In eukaryotes, mRNA is in constant flux between an actively translating state and translationally repressed states. Specifically, mRNA degradation and repression factors compete with translation factors to direct mRNAs out of translation for storage or decay. This process often leads to formation of cytoplasmic aggregates. P-bodies are granules that contain mRNA and degradation factors, suggesting they are sites of mRNA decay or storage. Stress granules form in response to stress conditions and contain mRNAs and translation factors.P-bodies and stress granules consist of mRNPs of different compositions, believed to mature and transition between the states. It is proposed that mRNAs transition between the two granules. In the work described below, we use <italic>Saccharomyces cerevisiae</italic> to demonstrate that a decay factor, Dhh1 is capable of existing in both P-body and stress granule mRNPs. This suggests that a decay factor can be part of two different mRNP complexes. Additionally, we identify two novel components of the stress granule mRNPs, Pbp4 and Lsm12, and determine that they are not essential for stress granule formation. Lastly, we show that the stress granule mRNP factor, Pab1, is not absolutely required for stress granule formation.An important aspect of cytoplasmic mRNA regulation is mRNA quality control. One example of this is nonsense-mediated mRNA decay (NMD), whereby aberrant mRNAs containing premature termination codons are targeted for decay, and can be localized to P-bodies. Upf1-3 and the mRNA decapping complex, Dcp2/Dcp1 are essential for NMD, which requires Upf1 interaction with stalled ribosomal/mRNA complexes to target aberrant mRNA for decapping and degradation. How Dcp2/Dcp1 is recruited to aberrant mRNA is poorly understood.Here, we show by yeast two-hybrid assays that an interaction between Dcp2 and Upf1 is mediated by the decapping stimulator Edc3. Interestingly, Edc3 and Upf2 share overlapping binding sites on the Upf1 N-terminal domain. The decapping stimulator, Pat1, also interacts on the Upf1 N-terminus, but Edc3 and Pat1 are not essential for NMD. Surprisingly, the Upf1-Edc3 interaction does not promote or negatively regulate NMD. Thus, the Upf1-Edc3 and Upf1-Pat1 interactions likely regulate a subset of mRNA transcripts, or are essential for proper NMD under different environmental conditions.

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Post-transcriptional control of gene expression in Eukaryotes plays a pivotal role in determining intricated networks defining physiological and pathological conditions among each organism. RNA Binding Proteins (RBPs), by exploiting RNA-protein and protein-protein interactions, have been recognized as the main actors in modulating these processes. As a consequence, RBPs aberrant expression, modulation or mis-localization, leads to the insurgence of complex phenotypes and diseases. Therefore, targeting and modulating the activity of RBPs found associated to different pathologies represents a new promising therapeutic strategy. During my PhD I aimed at identify, characterize and refine inhibitors targeting the RNA binding protein HuR. HuR belongs to the ELAVL protein family, it is ubiquitously expressed in the cells and among tissues and highly conserved throughout mammalian evolution. By binding AU/U rich elements (ARE) in the 3’UTRs of mRNAs, HuR mainly stabilizes its target transcripts, enhancing their translation. ARE sequences are found in 7% of the human mRNAs, coding for protein involved in key cellular processes as: immune response and inflammation, cell division and proliferation, angiogenesis, senescence and apoptosis. Hence, dysregulation in HuR expression and in its subcellular localization have been associated with the insurgence of several pathologies, mostly cancers and inflammation diseases. Notably, malignant transformations and poor prognosis in patients have been found characterized by highly nuclear or cytosolic HuR expression in a significant number of human cancers. Indeed, the majority of HuR regulated transcripts encode for protein responsible for the appearance of several cancerogenic traits. In particular, critical crosstalk established between cancer cells and inflammation processes play a pivotal role in worsening and compromising cancers development and onset. Moreover, considering that 90% of mRNAs coding for cytokines and chemokines contains repeated AREs sites in the 3’UTR, HuR plays a strong regulatory role in immune system (innate and adaptive) development and homeostasis as well as in pathogenic mechanisms. The searching for HuR inhibitors represents a challenging area, in the drug discovery field, due to its pleiotropic functions and its intrinsic structural complexity, which presents unfolded regions and sequences prone to aggregation. HuR disruptors have been reported in the literature, but without systematic studies, thus the identification of a new class of small molecules is still at the beginning. Among the molecules discovered so far, in 2015 our group identified through a High-throughput Screening a natural compound, DHTS, as a bona fide HuR inhibitor. Following that finding, we, me included, ascribed to the molecule a well-defined mechanism of action, identifying the specific binding sites on which HuR:DHTS interaction is based, defining that upon the mRNA binding DHTS interplays with HuR maintaining the protein in a closed conformation, thus inhibiting its function. Furthermore, we demonstrated DHTS anti-cancer activity in vitro, in cellular context and in vivo, in an HuR-dependent manner. In this way, DHTS represented the molecular scaffold, for the generation of a new class of highly potent HuR inhibitors, called Tanshinone Mimics (TMs). A functional oriented approach was applied for the synthesis of new molecules harboring only DHTS chemical elements responsible for HuR targeting, leading to a completely new molecular scaffold, not previously described in the literature, with respect to the ancestor molecule. I have characterized and identified more potent molecules, describing their anticancer properties, through the evaluation of their capabilities of downregulating the total expression level of well-known HuR targets, coding for proteins involved in tumor insurgence and progression, as VEGF, ERBB2 and CTNNB1, and reducing cancer cell migration, cell cycle progression in a minor extent. On the other end, I have explored TMs anti-inflammatory properties, counteracting the inflammatory response mediated by macrophages, directly impairing the binding between HuR and its pro-inflammatory targets, diminishing their expression and related protein secretion. Moreover, I have put evidences on TMs activity in vivo in acute inflammation mouse models. Lastly, I have evaluated TMs activity in affecting T-cells proliferation, on which HuR it is known to play a regulatory role. In conclusion, we identified TMs with Structure-Activity Relationships (SARs) towards HuR inhibition and its biological implications, aimed at ameliorating their specificity and bioavailability suitable for in vivo therapeutic strategies.

