Optimization of carbon and energy utilization through differential translational efficiency.

Control of translation is vital to all species. Here we employ a multi-omics approach to decipher condition-dependent translational regulation in the model acetogen Clostridium ljungdahlii. Integration of data from cells grown autotrophically or heterotrophically revealed that pathways critical to carbon and energy metabolism are under strong translational regulation. Major pathways involved in carbon and energy metabolism are not only differentially transcribed and translated, but their translational efficiencies are differentially elevated in response to resource availability under different growth conditions. We show that translational efficiency is not static and that it changes dynamically in response to mRNA expression levels. mRNAs harboring optimized 5'-untranslated region and coding region features, have higher translational efficiencies and are significantly enriched in genes encoding carbon and energy metabolism. In contrast, mRNAs enriched in housekeeping functions harbor sub-optimal features and have lower translational efficiencies. We propose that regulation of translational efficiency is crucial for effectively controlling resource allocation in energy-deprived microorganisms

An E2-guided E3 Screen Identifies the RNF17-UBE2U Pair as Regulator of the Radiosensitivity, Immunodeficiency, Dysmorphic Features, and Learning Difficulties (RIDDLE) Syndrome Protein RNF168

Protein ubiquitination has emerged as a pivotal regulatory reaction that promotes cellular responses to DNA damage. With a goal to delineate the DNA damage signal transduction cascade, we systematically analyzed the human E2 ubiquitin- and ubiquitin-like-conjugating enzymes for their ability to mobilize the DNA damage marker 53BP1 onto ionizing radiation-induced DNA double strand breaks. An RNAi-based screen identified UBE2U as a candidate regulator of chromatin responses at double strand breaks. Further mining of the UBE2U interactome uncovered its cognate E3 RNF17 as a novel factor that, via the radiosensitivity, immunodeficiency, dysmorphic features, and learning difficulties (RIDDLE) syndrome protein RNF168, enforces DNA damage responses. Our screen allowed us to uncover new players in the mammalian DNA damage response and highlights the instrumental roles of ubiquitin machineries in promoting cell responses to genotoxic stress.published_or_final_versio

Ubiquitin Ligase RNF146 Regulates Tankyrase and Axin to Promote Wnt Signaling

Canonical Wnt signaling is controlled intracellularly by the level of β-catenin protein, which is dependent on Axin scaffolding of a complex that phosphorylates β-catenin to target it for ubiquitylation and proteasomal degradation. This function of Axin is counteracted through relocalization of Axin protein to the Wnt receptor complex to allow for ligand-activated Wnt signaling. AXIN1 and AXIN2 protein levels are regulated by tankyrase-mediated poly(ADP-ribosyl)ation (PARsylation), which destabilizes Axin and promotes signaling. Mechanistically, how tankyrase limits Axin protein accumulation, and how tankyrase levels and activity are regulated for this function, are currently under investigation. By RNAi screening, we identified the RNF146 RING-type ubiquitin E3 ligase as a positive regulator of Wnt signaling that operates with tankyrase to maintain low steady-state levels of Axin proteins. RNF146 also destabilizes tankyrases TNKS1 and TNKS2 proteins and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels. We show that RNF146, tankyrase, and Axin form a protein complex, and that RNF146 mediates ubiquitylation of all three proteins to target them for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase protein aggregation at a centrosomal location. Tankyrase auto-PARsylation and PARsylation of Axin is known to lead to proteasome-mediated degradation of these proteins, and we demonstrate that, through ubiquitylation, RNF146 mediates this process to regulate Wnt signaling

