Browsing by Subject "REPRESSION"

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  • Swarnalok, De; Pollari, Maija; Varjosalo, Markku; Mäkinen, Kristiina (2020)
    In this study, we investigated the significance of a conserved five-amino acid motif 'AELPR' in the C-terminal region of helper component-proteinase (HCPro) for potato virus A (PVA; genusPotyvirus) infection. This motif is a putative interaction site for WD40 domain-containing proteins, including VARICOSE (VCS). We abolished the interaction site in HCPro by replacing glutamic acid (E) and arginine (R) with alanines (A) to generate HCPro(WD). These mutations partially eliminated HCPro-VCS co-localization in cells. We have earlier described potyvirus-induced RNA granules (PGs) in which HCPro and VCS co-localize and proposed that they have a role in RNA silencing suppression. We now demonstrate that the ability of HCPro(WD)to induce PGs, introduce VCS into PGs, and suppress RNA silencing was impaired. Accordingly, PVA carrying HCPro(WD)(PVA(WD)) infectedNicotiana benthamianaless efficiently than wild-type PVA (PVA(WT)) and HCPro(WD)complemented the lack of HCPro in PVA gene expression only partially. HCPro was purified from PVA-infected leaves as part of high molecular weight (HMW) ribonucleoprotein (RNP) complexes. These complexes were more stable when associated with wild-type HCPro than with HCPro(WD). Moreover, VCS and two viral components of the HMW-complexes, viral protein genome-linked and cylindrical inclusion protein were specifically decreased in HCPro(WD)-containing HMW-complexes. A boost in translation of replication-deficient PVA (PVA(Delta GDD)) was observed only if viral RNA expressed wild-type HCPro. The role of VCS-VPg-HCPro coordination in PVA translation was further supported by results from VCS silencing and overexpression experiments and by significantly elevated PVA-derivedRenillaluciferase vs PVA RNA ratio upon VPg-VCS co-expression. Finally, we found that PVA(WD)was unable to form virus particles or to spread systemically in the infected plant. We highlight the role of HCPro-VCS containing multi-protein assemblies associated with PVA RNA in protecting it from degradation, ensuring efficient translation, formation of stable virions and establishment of systemic infection. Author summary This study revealed that the potyviral helper component proteinase (HCPro) and the host protein VARICOSE (VCS) are linked in a manner that is important for suppression of RNA silencing, formation of potyvirus-induced RNA granules, translation of viral proteins, stability of virions, and development of systemic potato virus A (PVA) infection. The results suggest that HCPro and VCS belong to the core components of large RNP complexes regulating PVA infection. We suggest that these complexes protect viral RNAs in the cytoplasm after release from the replication complex and direct them to translation and intact to the viral particles.
  • Kun, Roland S.; Meng, Jiali; Salazar-Cerezo, Sonia; Makela, Miia R.; de Vries, Ronald P.; Garrigues, Sandra (2020)
    The CRISPR/Cas9 system has been successfully applied for gene editing in filamentous fungi. Previous studies reported that single stranded oligonucleotides can be used as repair templates to induce point mutations in some filamentous fungi belonging to genus Aspergillus. In Aspergillus niger, extensive research has been performed on regulation of plant biomass degradation, addressing transcription factors such as XlnR or GaaR, involved in (hemi-)cellulose and pectin utilization, respectively. Single nucleotide mutations leading to constitutively active forms of XlnR and GaaR have been previously reported. However, the mutations were performed by the introduction of versions obtained through site-directed or UV-mutagenesis into the genome. Here we report a more time- and cost-efficient approach to obtaining constitutively active versions by application of the CRISPR/Cas9 system to generate the desired mutation on-site in the A. niger genome. This was also achieved using only 60-mer single stranded oligonucleotides, shorter than the previously reported 90-mer strands. In this study, we show that CRISPR/Cas9 can also be used to efficiently change functional properties of the proteins encoded by the target gene by on-site genomic mutations in A. niger. The obtained strains with constitutively active XlnR and GaaR versions resulted in increased production of plant biomass degrading enzymes and improved release of D-xylose and L-arabinose from wheat bran, and D-galacturonic acid from sugar beet pulp.