<p>The interest for the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis and its role in the central nervous system (CNS) has grown during the past decade. GH has been associated with psychological functions as sleep, mood, general well-being and learning and memory. The present thesis is a contribution to clarify the functions and mechanisms involved in the actions of GH and IGF-1 in the CNS. A variant of the GH receptor (GHR) gene transcript lacking exon 3 (GHR3-) was cloned from ovine choroid plexus epithelial cells and tissue. The GHR3- transcript has previously only been identified in human tissue. Further, an anatomical study of the localization of GHR mRNA in the rat brain stem and spinal cord was carried out by the use of in situ hybridization. High densities of GHRs were found in areas associated with the regulation of food intake, sleep and nociception, functions known to be influenced by the GH/IGF-1 axis. The interaction with the opioid system was studied by an acute treatment with morphine. The levels of the transcripts for GHR and GHBP in the rat hippocampus and spinal cord were decreased 4 h after the injection of the opiate and restored to normal levels after 24 h. Young and aged rats injected with GH or IGF-1 showed differential gene regulation of subunits of the NMDA subtype of glutamate receptor in the hippocampus. This indicates an age-related difference in the sensitivity to GH/IGF-1 mediated effects on memory functions. Moreover, hypophysectomized rats treated with GH showed improved performance in the Morris water maze, a spatial memory task. The effect was accompanied with an increase in transcripts for NMDA receptor subunits and its associated membrane anchoring PSD-95 protein. Taken together, the results suggest that GH and/or IGF-1 play important roles in mechanisms associated with cognitive functions.</p>

The interest for the growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis and its role in the central nervous system (CNS) has grown during the past decade. GH has been associated with psychological functions as sleep, mood, general well-being and learning and memory. The present thesis is a contribution to clarify the functions and mechanisms involved in the actions of GH and IGF-1 in the CNS. A variant of the GH receptor (GHR) gene transcript lacking exon 3 (GHR3-) was cloned from ovine choroid plexus epithelial cells and tissue. The GHR3- transcript has previously only been identified in human tissue. Further, an anatomical study of the localization of GHR mRNA in the rat brain stem and spinal cord was carried out by the use of in situ hybridization. High densities of GHRs were found in areas associated with the regulation of food intake, sleep and nociception, functions known to be influenced by the GH/IGF-1 axis. The interaction with the opioid system was studied by an acute treatment with morphine. The levels of the transcripts for GHR and GHBP in the rat hippocampus and spinal cord were decreased 4 h after the injection of the opiate and restored to normal levels after 24 h. Young and aged rats injected with GH or IGF-1 showed differential gene regulation of subunits of the NMDA subtype of glutamate receptor in the hippocampus. This indicates an age-related difference in the sensitivity to GH/IGF-1 mediated effects on memory functions. Moreover, hypophysectomized rats treated with GH showed improved performance in the Morris water maze, a spatial memory task. The effect was accompanied with an increase in transcripts for NMDA receptor subunits and its associated membrane anchoring PSD-95 protein. Taken together, the results suggest that GH and/or IGF-1 play important roles in mechanisms associated with cognitive functions.