TRIP/NOPO E3 Ubiquitin Ligase Promotes Ubiquitylation of DNA Polymerase η

We previously identified a Drosophila maternal effect-lethal mutant named ‘no poles’ (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid cycles of syncytial embryogenesis because of activation of a Chk2-mediated DNA checkpoint. NOPO is the Drosophila homolog of human TNF receptor associated factor (TRAF)-interacting protein (TRIP), which has been implicated in TNF signaling. NOPO and TRIP contain RING domains closely resembling those of known E3 ubiquitin ligases. We herein sought to elucidate the mechanism by which TRIP/NOPO promotes genomic stability by performing a yeast two-hybrid screen to identify potential substrates/interactors. We identified members of the Y-family of DNA polymerases that facilitate replicative bypass of damaged DNA (translesion synthesis) as TRIP interactors. We show that TRIP and NOPO co-immunoprecipitate with human and Drosophila Polη, respectively, from cultured cells. We generated a null mutation in Drosophila Polη (dPolη) and found that dPolη-derived embryos have increased sensitivity to ultraviolet irradiation and exhibit nopo-like mitotic spindle defects. dPolη and nopo interact genetically in that overexpression of dPolηn hypomorphic nopo-derived embryos suppresses nopo phenotypes. We observed enhanced ubiquitylation of Polη by TRIP and NOPO E3 ligases in human cells and Drosophila embryos, respectively, and show that TRIP promotes hPolη localization to nuclear foci in human cells. We present a model in which TRIP/NOPO ubiquitylates Polη to positively regulate its activity in translesion synthesis

Fission yeast 26S proteasome mutants are multi-drug resistant due to stabilization of the pap1 transcription factor

Here we report the result of a genetic screen for mutants resistant to the microtubule poison methyl benzimidazol-2-yl carbamate (MBC) that were also temperature sensitive for growth. In total the isolated mutants were distributed in ten complementation groups. Cloning experiments revealed that most of the mutants were in essential genes encoding various 26S proteasome subunits. We found that the proteasome mutants are multi-drug resistant due to stabilization of the stress-activated transcription factor Pap1. We show that the ubiquitylation and ultimately the degradation of Pap1 depend on the Rhp6/Ubc2 E2 ubiquitin conjugating enzyme and the Ubr1 E3 ubiquitin-protein ligase. Accordingly, mutants lacking Rhp6 or Ubr1 display drug-resistant phenotypes

PCI proteins eIF3e and eIF3m define distinct translation initiation factor 3 complexes

BACKGROUND: PCI/MPN domain protein complexes comprise the 19S proteasome lid, the COP9 signalosome (CSN), and eukaryotic translation initiation factor 3 (eIF3). The eIF3 complex is thought to be composed of essential core subunits required for global protein synthesis and non-essential subunits that may modulate mRNA specificity. Interactions of unclear significance were reported between eIF3 subunits and PCI proteins contained in the CSN. RESULTS: Here, we report the unexpected finding that fission yeast has two distinct eIF3 complexes sharing common core subunits, but distinguished by the PCI proteins eIF3e and the novel eIF3m, which was previously annotated as a putative CSN subunit. Whereas neither eIF3e nor eIF3m contribute to the non-essential activities of CSN in cullin-RING ubiquitin ligase control, eif3m, unlike eif3e, is an essential gene required for global cellular protein synthesis and polysome formation. Using a ribonomic approach, this phenotypic distinction was correlated with a different set of mRNAs associated with the eIF3e and eIF3m complexes. Whereas the eIF3m complex appears to associate with the bulk of cellular mRNAs, the eIF3e complex associates with a far more restricted set. The microarray findings were independently corroborated for a random set of 14 mRNAs by RT-PCR analysis. CONCLUSION: We propose that the PCI proteins eIF3e and eIF3m define distinct eIF3 complexes that may assist in the translation of different sets of mRNAs

PCI proteins eIF3e and eIF3m define distinct translation initiation factor 3 complexes