  • Chmielarz, Piotr; Konovalova, Julia; Najam, Syeda Sadia; Alter, Heike; Piepponen, Timo Petteri; Erfle, Holger; Sonntag, Kai C.; Schutz, Gunther; Vinnikov, Ilya A.; Domanskyi, Andrii (2017)
    MicroRNAs (miRs) are important post-transcriptional regulators of gene expression implicated in neuronal development, differentiation, aging and neurodegenerative diseases, including Parkinson's disease (PD). Several miRs have been linked to PD-associated genes, apoptosis and stress response pathways, suggesting that deregulation of miRs may contribute to the development of the neurodegenerative phenotype. Here, we investigate the cell-autonomous role of miR processing RNAse Dicer in the functional maintenance of adult dopamine (DA) neurons. We demonstrate a reduction of Dicer in the ventral midbrain and altered miR expression profiles in laser-microdissected DA neurons of aged mice. Using a mouse line expressing tamoxifen-inducible CreERT2 recombinase under control of the DA transporter promoter, we show that a tissue-specific conditional ablation of Dicer in DA neurons of adult mice led to decreased levels of striatal DA and its metabolites without a reduction in neuronal body numbers in hemizygous mice (Dicer(HET)) and to progressive loss of DA neurons with severe locomotor deficits in nullizygous mice (Dicer(CKO)). Moreover, we show that pharmacological stimulation of miR biosynthesis promoted survival of cultured DA neurons and reduced their vulnerability to thapsigargin-induced endoplasmic reticulum stress. Our data demonstrate that Dicer is crucial for maintenance of adult DA neurons, whereas a stimulation of miR production can promote neuronal survival, which may have direct implications for PD treatment.
  • Vuorinen, Katariina; Zamora, Olena; Vaahtera, Lauri; Overmyer, Kirk; Brosché, Mikael (2021)
    Plants require interaction between signaling pathways to differentiate and integrate stress responses and deploy appropriate defenses. The hormones ethylene, salicylic acid (SA), and jasmonic acid (JA) are important regulators of plant defenses. Numerous interactions between these signaling pathways are the cornerstone of robust plant immunity. Additionally, during the early response to pathogens, reactive oxygen species (ROS) act as signaling molecules. Here, we examined the extent of signal interaction in the early stages of Botrytis cinerea infection. To enable a comparison between B. cinerea infection with ROS signaling, we subjected plants to ozone treatment, which stimulates an apoplastic ROS burst. We used a collection of single, double, and triple signaling mutants defective in hormone signaling and biosynthesis and subjected them to B. cinerea infection and ozone treatment at different timepoints. We examined lesion size, cell death, and gene expression (both quantitatively and spatially). The two treatments shared many similarities, especially in JA-insensitive mutants, which were sensitive to both treatments. Unexpectedly, a B. cinerea- susceptible JA-insensitive mutant (coil), became tolerant when both SA biosynthesis and signaling was impaired (coil npr1 sid2), demonstrating that JA responses may be under the control of SA. Extensive marker gene analysis indicated JA as the main regulator of both B. cinerea and ozone defenses. In addition, we identified the transcription factor SRI_ as a crucial regulator of PLANT DEFENSIN expression and cell-death regulation, which contributes to resistance to B. cinerea. Overall, our work further defines the context of ROS in plant defense signaling.
  • Evsyukov, Valentin; Domanskyi, Andrii; Bierhoff, Holger; Gispert, Suzana; Mustafa, Rasem; Schlaudraff, Falk; Liss, Birgit; Parlato, Rosanna (2017)
    Genetic mutations underlying neurodegenerative disorders impair ribosomal DNA (rDNA) transcription suggesting that nucleolar dysfunction could be a novel pathomechanism in polyglutamine diseases and in certain forms of amyotrophic lateral sclerosis/frontotemporal dementia. Here, we investigated nucleolar activity in pre-symptomatic digenic models of Parkinson's disease (PD) that model the multifactorial aetiology of this disease. To this end, we analysed a novel mouse model mildly overexpressing mutant human alpha-synuclein (hA53T-SNCA) in a PTEN-induced kinase 1 (PINK1/ PARK6) knockout background and mutant mice lacking both DJ-1 (also known as PARK7) and PINK1. We showed that overexpressed hA53T-SNCA localizes to the nucleolus. Moreover, these mutants show a progressive reduction of rDNA transcription linked to a reduced mouse lifespan. By contrast, rDNA transcription is preserved in DJ-1/PINK1 double knockout (DKO) mice. mRNA levels of the nucleolar transcription initiation factor 1A (TIF-IA, also known as RRN3) decrease in the substantia nigra of individuals with PD. Because loss of TIF-IA, as a tool to mimic nucleolar stress, increases oxidative stress and because DJ-1 and PINK1 mutations result in higher vulnerability to oxidative stress, we further explored the synergism between these PD-associated genes and impaired nucleolar function. By the conditional ablation of TIF-IA, we blocked ribosomal RNA (rRNA) synthesis in adult dopaminergic neurons in a DJ-1/PINK1 DKO background. However, the early phenotype of these triple knockout mice was similar to those mice exclusively lacking TIF-IA. These data sustain a model in which loss of DJ-1 and PINK1 does not impair nucleolar activity in a pre-symptomatic stage. This is the first study to analyse nucleolar function in digenic PD models. We can conclude that, at least in these models, the nucleolus is not as severely disrupted as previously shown in DA neurons from PD patients and neurotoxin-based PD mouse models. The results also show that the early increase in rDNA transcription and nucleolar integrity may represent specific homeostatic responses in these digenic pre-symptomatic PD models.