Role of cki-2 during development in C. elegans

Rapid progress has been made toward understanding the significance of CDK inhibitor proteins (CKIs) in the regulation of cell cycle progression. The overall goal of this study has been targeted to further expand our knowledge of CKI function through the investigation of a previously uncharacterized CKI named cki-2 during development in C. elegans. The characterization of cki-2 using a reverse genetic approach called co-suppression has revealed a novel mechanism that cki-2 and its related cell cycle regulators are required for the appropriate elimination of centrioles during oogenesis. Loss of cki-2 in the germ line caused perdurance of centrioles into the one-cell embryo, resulting in supernumerary centrosomes and aberrant cell divisions in the first cell cycle. This was significantly suppressed by reduction of cyclin E and a Cdk2 homologue, indicating that these cell cycle regulators are involved in this critical developmental process. In order to further understand the function of cki-2, a yeast two-hybrid screen was conducted which allowed us to identify three CKI-2 interacting proteins: orthologues of PCNA (PCN-1), SUMO (SMO-1), and a RING finger protein called RNF-1. CKI-2 has functionally separable domains in its amino (Cyclin/Cdk binding)- and carboxy (PCNA binding)-terminus and they exert distinct roles in cell cycle progression. It was observed that CKI-2 is covalently modified by SUMO on its N-terminus and this causes CKI-2 to relocalize to thr nucleolus, which is associated with its rapid degradation. Since many RING finger proteins act as components of the multi-subunit E3 ubquitin ligases, we speculated that RNF-1 might be involved in the CKI-2 degradation. This possibility was tested by co-expression of RNF-1 with CKI-2, revealing that co-expression of RNF-1 suppresses the embryonic lethality caused by the CKI-2 overexpression and moreover, this is correlated with an increased rate of CKI-2 degradation. In addition, western blot analyses performed on different genetic backgrounds suggested that the CKI-2 degradation occurs in an ubiquitin-dependent manner through the proteasome-mediated proteolysis pathway. Furthermore, a yeast-based assay developed to test a possible role of SUMO in modulating the CKI-2/RNF-1 interaction demonstrated that SUMO may antagonize the interaction between CKI-2 and RNF-l, these highlighting an intriguing model that appropriate levels of CKI-2 are regulated through ubiquitin-dependent proteolysis mediated by RNF-l, and which maybe modulated by SUMO

Ring finger protein 126 (RNF126) suppresses ionizing radiation-induced p53-binding protein 1 (53BP1) focus formation

Cells have evolved sophisticated mechanisms to maintain genomic integrity in response to DNA damage. Ionizing radiation (IR)-induced DNA damage results in the formation of IR-induced foci (iRIF) in the nucleus. The iRIF formation is part of the DNA damage response (DDR), which is an essential signaling cascade that must be strictly regulated because either the loss of or an augmented DDR leads to loss of genome integrity. Accordingly, negative regulation of the DDR is as critical as its activation. In this study, we have identified ring finger protein 126 (RNF126) as a negative regulator of the DDR from a screen of iRIF containing 53BP1. RNF126 overexpression abolishes not only the formation of 53BP1 iRIF but also of RNF168, FK2, RAP80, and BRCA1. However, the iRIF formation of H2AX, MDC1, and RNF8 is maintained, indicating that RNF126 acts between RNF8 and RNF168 during the DDR. In addition, RNF126 overexpression consistently results in the loss of RNF168-mediated H2A monoubiquitination at lysine 13/15 and inhibition of the non-homologous end joining capability. Taken together, our findings reveal that RNF126 is a novel factor involved in the negative regulation of DDR, which is important for sustaining genomic integrity

Translational quality control mechanisms that mitigate stop codon readthrough and ensure protein homeostasis

