DOF AFFECTING GERMINATION 2 is a positive regulator of light-mediated seed germination and is repressed by DOF AFFECTING GERMINATION 1

Abstract BACKGROUND: The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of the light-mediated seed germination process. DAG1 acts downstream PHYTOCHROME INTERACTING FACTOR3-LIKE 5 (PIL5), the master repressor, and negatively regulates gibberellin biosynthesis by directly repressing the biosynthetic gene AtGA3ox1. The Dof protein DOF AFFECTING GERMINATION (DAG2) shares a high degree of aminoacidic identity with DAG1. While DAG1 inactivation considerably increases the germination capability of seeds, the dag2 mutant has seeds with a germination potential substantially lower than the wild-type, indicating that these factors may play opposite roles in seed germination. RESULTS: We show here that DAG2 expression is positively regulated by environmental factors triggering germination, whereas its expression is repressed by PIL5 and DAG1; by Chromatin Immuno Precipitation (ChIP) analysis we prove that DAG1 directly regulates DAG2. In addition, we show that Red light significantly reduces germination of dag2 mutant seeds. CONCLUSIONS: In agreement with the seed germination phenotype of the dag2 mutant previously published, the present data prove that DAG2 is a positive regulator of the light-mediated seed germination process, and particularly reveal that this protein plays its main role downstream of PIL5 and DAG1 in the phytochrome B (phyB)-mediated pathway

Characterization of the lncRNA transcriptome in mESC-derived motor neurons: Implications for FUS-ALS

Long non-coding RNAs (lncRNAs) are currently recognized as crucial players in nervous system development, function and pathology. In Amyotrophic Lateral Sclerosis (ALS), identification of causative mutations in FUS and TDP-43 or hexanucleotide repeat expansion in C9ORF72 point to the essential role of aberrant RNA metabolism in neurodegeneration. In this study, by taking advantage of an in vitro differentiation system generating mouse motor neurons (MNs) from embryonic stem cells, we identified and characterized the long non-coding transcriptome of MNs. Moreover, by using mutant mouse MNs carrying the equivalent of one of the most severe ALS-associated FUS alleles (P517L), we identified lncRNAs affected by this mutation. Comparative analysis with humanMNs derived in vitro frominduced pluripotent stemcells indicated that candidate lncRNAs are conserved between mouse and human. Our work provides a global view of the long non-coding transcriptome of MN, as a prerequisite toward the comprehension of the still poorly characterized non-coding side ofMNphysiopatholog

A Regulatory Circuitry Between Gria2, miR-409, and miR-495 Is Affected by ALS FUS Mutation in ESC-Derived Motor Neurons

Mutations in fused in sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS). FUS is a multifunctional protein involved in the biogenesis and activity of several types of RNAs, and its role in the pathogenesis of ALS may involve both direct effects of disease-associated mutations through gain- and loss-of-function mechanisms and indirect effects due to the cross talk between different classes of FUS-dependent RNAs. To explore how FUS mutations impinge on motor neuron-specific RNA-based circuitries, we performed transcriptome profiling of small and long RNAs of motor neurons (MNs) derived from mouse embryonic stem cells carrying a FUS-P517L knock-in mutation, which is equivalent to human FUS-P525L, associated with a severe and juvenile-onset form of ALS. Combining ontological, predictive and molecular analyses, we found an inverse correlation between several classes of deregulated miRNAs and their corresponding mRNA targets in both homozygous and heterozygous P517L MNs. We validated a circuitry in which the upregulation of miR-409-3p and miR-495-3p, belonging to a brainspecific miRNA subcluster implicated in several neurodevelopmental disorders, produced the downregulation of Gria2, a subunit of the glutamate α‐amino‐3‐hydroxy‐5‐methyl-4-isoxazole propionic acid (AMPA) receptor with a significant role in excitatory neurotransmission. Moreover, we found that FUS was involved in mediating such miRNA repression. Gria2 alteration has been proposed to be implicated in MN degeneration, through disturbance of Ca2+ homeostasis, which triggers a cascade of damaging “excitotoxic” events. The molecular cross talk identified highlights a role for FUS in excitotoxicity and in miRNA-dependent regulation of Gria2. This circuitry also proved to be deregulated in heterozygosity, which matches the human condition perfectly

Drosophila CG3303 is an essential endoribonuclease linked to TDP-43-mediated neurodegeneration

