Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS

Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide-and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development.

The function and regulation of the U11-48K protein in U12-dependent splicing

The removal of noncoding sequences, or introns, from the eukaryotic messenger RNA precursors is catalyzed by a ribonucleoprotein complex known as the spliceosome. In most eukaryotes, two distinct classes of introns exist, each removed by a specific type of spliceosome. The major, U2-type introns account for over 99 % of all introns, and are almost ubiquitous. The minor, U12-type introns are found in most but not all eukaryotes, and reside in conserved locations in a specific set of genes. Due to their slow excision rates, the U12-type introns are expected to be involved in the regulation of the genes containing them by inhibiting the maturation of the messenger RNAs. However, little information is currently available on how the activity of the U12-dependent spliceosome itself is regulated. The levels of many known splicing factors are regulated through unproductive alternative splicing events, which lead to inclusion of premature STOP codons, targeting the transcripts for destruction by the nonsense-mediated decay pathway. These alternative splice sites are typically found in highly conserved sequence elements, which also contain binding sites for factors regulating the activation of the splice sites. Often, the activation is achieved by binding of products of the gene in question, resulting in negative feedback loops. In this study, I show that U11-48K, a protein factor specific to the minor spliceosome, specifically recognizes the U12-type 5' splice site sequence, and is essential for proper function of the minor spliceosome. Furthermore, the expression of U11-48K is regulated through a feedback mechanism, which functions through conserved sequence elements that activate alternative splicing and nonsense-mediated decay. This mechanism is conserved from plants to animals, highlighting both the importance and early origin of this mechanism in regulating splicing factors. I also show that the feedback regulation of U11-48K is counteracted by a component of the major spliceosome, the U1 small nuclear ribonucleoprotein particle, as well as members of the hnRNP F/H protein family. These results thus suggest that the feedback mechanism is finely tuned by multiple factors to achieve precise control of the activity of the U12-dependent spliceosome.

Importance of COMT in the regulation of prefrontal dopamine

The prefrontal cortex (PFC), located in the anterior region of the frontal lobe, is considered to have several key roles in higher cognitive and executive functions. In general, the PFC can be seen as a coordinator of thought and action allowing subjects to behave in a goal-directed manner. Due to its anatomical connections with a variety of cortical and subcortical structures, several neurotransmitters, including dopamine, are involved in the regulation of PFC activity. In general, the majority of released dopamine is cleared by the dopamine transporter (DAT). In the PFC however, the number of presynaptic DAT is diminished, emphasizing the relative importance of catechol-O-methyltransferase (COMT) in dopamine metabolism. As a result, the role of COMT in the etiology of psychotic disorders is under constant debate. The present study investigated the role of COMT in prefrontal cortical dopamine metabolism by different neurochemical methods in COMT knockout (COMT-KO) mice. Pharmacological tools to inhibit other dopamine clearing mechanisms were also used for a more comprehensive and collective picture. In addition, this study investigated how a lack of the soluble (S-) COMT isoform affects the total COMT activity as well as the pharmacokinetics of orally administered L-dopa using mutant mice expressing only the membrane-bound (MB-) COMT isoform. Also the role of COMT in striatal and accumbal dopamine turnover during Δ9-tetrahydrocannabinol (THC) challenge was studied. We found markedly increased basal dopamine concentrations in the PFC, but not the striatum or nucleus accumbens (NAcc), of mice lacking COMT. Pharmacological inhibition of the noradrenaline transporter (NET) and monoamine oxidase (MAO) elevated prefrontal cortical dopamine levels several-fold, whereas inhibition of DAT did not. The lack of COMT doubled the dopamine raising effects of NET and MAO inhibition. No compensatory expression of either DAT or NET was found in the COMT-KO mice. The lack of S-COMT decreased the total COMT activity by 50-70 % and modified dopamine transmission and the pharmacokinetics of exogenous Ldopa in a sex and tissue specific manner. Finally, we found that subsequent tolcapone and THC increased dopamine levels in the NAcc, but not in the striatum. Conclusively, this study presents neurochemical evidence for the important role of COMT in the PFC and shows that COMT is responsible for about half of prefrontal cortical dopamine metabolism. This study also highlights the previously underestimated proportional role of MB-COMT and supports the clinical evidence of a gene x environment interaction between COMT and cannabis.