A change in taste: The role of MicroRNAs in altering hedonic value

The mechanisms associated with neophobia, and anhedonia remain largely unknown. Neuropsychological disorders such as depression and schizophrenia are associated with excessive fear and anhedonia and have been linked to microRNA 137. We hypothesized that microRNAs (miRNAs) in the snail Lymnaea stagnalis are important for regulating feeding behaviour through either preventing neophobia or establishing hedonic value. To test these hypotheses, we used an injection of Poly-L-Lysine (PLL) to inhibit miRNA biogenesis and observed its effects on feeding behaviour. We repeated these experiments with pre-exposure to novel stimuli capable of eliciting neophobia to disentangle the processes predicted to regulate feeding behaviour. Next, we exposed snails to food stimuli of high hedonic value after PLL injection to reset their hedonic value for that food. Finally, we consolidated our results with previous research by examining the effect of PLL injection on a one trial appetitive classical conditioning procedure (1TT) to induce long term memory (LTM). We found that miRNAs are likely not required for preventing neophobia. Moreover, we discovered that snails experienced anhedonia in response to inhibition of miRNA biogenesis, resulting in diminished feeding behaviour for food stimuli with a previously high hedonic value. Snails showed diminished feeding behaviour for multiple food stimuli of high hedonic value post 1TT with PLL injection. This finding suggested that PLL causes anhedonia rather than an impairment of LTM formation following the 1TT procedure. This is the first evidence suggesting that inhibiting the biogenesis of miRNAs contributes to anhedonia in Lymnaea.

Conservation of Differential Animal MicroRNA Processing by Drosha and Dicer

In complex biochemical systems, an enzyme, protein, or RNA, symbolized as E, has hundreds or thousands of substrates or interacting partners. The relative specificity hypothesis proposes that such an E would differentially interact with and influence its many distinct, downstream substrates, thereby regulating the underlying biological process (es). The importance of relative specificity has been underappreciated, and evidence of its physiological consequences particularly lacking. Previously we showed that human Drosha and Dicer ribonucleases (RNases) both discriminate their respective microRNA (miRNA) substrates, and that differential cleavage by Drosha contributes to global differential miRNA expression. If relative specificity is an important biological mechanism, it should be evolutionarily conserved. To test this hypothesis, we hereby examined the cleavage of hundreds of zebrafish and fruitfly miRNA intermediates by Drosha and Dicer and the impact on miRNA biogenesis in these organisms. We showed that Drosha action regulates differential miRNA expression in zebrafish and fruitflies and identified the conserved secondary structure features and sequences in miRNA transcripts that control Drosha activity and miRNA expression. Our results established the conservation of miRNA processing mechanisms and regulatory functions by Drosha and Dicer, greatly strengthened the evidence for the physiological consequences of relative specificity as well as demonstrated its evolutionary significance.

MicroRNA biogenesis and activity in plant cell dedifferentiation stimulated by cell wall removal

Background Despite the frequent use of protoplast-to-plant system in in vitro cultures of plants, the molecular mechanisms regulating the first and most limiting stages of this process, i.e., protoplast dedifferentiation and the first divisions leading to the formation of a microcallus, have not been elucidated. Results In this study, we investigated the function of miRNAs in the dedifferentiation of A. thaliana mesophyll cells in a process stimulated by the enzymatic removal of the cell wall. Leaf cells, protoplasts and CDPs (cells derived from protoplasts) cultured for 24, 72 and 120 h (first cell division). In protoplasts, a strong decrease in the amount of AGO1 in both the nucleus and the cytoplasm, as well as dicing bodies (DBs), which are considered to be sites of miRNA biogenesis, was shown. However during CDPs division, the amounts of AGO1 and DBs strongly increased. MicroRNA transcriptome studies demonstrated that lower amount of differentially expressed miRNAs are present in protoplasts than in CDPs cultured for 120 h. Then analysis of differentially expressed miRNAs, selected pri-miRNA and mRNA targets were performed. Conclusion This result indicates that miRNA function is not a major regulation of gene expression in the initial but in later steps of dedifferentiation during CDPs divisions. miRNAs participate in organogenesis, oxidative stress, nutrient deficiencies and cell cycle regulation in protoplasts and CDPs. The important role played by miRNAs in the process of dedifferentiation of mesophyll cells was confirmed by the increased mortality and reduced cell division of CDPs derived from mutants with defective miRNA biogenesis and miR319b expression.

The light response in chickens divergently selected for feather pecking behavior reveals mechanistic insights towards psychiatric disorders

Background Feather pecking is a serious behavioral disorder in chickens that has a considerable impact on animal welfare and poses an economic burden for poultry farming. To study the underlying genetics of feather pecking animals were divergently selected for feather pecking over 15 generations based on estimated breeding values for the behavior. Methods and results By characterizing the transcriptomes of whole brains isolated from high and low feather pecking chickens in response to light stimulation we discovered a putative dysregulation of micro RNA processing caused by a lack of Dicer1. This results in a prominent downregulation of the GABRB2 gene and other GABA receptor transcripts, which might cause a constant high level of excitation in the brains of high feather pecking chickens. Moreover, our results point towards an increase in immune system-related transcripts that may be caused by higher interferon concentrations due to Dicer1 downregulation. Conclusion Based on our results, we conclude that feather pecking in chickens and schizophrenia in humans have numerous common features. For instance, a Dicer1 dependent disruption of miRNA biogenesis and the lack of GABRB2 expression have been linked to schizophrenia pathogenesis. Furthermore, disturbed circadian rhythms and dysregulation of genes involved in the immune system are common features of both conditions.

