Yi Zhang 1,2 , Julie Leclercq 1,2 , Shuangyang Wu 1,2,3,4 , Enrique ortega-Abboud 1,2 ,
stéphanie pointet 1,2 , Chaorong tang 5 , Songnian Hu 3 & pascal Montoro 1,2
MicroRNA-mediated post-transcriptional regulation has been reported on Ros production and
scavenging systems. Although microRNAs first appeared highly conserved among plant species, several aspects of biogenesis, function and evolution of microRNAs were shown to differ. High throughput transcriptome and degradome analyses enable to identify small RNAs and their mRNA targets. A non- photosynthetic tissue particularly prone to redox reactions, laticifers from Hevea brasiliensis, revealed species-specific post-transcriptionalregulations. This paper sets out to identify the 407 genes of the thirty main redox-related gene families harboured by the Hevea genome. There are 161 redox-related genes expressed in latex. Thirteen of these redox-related genes were targeted by 11 microRNAs. To our knowledge, this is the first report on a mutation in the miR398 binding site of the cytosolic CuZnSOD. A working model was proposed for transcriptional and post-transcriptional regulation with respect to the predicted subcellular localization of deduced proteins.
This study provided several new insights into Hevea concerning miRNA biogenesis and activity, tissue-specific post-transcriptional regulation in response to abiotic stress, and the impact of Hevea speciation with the whole genome duplication shared with cassava. The divergence observed from annual plant species suggests enhanced adaptation to stress in Hevea, particularly in laticifers, which are subjected to recurrent combinatorial stresses. Concerning the post-transcriptional regulation observed for sucrose metabolism in bark and in each step of the natural rubber biosynthesis pathway ( Fig. 11 ), intra- and interspecific comparison of rubber-producing plants is needed to decipher the impact of post-transcriptionalregulations in response to stress in the context of rubber production.
PART III : Project
The cellular and molecular mechanisms involving ARE-containing mRNAs and AU-BPs have been intensively studied in cultured cells. However how and when AREs are involved in the processes of proliferation and differentiation that sustain development remain largely unknown. In this regard, germ cell development represents an excellent system for addressing these issues. Spermatogenesis is one of the few developmental situations where transcription ceases before the end of differentiation, relying thus on pre-existing mRNAs to complete spermatozoa formation. It has been shown that extensive post-transcriptionalregulations requiring RBPs are necessary for this event. Among others, I focused on HuR and AUF1. From analyses using somatic tissues or in vitro cultured cells, Hu proteins were shown to be the only AU-BPs able to stabilize their target mRNAs whatever the cellular context (Brennan and Steitz, 2001) and to regulate the translation of a variety of mRNAs, possibly by selective transport from translation repression sites to polysomes (Lal et al., 2004). Conversely, AUF1, first isolated for its destabilizing function (DeMaria and Brewer, 1996), has later on been shown to control mRNA stabilization and translation (Gouble et al., 2002; Xu et al., 2001). Although expressed in various somatic tissues, HuR and AUF1 are also strongly expressed in the testis, suggesting their involvement in spermatogenesis (Lafon et al., 1998; Lu and Schneider, 2004). This hypothesis was supported, at least for HuR, because its overexpression in the testis impairs differentiation of HuR transgenic gametes (Levadoux-Martin et al., 2003).
Figure 5. Cis- and trans-acting elements of the mitochondrial cycle: transcriptional and post-transcriptionalregulations. The upper line shows the correspondence between the previously defined metabolic phases (R/B, R/C, Ox, ) and the phases A to F defined in this work as relevant to the 626 oscillating nuclear genes, which code for mitochondrial proteins. The colors of the bar reflect the corresponding phases of the metabolic cycle (green = R/B; blue = R/C; red/brown = Ox). A and B show the percentage of genes in each phases A to F that contain cis-acting regulatory motif in 59 and 39 UTR regions, respectively. The significant motifs were identified using various bioinformatic tools (MatrixREDUCE , YEASTRACT ) or from published motifs whose consensus sequences are P3E = CCUGUAAAUACCC, PRSE = UAUAUAUUCUUA, NRSE1 = UUU- GAUAGACUC . C: Oscillating concentrations of HAP4 and HAP1 mRNAs analyzed with EDPM. They were found to peak during phases A and C, respectively. HAP1 mRNA peaks when the dissolved oxygen concentration is maximum (phase R/C of Tu et al. ). This is in agreement with observed oxygen-dependent transcription regulation of HAP1 (see the main text). The only known trans-acting factor recognizing the 39 motif P3E is Puf3p [10,12,17]. PUF3 mRNA does not significantly oscillate and could not be precisely assigned to a particular phase of the mitochondrial cycle (data not shown). D: Schematic summary of the above data. The % of Puf3p target mRNAs and the abundance of the mRNAs coding for the two transcription factors Hap4p and Hap1p are represented along one oscillatory period. The HAP4 mRNA variations coincide with the abundance of genes with a Hap4p binding site in their promoter and the HAP1 mRNA follows the variations of dissolved O 2 . This is in agreement with the property of O 2 sensor
of cells in G0, suggesting that Dll4-expressing endothelial cells participate to the maintenance of HSPC out of the cell cycle.