Endoribonucleases participate in almost every step of eukaryotic RNA metabolism, acting either as degradative or biosynthetic enzymes. We previously identified the founding member of the Eukaryotic EndoU ribonuclease family, whose components display unique biochemical features and are flexibly involved in important biological processes, such as ribosome biogenesis, tumorigenesis and viral replication. Here we report the discovery of the CG3303 gene product, which we named DendoU, as a novel family member in Drosophila. Functional characterisation revealed that DendoU is essential for Drosophila viability and nervous system activity. Pan-neuronal silencing of dendoU resulted in fly immature phenotypes, highly reduced lifespan and dramatic motor performance defects. Neuron-subtype selective silencing showed that DendoU is particularly important in cholinergic circuits. At the molecular level, we unveiled that DendoU is a positive regulator of the neurodegeneration-associated protein dTDP-43, whose downregulation recapitulates the ensemble of dendoU-dependent phenotypes. This interdisciplinary work, which comprehends in silico, in vitro and in vivo studies, unveils a relevant role for DendoU in Drosophila nervous system physio-pathology and highlights that DendoU-mediated neurotoxicity is, at least in part, contributed by dTDP-43 loss-of-function

FUS affects circular RNA expression in murine embryonic stem cell-derived motor neurons

The RNA-binding protein FUS participates in several RNA biosynthetic processes and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Here we report that FUS controls back-splicing reactions leading to circular RNA (circRNA) production. We identified circRNAs expressed in in vitro -derived mouse motor neurons (MNs) and determined that the production of a considerable number of these circRNAs is regulated by FUS. Using RNAi and overexpression of wild-type and ALS-asso- ciated FUS mutants, we directly correlate the modulation of circRNA biogenesis with alteration of FUS nuclear levels and with putative toxic gain of function activities. We also demonstrate that FUS regulates circRNA biogenesis by binding the introns flanking the back-splicing junctions and that this control can be reproduced with artificial constructs. Most circRNAs are conserved in humans and specific ones are deregulated in human-induced pluripotent stem cell-derived MNs carrying the FUS P525L mutation associated with AL

FUS Alters circRNA Metabolism in Human Motor Neurons Carrying the ALS-Linked P525L Mutation

Deregulation of RNA metabolism has emerged as one of the key events leading to the degeneration of motor neurons (MNs) in Amyotrophic Lateral Sclerosis (ALS) disease. Indeed, mutations on RNA-binding proteins (RBPs) or on proteins involved in aspects of RNA metabolism account for the majority of familiar forms of ALS. In particular, the impact of the ALS-linked mutations of the RBP FUS on many aspects of RNA-related processes has been vastly investigated. FUS plays a pivotal role in splicing regulation and its mutations severely alter the exon composition of transcripts coding for proteins involved in neurogenesis, axon guidance, and synaptic activity. In this study, by using in vitro-derived human MNs, we investigate the effect of the P525L FUS mutation on non-canonical splicing events that leads to the formation of circular RNAs (circRNAs). We observed altered levels of circRNAs in FUSP525L MNs and a preferential binding of the mutant protein to introns flanking downregulated circRNAs and containing inverted Alu repeats. For a subset of circRNAs, FUSP525L also impacts their nuclear/cytoplasmic partitioning, confirming its involvement in different processes of RNA metabolism. Finally, we assess the potential of cytoplasmic circRNAs to act as miRNA sponges, with possible implications in ALS pathogenesis

The DOF protein DAG1 and the della protein GAI cooperate in negatively regulating the AtGA3ox1 gene

