Conceptual modeling of mRNA decay provokes new hypotheses.

Biologists are required to integrate large amounts of data to construct a working model of the system under investigation. This model is often informal and stored mentally or textually, making it prone to contain undetected inconsistencies, inaccuracies, or even contradictions, not much less than a representation in free natural language. Using Object-Process Methodology (OPM), a formal yet visual and humanly accessible conceptual modeling language, we have created an executable working model of the mRNA decay process in Saccharomyces cerevisiae, as well as the import of its components to the nucleus following mRNA decay. We show how our model, which incorporates knowledge from 43 articles, can reproduce outcomes that match the experimental findings, evaluate hypotheses, and predict new possible outcomes. Moreover, we were able to analyze the effects of the mRNA decay model perturbations related to gene and interaction deletions, and predict the nuclear import of certain decay factors, which we then verified experimentally. In particular, we verified experimentally the hypothesis that Rpb4p, Lsm1p, and Pan2p remain bound to the RNA 3'-untranslated region during the entire process of the 5' to 3' degradation of the RNA open reading frame. The model has also highlighted erroneous hypotheses that indeed were not in line with the experimental outcomes. Beyond the scientific value of these specific findings, this work demonstrates the value of the conceptual model as an in silico vehicle for hypotheses generation and testing, which can reinforce, and often even replace, risky, costlier wet lab experiments.

Promoter elements regulate cytoplasmic mRNA decay.

Promoters are DNA elements that enable transcription and its regulation by trans-acting factors. Here, we demonstrate that yeast promoters can also regulate mRNA decay after the mRNA leaves the nucleus. A conventional yeast promoter consists of a core element and an upstream activating sequence (UAS). We find that changing UASs of a reporter gene without altering the transcript sequence affects the transcript's decay kinetics. A short cis element, comprising two Rap1p-binding sites, and Rap1p itself, are necessary and sufficient to induce enhanced decay of the reporter mRNA. Furthermore, Rap1p stimulates both the synthesis and the decay of a specific population of endogenous mRNAs. We propose that Rap1p association with target promoter in the nucleus affects the composition of the exported mRNP, which in turn regulates mRNA decay in the cytoplasm. Thus, promoters can play key roles in determining mRNA levels and have the capacity to coordinate rates of mRNA synthesis and decay.

Exposure to stressors during juvenility disrupts development-related alterations in the PSA-NCAM to NCAM expression ratio: potential relevance for mood and anxiety disorders.

Childhood trauma is associated with higher rates of both mood and anxiety disorders in adulthood. The exposure of rats to stressors during juvenility has comparable effects, and was suggested as a model of induced predisposition for these disorders. The neural cell adhesion molecule (NCAM) and its polysialylated form PSA-NCAM are critically involved in neural development, activity-dependent synaptic plasticity, and learning processes. We examined the effects of exposure to stressors during juvenility on coping with stressors in adulthood and on NCAM and PSA-NCAM expression within the rat limbic system both soon after the exposure and in adulthood. Exposure to stressors during juvenility reduced novel-setting exploration and impaired two-way shuttle avoidance learning in adulthood. Among naive rats, a development-related decrease of about 50% was evident in the PSA-NCAM to NCAM expression ratio in the basolateral amygdala, in the CA1 and dentate gyrus regions of the hippocampus, and in the entorhinal cortex. In juvenile-stressed rats, we found no such decrease, but rather an increase in the polysialylation of NCAM ( approximately 50%), evident soon after the exposure to juvenile stress and also in adulthood. Our results suggest that exposure to stressors during juvenility alters the maturation of the limbic system, and potentially underlies the predisposition to exhibit stress-related symptoms in adulthood.

Neuromodulators of LTP and NCAMs in the amygdala and hippocampus in response to stress.

Possibly, at the onset of an emotional event the stress hormones permissively mediate plasticity. Specifically, CORT and NE stress hormones participate in modulation of memory consolidation processes in both the amygdala and the hippocampus. In addition, glucocorticoids and norepinephrin bound to adrenoceptors are also involved in modulating the regulation of NCAM polysialylation both in the amygdala and in the hippocampus. PSA-related synaptic remodeling is mobilized for memory formation in particularly challenging circumstances.

The zinc finger of the CSN-associated deubiquitinating enzyme USP15 is essential to rescue the E3 ligase Rbx1.