To get insight into the mechanisms of Dll4 actions, we have analyzed the molecular regulation of the cell cycle machinery after the onset of culture onto Dll4Fc. The role of p21 Cip1 was assessed using p21 Cip1/Waf1 -deficient mice and showed unambiguously that p21 Cip1/Waf1 is not involved in mbDll4 activity. We next examined the transcriptional modulation of some cell cycle genes. The D-cyclin family, cyclin D1, cyclin D2, and cyclin D3, were all expressed, albeit at different levels, in HSPCs (31). Cyclin D1-/-D2-/-D3-/- embryos displayed a reduced numbers of fetal HSPCs, with impairment in their ability to proliferate, revealing a unique requirement for the D-cyclins in the hematopoietic lineage (32). The downmodulation of the 3 D-cyclins observed in response to Dll4 is therefore in agreement with the key role of D-cyclins in HSPC proliferation. E2F4, which can bind to all 3 pRB family members to form the majority of cellular pRB family complexes, has been proposed to play a critical role in coordinating cell cycle exit (33). The retinoblastoma (Rb) family of transcriptional repressors, including the pRb, p107, and p130 proteins, restricts cell cycle entry by regulating E2F gene transcription of positive cell cycle regulators. Conditional deletion of all three Rb family members in adult mice resulted in a robust cell-intrinsic myeloproliferation phenotype, with an increase in HSPC proliferation, and severe defects in self-renewal (34). Therefore, the upregulation of E2F4 and Rb in response to Dll4 is consistent with their role in the cell cycle. Taken together, these findings indicate that Cyclins D and Rb family members may play a critical role in the activity of the Dll4/Notch pathway for the maintenance of the quiescent state.
A search was performed on the official, public websites of the identified municipalities for regulations related to noise, nuisances, peace and order, and environmental protection. The condition for inclusion in this review was that the regulation directly and explicitly covered noise. Thirteen municipalities did not have publicly available regulations governing noise and were excluded from further consideration. This resulted in a total of 74 Quebec municipalities.
, the present study describes their expression pattern, their post-transcriptional regulation and their ability to activate or repress transcriptional activity on synthetic or native auxin-responsive promoters. Transactivation assays revealed that 36% of tomato ARFs are strong repressors of transcriptional activity while only 22% are transcriptional activators. The repressor/activator ratio among ARFs is more than twice higher in tomato (3.6) compared to Arabidopsis (1.7), yet, it remains to be elucidated whether this feature may account for differences in developmental and growth behaviour between the two species. In contrast to repressor ARFs, most activator Sl- ARFs promote transcription of target genes only upon exogenous auxin treatment thus suggesting that activator ARFs require some input from a highly activated auxin signalling pathway in order to potentiate transcriptional activity. It is conceivable that when the auxin level is low, the amount of Aux/IAA proteins available is sufficient to block ARFs at the protein level thus preventing these latter from activating the transcription of the target genes. In this perspective, it has to be postulated that Aux/IAAs are present in excess in the cell when the tissue is not subjected to auxin treatment.
progenitors , which represent a proliferating population of muscle stem cells . This implies that Rbm24 functions in the cytoplasmic compartment during the differentiation step of muscle development, but its post-transcriptional regulatory roles remain to be explored. Interestingly, and consistent with its function in regulating alternative splicing of those mRNAs encoding muscle-specific contractile proteins, the localization of Rbm24 protein in myofibers of adult muscles is only restricted to the nucleus. This translocation as a function of cell differentiation state can be also observed in C2C12 cell line expressing Rbm24-GFP. The fusion protein is first expressed in the cytoplasm of mononucleated myoblast cells, and then accumulates in the nucleus of multinucleated myotubes (Fig. 3). These in vivo and in vitro observations suggest that Rbm24 may exert distinct post-transcriptional activities in differentiating myoblasts and terminally differentiated myofibers. Thus, there is a possibility that its regulatory roles on muscle cell differentiation and muscular functionality may be cell-type specific and may depend on its subcellular localization and the presence or absence of its co- factors.