International audienceDear Editor, Seed germination is controlled by multiple endo­ genous and environmental factors, which are integrated to trigger this developmental process at the right time. Gibberellins (GAs) are known to induce this process, and the levels of GAs are modulated by light—one of the most important environmental factors affecting seed germina­ tion. The bHLH transcription factor PIL5 (PHYTOCHROME INTERACTING FACTOR 3­LIKE 5) is the master repressor of light­mediated seed germination in Arabidopsis (Oh et al., 2004). In seeds kept in the dark, PIL5 activates transcrip­ tion of the GAI (GA INSENSITIVE) gene (Peng et al., 1997; Oh et al., 2007), a DELLA transcriptional regulator that represses GA­mediated processes (Sun, 2011). GAI plays a role in many growth processes with both unique and overlapping functions with another DELLA protein: RGA (REPRESSOR OF ga1-3) (Dill and Sun, 2001). Also, the DOF transcription factor DAG1 (DOF AFFECTING GERMINATION1) acts in the light­mediated seed germination pathway downstream of PIL5: DAG1 expression is reduced in seeds irradiated for 24 h with red (R) light, and this reduction is dependent on PIL5 as, in pil5 mutant seeds, DAG1 expression is reduced irrespective of light conditions (Gabriele et al., 2010). Null mutant seeds dag1 need a fluence rate six times lower than wild­type to germinate (Papi et al., 2000, 2002); similarly, gai–t6rga28 double mutant seeds require less R light fluences than wild­type ones to germinate (Oh et al., 2007). To further clarify the role of DAG1 in light­mediated seed germination, we focus here on the functional rela­ tionship between DAG1 and GAI in the control of this process. We have recently demonstrated that DAG1 specifi­ cally represses AtGA3ox1 expression. In dag1 mutant seeds, only this GA biosynthetic gene was up­regulated, while the level of expression of AtGA3ox2 and AtGA2ox2 were unchanged compared to the wild­type (Gabriele et al., 2010). A very similar expression profile of AtGA3ox1 was shown by Oh et al. (2007) in gai–t6rga28 double mutant seeds. To verify whether GAI plays a role in the regulation of GA metabolic genes, and in particular of AtGA3ox1, we performed a quantitative RT–PCR (RT–qPCR) analysis on gai-t6 mutant seeds. The level of the AtGA3ox1 transcript was highly increased in the gai-t6 null mutant compared to the wild­type, both in seeds imbibed in the dark and those exposed to R light (Figure 1A), while expression of AtGA3ox2 and AtGA2ox2 was not significantly altered. Since—similarly to DAG1 inactivation—GAI inactivation specifically affected AtGA3ox1 expression, we decided to verify whether the presence of GAI is necessary for DAG1­ mediated repression of AtGA3ox1. In agreement with our hypothesis, promoter analysis of GAI­regulated genes revealed a significant enrichment of DOF­binding sites (Gallego­Bartolomé et al., 2011), suggesting that these transcription factors may mediate GAI activity. We used the dag1DAG1–HA (Gabriele et al., 2010) and the dag1gai-t6DAG1–HA lines, which overexpress DAG1 respectively in the dag1 and dag1gai-t6 mutant backgrounds. Both these lines expressed the DAG1–HA chimeric protein as revealed by immunoblot analysis (Supplemental Figure 1). As expected, the expression of AtGA3ox1 in dag1DAG1–HA seeds was highly reduced compared to wild­type both in the dark and under R light, due to overexpression of DAG1–HA, whereas AtGA3ox1 was strongly overexpressed in dag1gai-t6DAG1–HA seeds (Figure 1B and 1C), suggesting that both DAG1 and GAI are involved in the regulation of AtGA3ox1. Since inactivation of GAI makes DAG1 unable to repress AtGA3ox1 expression, we set to assess whether these two factors directly collaborate in regulating this GA biosynthetic gene. We performed chromatin immunopre­ cipitation (ChIP) assays using the GAI–MYC transgenic line constructed by Oh et al. (2007), and the dag1DAG1–HA line (Gabriele et al., 2010) as a positive control. Cross­linked and sonicated protein–DNA complexes were precipitated with anti­MYC and anti­HA antibodies, respectively. We amplified by real­time PCR (qPCR) three regions of the AtGA3ox1 promoter containing different numbers of cop­ ies of DOF­binding sites (0, 2, and 15) (Figure 1D). As a nega­ tive control, we performed the same assays without adding the antibody, or with both antibodies on wild­type seeds (Supplemental Figure 2). The relative amounts of precipi­ tated promoter fragments of AtGA3ox1 by DAG1–HA are higher than the negative control, and the enrichment of the target fragment is proportional to the number of DOF sites present in the region. By contrast, the enrichment of precipitated promoter fragments of AtGA3ox1 was ver

Independent and interactive effects of DOF AFFECTING GERMINATION 1 (DAG1) and the DELLA proteins GA INSENSITIVE (GAI) and REPRESSOR OF ga1-3 (RGA) in embryo development and seed germination

BACKGROUND: The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of seed germination acting downstream of the master repressor PHYTOCROME INTERACTING FACTOR3-LIKE 5 (PIL5). Among others, PIL5 induces the expression of the genes encoding the two DELLA proteins GA INSENSITIVE 1 (GAI) and REPRESSOR OF ga1-3 (RGA). RESULTS: Based on the properties of gai-t6 and rga28 mutant seeds, we show here that the absence of RGA severely increases dormancy, while lack of GAI only partially compensates RGA inactivation. In addition, the germination properties of the dag1rga28 double mutant are different from those of the dag1 and rga28 single mutants, suggesting that RGA and DAG1 act in independent branches of the PIL5-controlled germination pathway. Surprisingly, the dag1gai-t6 double mutant proved embryo-lethal, suggesting an unexpected involvement of (a possible complex between) DAG1 and GAI in embryo development. CONCLUSIONS: Rather than overlapping functions as previously suggested, we show that RGA and GAI play distinct roles in seed germination, and that GAI interacts with DAG1 in embryo development.Background: The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of seed germination acting downstream of the master repressor PHYTOCROME INTERACTING FACTOR3-LIKE 5 (PIL5). Among others, PIL5 induces the expression of the genes encoding the two DELLA proteins GA INSENSITIVE 1 (GAI) and REPRESSOR OF ga1-3 (RGA). Results: Based on the properties of gai-t6 and rga28 mutant seeds, we show here that the absence of RGA severely increases dormancy, while lack of GAI only partially compensates RGA inactivation. In addition, the germination properties of the dag1rga28 double mutant are different from those of the dag1 and rga28 single mutants, suggesting that RGA and DAG1 act in independent branches of the PIL5-controlled germination pathway. Surprisingly, the dag1gai-t6 double mutant proved embryo-lethal, suggesting an unexpected involvement of (a possible complex between) DAG1 and GAI in embryo development. Conclusions: Rather than overlapping functions as previously sug