The COP9 signalosome (CSN) is a conserved protein complex found in all eukaryotic cells and involved in the regulation of the ubiquitin (Ub)/26S proteasome system. It binds numerous proteins, including the Ub E3 ligases and the deubiquitinating enzyme Ubp12p, the S. pombe ortholog of human USP15. We found that USP15 copurified with the human CSN complex. Isolated CSN complex exhibited protease activity that deubiquitinated poly-Ub substrates and was completely inhibited by o-phenanthroline (OPT), a metal-chelating agent. Surprisingly, the recombinant USP15 was also not able to cleave isopeptide bonds of poly-Ub chains in presence of OPT. Detailed analysis of USP sequences led to the discovery of a novel zinc (Zn) finger in USP15 and related USPs. Mutation of a single conserved cysteine residue in the predicted Zn binding motif resulted in the loss of USP15 capability to degrade poly-Ub substrates, indicating that the Zn finger is essential for the cleavage of poly-Ub chains. Moreover, pulldown experiments demonstrated diminished binding of tetra-Ub to mutated USP15. Cotransfection of USP15 and the Ub ligase Rbx1 revealed that the wild-type deubiquitinating enzyme, but not the USP15 mutant with a defective Zn finger, stabilized Rbx1 toward the Ub system, most likely by reversing poly/autoubiquitination. In summary, a functional Zn finger of USP15 is needed to maintain a conformation essential for disassembling poly-Ub chains, a prerequisite for rescuing the E3 ligase Rbx1.

Deubiquitinating enzymes are IN/(trinsic to proteasome function).

Covalent conjugation of the ubiquitin tag to cellular proteins plays a central role in a number of processes, the most notable among them being degradation by the 26S proteasome. A fundamental property of this process is that ubiquitination, in contrast to subsequent degradation, is reversible due to a number of deubiquitinating enzymes that mediate the disassembly of ubiquitin-protein conjugates. The uniqueness of ubiquitin as a reversible tag necessitates mechanisms to guarantee its efficiency. Interestingly, some deubiquitinating enzymes are associated with the 26S proteasome itself. We include a brief overview of the key proteasome-associated deubiquitinating enzymes such as Rpn11/POH1, UCH37/Uch2, Ubp6/Usp14 and Doa4/Ubp4. We go on to discuss how these enzymes may contribute to, or possibly counteract, proteolysis by the proteasome. For example, cumulative evidence points to a partitioning of proteasome action between proteolysis and deubiquitination. On the one hand, inhibition of proteolysis promotes deubiquitination, while on the other hand, inhibition of deubiquitination can promote proteolysis. The plethora of deubiquitinating enzymes may serve as proof reading devices altering the equilibrium between these two processes and allowing for reversal of fortune at various stages of the process. To promote degradation over deubiquitination, certain polyubiquitin conformations could be stabilized or protected from deubiquitinating enzymes in order that they can serve as efficient targeting signals leading to the proteasome. We hypothesize that polvubiquitin chains could also serve as "timers": by slowing down chain disassembly, longer chains allow ample time for unfolding and proteolysis of the substrate.

Complementary roles for Rpn11 and Ubp6 in deubiquitination and proteolysis by the proteasome.

Substrates destined for degradation by the 26 S proteasome are labeled with polyubiquitin chains. These chains can be dismantled by deubiquitinating enzymes (DUBs). A number of reports have identified different DUBs that can hydrolyze ubiquitin from substrates bound to the proteasome. We measured deubiquitination by both isolated lid and base-core particle subcomplexes, suggesting that at least two different DUBs are intrinsic components of 26 S proteasome holoenzymes. In agreement, we find that highly purified proteasomes contain both Rpn11 and Ubp6, situated within the lid and base subcomplexes, respectively. To study their relative contributions, we purified proteasomes from a mutant in the putative metalloprotease domain of Rpn11 and from a ubp6 null. Interestingly, in both preparations we observed slower deubiquitination rates, suggesting that Rpn11 and Ubp6 serve complementary roles. In accord, the double mutant is synthetically lethal. In contrast to WT proteasomes, proteasomes lacking the lid subcomplex or those purified from the rpn11 mutant are less sensitive to metal chelators, supporting the prediction that Rpn11 may be a metalloprotein. Treatment of proteasomes with ubiquitin-aldehyde or with cysteine modifiers also inhibited deubiquitination but simultaneously promoted degradation of a monoubiquitinated substrate along with the ubiquitin tag. Degradation is unique to 26 S proteasome holoenzymes; we could not detect degradation of a ubiquitinated protein by "lidless" proteasomes, although they were competent for deubiquitination. The fascinating observation that a single ubiquitin moiety is sufficient for targeting an otherwise stable substrate to proteasomes exposes how rapid deubiquitination of poorly ubiquitinated substrates may counteract degradation.