Cells invest tremendously to maintain the fidelity for transcription and translation to ensure accurate transmission of the genetic code into proteins. Yet, errors may occur at each stage. During transcription, errors arise at a rate of ~10-5-10-4 per base, whereas the error rate during translation is around a magnitude higher with ~10-4-10-3 amino acid misincorporations per codon. Such errors become increasingly frequent with ageing, posing a sizable risk for the organism. In some instances, this leads to missing or misread stop codons, allowing translation to continue into the 3’UTRs of transcripts. Such C-terminal extensions may interfere with the folding of proteins, or worse, promote promiscuous interactions with other proteins, which in turn may disturb cellular processes and reduce overall fitness. Translation into the polyA-tail of transcripts leads to the activation of the ribosome quality control (RQC) complex, which clears both aberrant protein and mRNA. However, in most cases, translation would be terminated at stop codons within the 3’UTR before the ribosome reaches the polyA-tail. Such readthrough events would therefore not be recognized by the RQC. While previous studies suggested that such readthrough products are recognized and efficiently cleared by cells, the underlying mechanism remained unclear. Given the decline in translational fidelity during ageing, this clearance pathway is expected to become increasingly important to release the burden on the proteostasis network. Using the nematode C. elegans as a model for ageing, we aimed to identify the quality control mechanisms mitigating translational readthrough and investigated the consequences of their failure during ageing. Using this approach, we identified in C. elegans and human cells that readthrough proteins are cleared through a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay mediated by GCN1 and CCR4/NOT limits the accumulation of readthrough proteins. Selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes encounter non-optimal codons, a feature of 3′UTR sequences. Dysfunction of GCN1 results in mRNA and proteome imbalance, increasingly affecting transmembrane proteins and collagens during ageing. These results define GCN1 as a key factor acting during translation in maintaining protein homeostasis

Ubc13 dosage is critical for immunoglobulin gene conversion and gene targeting in vertebrate cells

In contrast to lower eukaryotes, most vertebrate cells are characterized by a moderate efficiency of homologous recombination (HR) and limited feasibility of targeted genetic modifications. As a notable exception, the chicken DT40 B cell line is distinguished by efficient homology-mediated repair of DNA lesions during Ig gene conversion, and also shows exceptionally high gene-targeting efficiencies. The molecular basis of these phenomena is elusive. Here we show that the activity levels of Ubc13, the E2 enzyme responsible for non-canonical K63-linked polyubiquitination, are critical for high efficiency of Ig gene conversion and gene targeting in DT40. Ubc13+/− cells show substantially lower homology-mediated repair, yet do not display changes in somatic hypermutation, overall DNA repair or cell proliferation. Our results suggest that modulation of the activity of K63-linked polyubiquitination may be used to customize HR efficiencies in vertebrate cells

HSV-1 ICP0: paving the way for viral replication

Herpes simplex virus type 1 (HSV-1) has two distinct phases of its viral life cycle: lytic and latent. One viral immediate-early protein that is responsible for determining the balance between productive lytic replication and reactivation from latency is infected cell protein 0 (ICP0). ICP0 is a 775-amino acid really interesting new gene (RING)-finger-containing protein that possesses E3 ubiquitin ligase activity, which is required for ICP0 to activate HSV-1 gene expression, disrupt nuclear domain (ND) 10 structures, mediate the degradation of cellular proteins, and evade the host cell’s intrinsic and innate antiviral defenses. This article examines our current understanding of ICP0’s transactivating, E3 ubiquitin ligase, and antihost defense activities and their inter-relationships to one another. Lastly, we will discuss how these properties of ICP0 may be utilized as possible targets for HSV-1 antiviral therapies

HORMONE EPIMERS REGULATE ER STRESS AND CORE REGULATORY GENES: NETWORK ANALYSIS WITH APPLICATIONS TO GLIOMA AND CHRONIC PRESSURE ULCERS