  • Aakko, Sofia; Straume, Anne Hege; Birkeland, Einar Elvbakken; Chen, Ping; Qiao, Xi; Lonning, Per Eystein; Kallio, Marko J. (2019)
    Taxanes are chemotherapeutic agents used in the treatment of solid tumors, particularly of breast, ovarian, and lung origin. However, patients show divergent therapy responses, and the molecular determinants of taxane sensitivity have remained elusive. Especially the signaling pathways that promote death of the taxane-treated cells are poorly characterized. Here we describe a novel part of a signaling route in which c-Myc enhances paclitaxel sensitivity through upregulation of miR-203b-3p and miR-203a-3p; two clustered antiapoptosis protein BcI-xL controlling microRNAs. In vitro, the miR-203b-3p decreases the expression of BcI-xL by direct targeting of the gene's mRNA 3'UTR. Notably, overexpression of the miR-203b-3p changed the fate of paclitaxel-treated breast and ovarian cancer cells from mitotic slippage to cell death. In breast tumors, high expression of the miR-203b-3p and MYC was associated with better therapy response and patient survival. Interestingly, in the breast tumors, MYC expression correlated negatively with BCL2L1 expression but positively with miR-203b-3p and miR-203a-3p. Finally, silencing of MYC suppressed the transcription of both miRNAs in breast tumor cells. Pending further validation, these results may assist in patient stratification for taxane therapy.
  • Morgunova, Ekaterina; Yin, Yimeng; Jolma, Arttu; Dave, Kashyap; Schmierer, Bernhard; Popov, Alexander; Eremina, Nadejda; Nilsson, Lennart; Taipale, Jussi (2015)
    The mammalian cell cycle is controlled by the E2F family of transcription factors. Typical E2Fs bind to DNA as heterodimers with the related dimerization partner (DP) proteins, whereas the atypical E2Fs, E2F7 and E2F8 contain two DNA-binding domains (DBDs) and act as repressors. To understand the mechanism of repression, we have resolved the structure of E2F8 in complex with DNA at atomic resolution. We find that the first and second DBDs of E2F8 resemble the DBDs of typical E2F and DP proteins, respectively. Using molecular dynamics simulations, biochemical affinity measurements and chromatin immunoprecipitation, we further show that both atypical and typical E2Fs bind to similar DNA sequences in vitro and in vivo. Our results represent the first crystal structure of an E2F protein with two DBDs, and reveal the mechanism by which atypical E2Fs can repress canonical E2F target genes and exert their negative influence on cell cycle progression.
  • Ahi, Ehsan Pashay; Tsakoumis, Emmanouil; Brunel, Mathilde; Schmitz, Monika (2021)
    The signal mediated by leptin hormone and its receptor is a major regulator of body weight, food intake and metabolism. In mammals and many teleost fish species, leptin has an anorexigenic role and inhibits food intake by influencing the appetite centres in the hypothalamus. However, the regulatory connections between leptin and downstream genes mediating its appetite-regulating effects are still not fully explored in teleost fish. In this study, we used a loss of function leptin receptor zebrafish mutant and real-time quantitative PCR to assess brain expression patterns of several previously identified anorexigenic genes downstream of leptin signal under different feeding conditions (normal feeding, 7-day fasting, 2 and 6-h refeeding). These downstream factors include members of cart genes, crhb and gnrh2, as well as selected genes co-expressed with them based on a zebrafish co-expression database. Here, we found a potential gene expression network (GRN) comprising the abovementioned genes by a stepwise approach of identifying co-expression modules and predicting their upstream regulators. Among the transcription factors (TFs) predicted as potential upstream regulators of this GRN, we found expression pattern of sp3a to be correlated with transcriptional changes of the downstream gene network. Interestingly, the expression and transcriptional activity of Sp3 orthologous gene in mammals have already been implicated to be under the influence of leptin signal. These findings suggest a potentially conserved regulatory connection between leptin and sp3a, which is predicted to act as a transcriptional driver of a downstream gene network in the zebrafish brain.