Sequencing of Argonaute-bound microRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a

MicroRNAs (miRNA) are short non-coding RNAs widely implicated in gene regulation. Most metazoan miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis does not require Drosha. The functional importance of the TSS-miRNA biogenesis is uncertain. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (AGO-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without DROSHA knockout. We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by AGO-qCLASH. Targets for miR-320a are enriched for the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and antagomir- transfected cells, differentially expressed gene products are associated with eIF2 signaling. Within the AGO-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone calnexin as a direct miR-320a down-regulated target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. In summary, our study investigates the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response.

The microRNA miR-202 prevents precocious spermatogonial differentiation and meiotic initiation during mouse spermatogenesis

ABSTRACT Spermatogonial differentiation and meiotic initiation during spermatogenesis are tightly regulated by a number of genes, including those encoding enzymes for miRNA biogenesis. However, whether and how single miRNAs regulate these processes remain unclear. Here, we report that miR-202, a member of the let-7 family, prevents precocious spermatogonial differentiation and meiotic initiation in spermatogenesis by regulating the timely expression of many genes, including those for key regulators such as STRA8 and DMRT6. In miR-202 knockout (KO) mice, the undifferentiated spermatogonial pool is reduced, accompanied by age-dependent decline of fertility. In KO mice, SYCP3, STRA8 and DMRT6 are expressed earlier than in wild-type littermates, and Dmrt6 mRNA is a direct target of miR-202-5p. Moreover, the precocious spermatogonial differentiation and meiotic initiation were also observed in KO spermatogonial stem cells when cultured and induced in vitro, and could be partially rescued by the knockdown of Dmrt6. Therefore, we have not only shown that miR-202 is a regulator of meiotic initiation but also identified a previously unknown module in the underlying regulatory network.

Tumor evolution selectively inactivates the core microRNA machinery for immune evasion

Cancer cells acquire genetic heterogeneity to escape from immune surveillance during tumor evolution, but a systematic approach to distinguish driver from passenger mutations is lacking. Here we investigate the impact of different immune pressure on tumor clonal dynamics and immune evasion mechanism, by combining massive parallel sequencing of immune edited tumors and CRISPR library screens in syngeneic mouse tumor model and co-culture system. We find that the core microRNA (miRNA) biogenesis and targeting machinery maintains the sensitivity of cancer cells to PD-1-independent T cell-mediated cytotoxicity. Genetic inactivation of the machinery or re-introduction of ANKRD52 frequent patient mutations dampens the JAK-STAT-interferon-γ signaling and antigen presentation in cancer cells, largely by abolishing miR-155-targeted silencing of suppressor of cytokine signaling 1 (SOCS1). Expression of each miRNA machinery component strongly correlates with intratumoral T cell infiltration in nearly all human cancer types. Our data indicate that the evolutionarily conserved miRNA pathway can be exploited by cancer cells to escape from T cell-mediated elimination and immunotherapy.

Association of Gem-associated protein 4 gene polymorphisms with the risk of colorectal cancer in the Polish population

Aim: Gem-associated protein 4 (GEMIN4), a member of the GEMIN gene family, is a key compound of the regulating factors responsible for miRNA biogenesis. Genetic variability within this gene can alter the risk for development of colorectal cancer (CRC) as was shown for other genes involved in miRNA biogenesis. Therefore, presented study was intended to identify genetic variants of three single nucleotide polymorphisms (SNPs) in the GEMIN4 gene (rs1062923, rs2740348 and rs910925) and their relationship with CRC. Methods: The study comprised 203 patients and 179 age and sex matched controls. Genotyping of GEMIN4 gene variants was done using Taqman® assay. The association of GEMIN4 variants with CRC was done by odds ratio analysis. Haplotype analysis was done to see the combined effect of studied variants on CRC. Results: Patients carrying all variant genotypes for GEMIN4 rs1062923 (odds ratio [OR]= 0.205; 95% confidence interval [CI]= 0.1034-0.4065 for CC variant and [OR] = 0.1436; [CI] = 0.0869-0.2373 for CT variant, respectively) and GEMIN4 rs2740348 (odds ratio [OR]= 0.4498; 95% confidence interval [CI]= 0.2342-0.8637for CC variant and [OR] = 0.3986; [CI] = 0.2043-0.7776 for CG variant, respectively) showed significant association in lower occurrence of cancer, whereas in case of GEMIN4 G/C rs910925 variant genotype, no significance correlation was found. Conclusions: Our study gives a substantive support for the association between the GEMIN4 gene variants/miRNA biogenesis and CRC risk.