DHEA has been determined to have medically significant activity and is the parent compound to the more active metabolites; 17α-AED, 17β-AED and 17β-AET, which exhibit strong biological activity that has been attributed to androgenic, estrogenic or anti-glucocorticoid activity in vivo and in vitro. This study compared DHEA, 17α-AED, 17β-AED and 17β-AET for their ability to activate the human AR, ER and GR receptors and determine the relative androgenicity, estrogenicity and glucocorticoid activity. The results show that, at the receptor level, these androstene hormones are weak AR and even weaker ER activators. Direct androstene hormone activation of the human AR, ERα, and ERβ may not be essential for their biological function. Similarly, these hormones indirectly activated the human GR receptor; only in the presence of high dexamethasone concentrations. These results underscore the major difference between androstene hormone interactions with these nuclear receptors. 17β-AED and 17α-AED, androstene epimers that produce either survival or death, were utilized to treat T98G Glioblastoma cells. We identified 26 genes oppositely regulated by 17β-AED and 17α-AED to directly affect the cellular life or death decision. Network analysis demonstrated that these 26 genes are essential to regulating three critical Glioblastoma pathways. This report, for the first time, demonstrates that naturally occurring, chemically identical adrenal hormones (17β-AED or 17α-AED) direct a cellular life or death decision through contrasting modulation of identical signaling pathways and core regulators. Chronic pressure ulcers represent a significant health problem and are characterized by hypoxia, bacterial infection, repetitive ischemia/reperfusion and altered cellular and systemic stress responses. Whole genome microarray analysis was utilized in conjunction with IPA® premiere networking software to analyze chronic wound edge tissue. IPA® network analysis identified Ubiquitin C (UBC) as the most significant network. Sixteen (16) ubiquitin C associated genes were identified to be different in the chronic pressure ulcer and normal skin control. Targeted network analysis associated core regulators to 8 UBC associated genes that are unique to chronic pressure ulcers. The identification of these genes will allow the establishment of more effective treatments for Spinal Cord Injury (SCI) patients with chronic pressure ulcers

The Function of Wnt/beta-catenin Signaling in Ewing Sarcoma and its Contribution to Pathogenesis

Ewing sarcoma is an aggressive bone and soft tissue tumor with a high propensity for metastasis; however, the mechanisms that contribute to this process are poorly understood. The Wnt/beta-catenin signaling pathway is critical for oncogenesis in numerous cancers, and although previous studies implicate a role for this pathway in Ewing sarcoma, its specific function and contribution is unknown. Previous work by our lab revealed that the Wnt-modulatory receptor LGR5 is highly expressed in patients with aggressive disease, and we hypothesized that LGR5 regulates activation of Wnt/beta-catenin signaling. Through investigation of primary tumors, we discovered that focal nuclear beta-catenin is detectable in a subset of Ewing sarcoma patients and strongly associated with LGR5 expression. Patients whose tumors have nuclear beta-catenin or high expression of the downstream Wnt/beta-catenin target LEF1, experienced worse clinical outcomes and overall survival. We next used in vitro and in vivo models to determine the function of Wnt/beta-catenin signaling in Ewing sarcoma. Importantly, we found that LGR5 expression and Wnt activation were highly heterogeneous. We then investigated the downstream effects of Wnt/beta-catenin activation in the most highly Wnt-responsive cells. RNA-sequencing revealed that Wnt/beta-catenin paradoxically inhibits EWS-ETS transcriptional activity, resulting in a phenotypic change from a proliferative to a migratory and metastatic state in vitro and in vivo. In addition, the metastasis-associated molecule Tenascin C was upregulated by Wnt/beta-catenin signaling, and was found to be a mediator of migration in vitro and metastasis in vivo. In the context of the tumor microenvironment, we further found that patient tumors with high Wnt/LEF1 expression had significant correlation with expression of stroma- and angiogenesis-related genes associated with a poor prognosis. Together, these data provide novel avenues of exploration for tumor-microenvironment interactions. In conclusion these findings implicate a critical role for Wnt/beta-catenin-singaling in mediating migration and metastasis. This occurs in part through antagonism of EWS/ETS fusion protein activity and by up-regulation of the metastasis-associated gene Tenascin C. In addition, tumor-microenvironment interactions modulated by Wnt/beta-catenin further contribute to pathogenesis. Together these findings provide exciting new venues for therapeutic investigation.PHDMolecular & Cellular Path PhDUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144020/1/easp_1.pd