BACKGROUND: PCI/MPN domain protein complexes comprise the 19S proteasome lid, the COP9 signalosome (CSN), and eukaryotic translation initiation factor 3 (eIF3). The eIF3 complex is thought to be composed of essential core subunits required for global protein synthesis and non-essential subunits that may modulate mRNA specificity. Interactions of unclear significance were reported between eIF3 subunits and PCI proteins contained in the CSN. RESULTS: Here, we report the unexpected finding that fission yeast has two distinct eIF3 complexes sharing common core subunits, but distinguished by the PCI proteins eIF3e and the novel eIF3m, which was previously annotated as a putative CSN subunit. Whereas neither eIF3e nor eIF3m contribute to the non-essential activities of CSN in cullin-RING ubiquitin ligase control, eif3m, unlike eif3e, is an essential gene required for global cellular protein synthesis and polysome formation. Using a ribonomic approach, this phenotypic distinction was correlated with a different set of mRNAs associated with the eIF3e and eIF3m complexes. Whereas the eIF3m complex appears to associate with the bulk of cellular mRNAs, the eIF3e complex associates with a far more restricted set. The microarray findings were independently corroborated for a random set of 14 mRNAs by RT-PCR analysis. CONCLUSION: We propose that the PCI proteins eIF3e and eIF3m define distinct eIF3 complexes that may assist in the translation of different sets of mRNAs

Global gene expression during muscle hypertrophy induced by myostatin suppression and/or beta-adrenergic agonist

Ph.D. University of Hawaii at Manoa 2011.Includes bibliographical references.Skeletal muscle growth is important for animal agriculture, particularly for meat-producing animals, and for human health as well. A better understanding of the mechanisms regulating skeletal muscle growth is expected to contribute to improving the efficiency of meat animal production and alleviating human suffereings caused by muscle atrorphic conditions. Currently, two molecules have been shown to have dramatic effects on skeletal muscle mass: myostatin and β-adrenergic agonists. Myostatin (Mstn), a member of the TGF-β superfamily proteins, acts as a potent negative regulator of skeletal muscle growth. Suppression of Mstn by varying means has shown to increase skeletal muscle mass of animals. Administration of β-adrenergic agonist (BAA) such as clenbuterol (CL) induces dramatic increase in skeletal muscle mass or inhibits muscle atrophy. Although there have been numerous demonstrations of the muscle growth--promoting effect of BAA administration or Mstn suppression, the effect of a combination of BAA administration and Mstn inhibition on skeletal muscle growth has not been investigated. Similarly, very little is known about the molecular signaling pathways leading to muscle hypertrophy induced by the two stimuli and the genes that are commonly regulated by both Mstn and BAA administration. Therefore, the objectives of this study were 1) to investigate the combined effect of Mstn-suppression and BAA administration on skeletal muscle growth, 2) to examine the role of the Akt/mTOR pathway in the two muscle hypertrophic models, 3) to examine global changes in gene expression in skeletal muscle undergoing hypertrophy induced by chronic suppression of Mstn or BAA administration , and 4) to compare the changes in gene expression between these two muscle hypertrophic models. We used a transgenic mouse strain that overexpresses the Mstn-prodomain (Mstn-pro) and exhibits a significant increase in skeletal muscle mass regardless of age and sex. Clenbuterol (CL) was used as a BAA compound. Heterozygous Mstn-pro and wild-type littermates were produced and were given 0 or 20 ppm of CL in their drinking water. Phosphorylation of molecules involved in the Akt/mTOR pathway was examined by using the Western blot analysis. RNA samples of the gastrocnemius muscle in each group were subjected to microarray analysis using the Affymetrix GeneChip Mouse 430-2.0 platform. CL increased body and muscle mass of male and female mice in both genotypes, indincating that the muscle-hypertrophic effect of CL is additive to the effect of Mstn suppression. Levels of phosphorylated muscle 4E-BP1 and p70S6k, two downstream effectors of the mTOR pathway, were higher in Mstn-pro mice than in wild type mice. Levels of phosphorylated muscle Akt, an upstream effector of the mTOR pathway, were also higher in Mstn-pro mice than in wild type mice, indicating that the Akt/mTOR anabolic pathway is involved in the regulation of muscle mass by Mstn. CL increased the phosphorylation of Akt, 4E-BP1 and p70S6k in both genotypes, resulting in the highest phosphorylation levels of Akt, 4E-BP1 and p70S6k in CL-fed Mstn-pro mice. This result suggests that like Mstn, BAA also regulates muscle hypertrophy through the Akt/mTOR pathway, and the pathways of Mstn and CL signaling converge to the Akt/mTOR anabolic pathway to regulate skeletal muscle hypertrophy. Microarray analysis of global gene expression showed that Mstn suppression and CL administration induced significant changes in the mRNA abundance of various genes associated with muscle contraction, initiation of translation, transcription, and muscle hypertrophic signal pathway, suggesting that increased protein synthesis is partly responsible for the hypetrophy induced by Mstn and CL. Additionally, the alteration of Igf2 obsderved in Mstn suppressed mice, and the alterations of eIF4e, Acvr2b, FoxO and PTEN observed in mice treated with CL indicate that the pathways of Mstn and CL signaling converge to the Akt/mTOR anabolic pathway to regulate skeletal muscle hypertrophy