accumulates glycogen (Timmermans and Van Melderen, 2009). We show here that attenuation of the essential CsrA post-transcriptional regulator creates a growth defect in presence of glucose in rich or minimal medium. The accumulation of glycogen was observed in the csrA51 strain, independently of the growth rate default. This suggests that glycogen accumulation is not the actual cause of the CsrA essentiality. The growth defect was not observed with all glycolytic substrates (i.e., not on fructose or fucose) and was related to the upper part of glycolysis. Using systematic analysis of CCM components in the wild-type and csrA51 strains, we discovered that most of the molecular discrepancies between the two strains are indeed located in the close vicinity of the point of glucose entry into the central car- bon metabolism. The most down-regulated enzyme of the glycolytic pathway was PfkA. Since substrates enter- ing the CCM after this metabolic step are not associated with a csrA51 growth defect, we hypothesized that down-regulation of PfkA creates an engorgement of the metabolic fluxes in the first glycolytic reaction that could lead to the growth deficiency. This hypothesis was sup- ported by the observed higher pools of F6P and S7P [i.e., the two PfkA substrates (Nakahigashi et al., 2009)] and more largely by the accumulation of metabolites in the top of glycolysis before the PfkA step. Restoring a wild-type level of PfkA in the csrA51 mutant prevent these metabolite accumulation and partially restore the growth rate. From all these elements, we conclude that PfkA plays a major role in the csrA51 phenotypes. How- ever, all the consequences of the csrA51 mutation on the CCM cannot be mediated through PfkA down regu- lation only. First, the pfkA deletion does not entirely pre- vent growth on glucose minimal medium as observed in a strain in which csrA is deleted (Nakahigashi et al., 2009; Timmermans and Van Melderen, 2010). Second, restoring the WT level of PfkA expression did not entirely restore a WT growth rate in the csrA51 strain (this work). Glycolytic flux control is known to be shared among its enzymes in microorganisms (Smallbone et al., 2013). This might explain why the influence of the CSR system is so dramatic since it controls the expres- sion of most of the glycolytic enzymes conjointly, among which PFK appears to be the most crucial metabolic step.
Experimental Procedures mES cell culture
V6.5 (C57BL/6-129) murine ES cells were grown under typical mES conditions on irradiated mouse embryonic fibroblasts (MEFs). For location analysis, cells were grown for two passages off of MEFs. For location analysis on mES cells following treatment with small molecule inhibitors, cells were grown two passages off feeders and prior to formaldehyde crosslinking, the cells were treated with the indicated final concentration of flavopiridol (1μM for 1 hour for ChIP-chip and ChIP-seq experiments, or the indicated concentration and time for Western blot analysis), or c-Myc/Max inhibitor 10058-F4 (50μM for 6 hours), both dissolved in DMSO, in the growth medium. As a control, DMSO alone was added at the same final volume as with drug. Small molecule inhibitors used were: Flavopiridol (Sigma cat #F3055), and c-Myc inhibitor 10058-F4 (Sigma cat #F3680). For location analysis following shRNA knockdown (OpenBiosystems), viral media was collected 48 hours after co-transfection in 293T cells and the mES cells were directly infected with the viral media 24 hours after initial plating of mES cells. The infection media was 1:2 viral media:mES media with 2mM polybrene. The efficiently infected cells were selected for 24 hours post infection with mES media containing 2μM puromycin. Cells were cross-linked 72 hours post selection. For location analysis following Oct4 shutdown, ZHBTc4 mES cells (Niwa et al., 2000) were grown under standard mES cell culture conditions and expanded for two passages off MEF feeders. mES media with 2 μg/ml doxycycline was added to the cells for 0 hours, 12 hours and 24 hours prior to formaldehyde crosslinking.
Enfotec, Ottawa, ON, K1T 2J7, Canada
Transport Canada has the responsibility for regulating Arctic shipping in Canada as part of the Arctic Shipping Pollution Prevention Regulations. A Zone-Date System (ZDS) is used North of the 60° latitude. The ZDS is based on historical data of ice conditions up to the early 1970’s and on the premise that the ice conditions are consistent from year-to-year. The ZDS consists of sixteen of geographic regions (Zones) and an associated Table that indicates the dates that each class of vessel is allowed in each geographical region. The Arctic Ice Regime Shipping System (AIRSS) is used by vessels wishing to access the Arctic Control Zones outside permissible dates for the vessels. The AIRSS, in contrast, allows shipping based on the actual, not historical, ice conditions.
The TOBM was first developed by the Tokyo metropolitan government in 1972 [122,123] and notified by the Japan Environment Agency in 1995. Since odour measure‐ ment is a crucial element of odour management and regulation, a quality control manual on the TOBM for laboratory use was published in 2002 to develop a reliable odour meas‐ urement method . Local authorities determine the odour index standard values within a range from 10 to 21 established by the government. After the amendment of the OOCL, local authorities became entitled to choose either of the two regulations: (1) based on the concentrations of odourants, or (2) based on the odour index. According to the OOCL, the range of the regulation standards of both the concentrations of odourants and the odour index at the site’s property line is equivalent to an odour intensity, which ranges from 2.5 to 3.5 on the six‐point odour intensity scale shown in Table 27.
Consistency is a property of regulations that has already been given some attention in the literature. For instance, as for confidentiality policies, consistency allows to avoid cases when the user has both the permission and the prohibition to know something . More generally, according to  which studies consistency of general kind of regulations, a regulation is consistent if there is no possible situation which leads an agent to normative contradictions or dilemmas (a given behaviour is prescribed and not prescribed, or prohibited and not prohibited) and contrary conflicts (a given behaviour is prescribed and prohibited).