we have studied the intracellular trafficking of RNASET2 in mammalian cells. RNASET2 co-localizes with markers for the trans-Golgi network (TGN), which is the central sorting and processing station of the secretory pathway. Moreover, using the temperature-sensitive vesicular stomatitis glycoprotein, we demonstrate that RNASET2 undergoes delivery to the plasma membrane. In contrast to other RNA-interacting proteins, RNASET2 does not accumulate in stress granules upon metabolic stress in mammalian cells. Surprisingly, RNASET2 shows co-localization with processing bodies (P-bodies), which increases upon metabolic stress. Finally, cells lacking RNASET2 show a reduced numbers of P-bodies. CONCLUSIONS: In this study, we have identified two distinct cellular pools of RNASET2-containing granules. One pool undergoes membrane delivery using the TGN, and it is released to the extracellular environment. The second pool is recruited into P-bodies, suggesting a possible involvement of RNASET2 in P-body formation in mammalian cells.