The ubiquitin-conjugating DNA repair enzyme is a maternal factor essential for early embryonic development in mice

The Saccharomyces cerevisiae RAD6 protein is required for a surprising diversity of cellular processes, including sporulation and replicational damage bypass of DNA lesions. In mammals, two RAD6-related genes, HR6A and HR6B, encode highly homologous proteins. Here, we describe the phenotype of cells and mice deficient for the mHR6A gene. Just like mHR6B knockout mouse embryonic fibroblasts, mHR6A-deficient cells appear to have normal DNA damage resistance properties, but mHR6A knockout male and female mice display a small decrease in body weight. The necessity for at least one functional mHR6A (X-chromosomal) or mHR6B (autosomal) allele in all somatic cell types is supported by the fact that neither animals lacking both proteins nor females with only one intact mHR6A allele are viable. In striking contrast to mHR6B knockout males, which show a severe spermatogenic defect, mHR6A knockout males are normally fertile. However, mHR6A knockout females fail to produce offspring despite a normal ovarian histology and ovulation. The absence of mHR6A in oocytes prevents development beyond the embryonic two-cell stage but does not result in an aberrant methylation pattern of histone H

Interaction of hHR23 with S5a. The ubiquitin-like domain of hHR23 mediates interaction with S5a subunit of 26 S proteasome

hHR23B is one of two human homologs of the Saccharomyces cerevisiae nucleotide excision repair (NER) gene product RAD23 and a component of a protein complex that specifically complements the NER defect of xeroderma pigmentosum group C (XP-C) cell extracts in vitro. Although a small proportion of hHR23B is tightly complexed with the XP-C responsible gene product, XPC protein, a vast majority exists as an XPC-free form, indicating that hHR23B has additional functions other than NER in vivo. Here we demonstrate that the human NER factor hHR23B as well as another human homolog of RAD23, hHR23A, interact specifically with S5a, a subunit of the human 26 S proteasome using the yeast two-hybrid system. Furthermore, hHR23 proteins were detected with S5a at the position where 26 S proteasome sediments in glycerol gradient centrifugation of HeLa S100 extracts. Intriguingly, hHR23B showed the inhibitory effect on the degradation of (125)I-lysozyme in the rabbit reticulocyte lysate. hHR23 proteins thus appear to associate with 26 S proteasome in vivo. From co-precipitation experiments using several series of deletion mutants, we defined the domains in hHR23B and S5a that mediate this interaction. From these results, we propose that part of hHR23 proteins are involved in the proteolytic pathway in cells

The conserved ubiquitin-like protein Hub1 plays a critical role in splicing in human cells

Different from canonical ubiquitin-like proteins, Hub 1 does not form covalent conjugates with substrates but binds proteins non- covalently. In Saccharomyces cerevisiae , Hub 1 associates with spliceosomes and mediates alternative splicing of SRC 1 , without affecting pre-mRNA splicing generally. Human Hub 1 is highly similar to its yeast homolog, but its cellular function remains largely unexplored. Here, we show that human Hub 1 binds to the spliceosomal protein Snu 66 as in yeast; however, unlike its S. cerevisiae homolog, human Hub 1 is essential for viability. Prolonged in vivo depletion of human Hub 1 leads to various cellular defects, including splicing speckle abnormalities, partial nuclear retention of mRNAs, mitotic catastrophe, and consequently cell death by apoptosis. Early consequences of Hub 1 depletion are severe splicing defects, however, only for specific splice sites leading to exon skipping and intron retention. Thus, the ubiquitin-like protein Hub 1 is not a canonical spliceosomal factor needed generally for splicing, but rather a modulator of spliceosome performance and facilitator of alternative splicing

CHARACTERIZATION OF PLANT POLYADENYLATION TRANSACTING FACTORS-FACTORS THAT MODIFY POLY(A) POLYMERSE ACTIVITY

Plant polyadenylation factors have proven difficult to purify and characterize, owing to the presence of excessive nuclease activity in plant nuclear extracts, thereby precluding the identification of polyadenylation signal-dependent processing and polyadenylation in crude extracts. As an alternative approach to identifying such factors, a screen was conducted for activities that inhibit the non-specific activity of plant poly(A) polymerases (PAP). One such factor (termed here as Putative Polyadenylation Factor B, or PPF-B) was identified in a screen of DEAE-Sepharose column fractions using a partially purified preparation of a plant nuclear poly(A) polymerase. This factor was purified to near homogeneity. Surprisingly, in addition to being an effective inhibitor of the nuclear PAP, PPF-B inhibited the activity of a chloroplast PAP. In contrast, this factor stimulated the activity of the yeast PAP. Direct assays of ATPase, proteinase, and nuclease activities indicated that inhibition of PAP activity was not due to depletion of substrates or degradation of products of the PAP reaction. The major polypeptide component of PPF-B proved to be a novel linker histone (RSP), which copurified with inhibitory activity by affinity chromatography on DNA-cellulose. The association of inhibitory activity with a linker histone and the spectrum of inhibitory activity, raise interesting possibilities regarding the role of PPF-B in nuclear RNA metabolism. These include a link between DNA damage and polyadenylation, as well as a role for limiting the polyadenylation of stable RNAs in the nucleus and nucleolus. The Arabidopsis genome possesses genes encoding probable homologs of most of the polyadenylation subunits that have been identified in mammals and yeast. Two of these reside on chromosome III and V and have the potential to encode a protein that is related to the yeast and mammalian Fip1 subunit (AtFip1-III and AtFip1-V). These genes are universally expressed in Arabidopsis tissues. AtFip1-V stimulates the non-specific activity of at least one Arabidopsis nuclear PAP, binds RNA, and interacts with other polyadenylation homologs AtCstF77 and AtCPSF30. These studies suggest that AtFip1- V is an authentic polyadenylation factor that coordinates other subunits and plays a role in regulating the activityof PAP in plants

Components of the Ubiquitin Proteasome System are Required for the Nonapoptotic Death of the C. Elegans Linker Cell

Cell death is a major cell fate that promotes tissue sculpting and morphogenesis during animal development. Many developmental cell-culling events cannot be accounted for solely by caspase-dependent apoptosis, yet, alternate pathways are poorly understood. Direct evidence that caspase-independent non-apoptotic cell death pathways operate during animal development is provided by studies of the C. elegans linker cell. Genetic studies of linker cell death have led to the identification of genes that promote this process, including pqn-41, which encodes a glutamine-rich protein, as well as tir-1/TIRdomain and sek-1/MAPKK, which may function in the same pathway as pqn-41. The let- 7 microRNA and its indirect target, the Zn-finger transcription factor LIN-29, also promote linker cell death, and may act early in the process. Our work suggests that components of the ubiquitin proteasome system (UPS) act to promote linker cell death. We show that LET-70, an E2 ubiquitin-conjugating enzyme, is required cell-autonomously for linker cell death. LET-70 levels, as well as those of ubiquitin and some proteasome components, increase just before linker cell death initiation. This rise is dependent on an MLL-type histone methyltransferase complex and a MAPK cascade, whose activities are required for linker cell death. The E3 ligase components SIAH-1, RBX-1, and CUL-3 are also required for linker cell death and appear to act in the same pathway as let-70. We also identify the PLZF transcription factor EOR-1, and its accessory protein, EOR-2 as major regulators of linker cell death. Our studies suggest that EOR-1/2, as well as all known regulators of the linker cell death pathway may act upstream of UPS components to promote cell death. Our studies reveal that activation of the ubiquitin proteasome system is an important event promoting linker cell death. Given the morphological similarities between linker cell death and non-apoptotic developmental and pathological cell death in vertebrates, we raise the possibility that the proteasome may be a key mediator of vertebrate cell death