beta-arrestin-1 competitively inhibits insulin-induced ubiquitination and degradation of insulin receptor substrate 1.

beta-arrestin-1 is an adaptor protein that mediates agonist-dependent internalization and desensitization of G-protein-coupled receptors (GPCRs) and also participates in the process of heterologous desensitization between receptor tyrosine kinases and GPCR signaling. In the present study, we determined whether beta-arrestin-1 is involved in insulin-induced insulin receptor substrate 1 (IRS-1) degradation. Overexpression of wild-type (WT) beta-arrestin-1 attenuated insulin-induced degradation of IRS-1, leading to increased insulin signaling downstream of IRS-1. When endogenous beta-arrestin-1 was knocked down by transfection of beta-arrestin-1 small interfering RNA, insulin-induced IRS-1 degradation was enhanced. Insulin stimulated the association of IRS-1 and Mdm2, an E3 ubiquitin ligase, and this association was inhibited to overexpression of WT beta-arrestin-1, which led by decreased ubiquitin content of IRS-1, suggesting that both beta-arrestin-1 and IRS-1 competitively bind to Mdm2. In summary, we have found the following: (i) beta-arrestin-1 can alter insulin signaling by inhibiting insulin-induced proteasomal degradation of IRS-1; (ii) beta-arrestin-1 decreases the rate of ubiquitination of IRS-1 by competitively binding to endogenous Mdm2, an E3 ligase that can ubiquitinate IRS-1; (iii) dephosphorylation of S412 on beta-arrestin and the amino terminus of beta-arrestin-1 are required for this effect of beta-arrestin on IRS-1 degradation; and (iv) inhibition of beta-arrestin-1 leads to enhanced IRS-1 degradation and accentuated cellular insulin resistance.

The Trim39 ubiquitin ligase inhibits APC/CCdh1-mediated degradation of the Bax activator MOAP-1.

Proapoptotic Bcl-2 family members, such as Bax, promote release of cytochrome c from mitochondria, leading to caspase activation and cell death. It was previously reported that modulator of apoptosis protein 1 (MOAP-1), an enhancer of Bax activation induced by DNA damage, is stabilized by Trim39, a protein of unknown function. In this paper, we show that MOAP-1 is a novel substrate of the anaphase-promoting complex (APC/C(Cdh1)) ubiquitin ligase. The influence of Trim39 on MOAP-1 levels stems from the ability of Trim39 (a RING domain E3 ligase) to directly inhibit APC/C(Cdh1)-mediated protein ubiquitylation. Accordingly, small interfering ribonucleic acid-mediated knockdown of Cdh1 stabilized MOAP-1, thereby enhancing etoposide-induced Bax activation and apoptosis. These data identify Trim39 as a novel APC/C regulator and provide an unexpected link between the APC/C and apoptotic regulation via MOAP-1.

Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells

The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells.

mRNA metabolism: nonsense mediated mRNA decay as a tool for gene therapy and the role of human DIS3L2 in transcript degradation

Tese de mestrado em Biologia Humana e Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015

HIV-1 Vpr induces the K48-linked polyubiquitination and proteasomal degradation of target cellular proteins to activate ATR and promote G2 arrest

HIV-1 viral protein R (Vpr) induces a cell cycle arrest at the G2/M phase by a mechanism involving the activation of the DNA damage sensor ATR. We and others recently showed that Vpr performs this function by subverting the activity of the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase. Vpr could thus act as a connector between the E3 ligase and an unknown cellular factor whose ubiquitination would induce G2 arrest. While attractive, this model is solely based on the indirect observation that some mutants of Vpr retain their interaction with the E3 ligase but fail to induce G2 arrest. Using a tandem affinity purification approach, we observed that Vpr interacts with ubiquitinated cellular proteins and that this association requires the recruitment of an active E3 ligase given that depletion of VPRBP by RNA interference or overexpression of a dominant-negative mutant of CUL4A decreased this association. Importantly, G2-arrest-defective mutants of Vpr in the C-terminal putative substrate-interacting domain displayed decreased association with ubiquitinated proteins. We also found that inhibition of proteasomal activity increased this association and that the ubiquitin chains were at least in part constituted of classical K48 linkages. Interestingly, inhibition of K48 polyubiquitination specifically impaired Vpr-induced phosphorylation of H2AX, an early target of ATR, but did not affect UV-induced H2AX phosphorylation. Overall, our results provide direct evidence that association of Vpr with the DDB1-CUL4A (VPRBP) E3 ubiquitin ligase induces the K48-linked polyubiquitination of yet-unknown cellular proteins resulting in their proteasomal degradation and ultimately leading to activation of ATR and G2 arrest.

Synergistic acceleration of thyroid hormone degradation by phenobarbital and the PPAR alpha agonist WY14643 in rat hepatocytes

Energy balance is maintained by controlling both energy intake and energy expenditure. Thyroid hormones play a crucial role in regulating energy expenditure. Their levels are adjusted by a tight feed back-control led regulation of thyroid hormone production/incretion and by their hepatic metabolism. Thyroid hormone degradation has previously been shown to be enhanced by treatment with phenobarbital or other antiepileptic drugs due to a CAR-dependent induction of phase 11 enzymes of xenobiotic metabolism. We have recently shown, that PPAR alpha agonists synergize with phenobarbital to induce another prototypical CAR target gene, CYP2B1. Therefore, it was tested whether a PPAR alpha agonist could enhance the phenobarbital-dependent acceleration of thyroid hormone elimination. In primary cultures of rat hepatocytes the apparent half-life of T3 was reduced after induction with a combination of phenobarbital and the PPARa agonist WY14643 to a larger extent than after induction with either Compound alone. The synergistic reduction of the half-life could be attributed to a synergistic induction of CAR and the CAR target genes that code for enzymes and transporters involved in the hepatic elimination of T3, such as OATP1A1, OATP1A3, UGT1A3 and UCT1A10. The PPAR alpha-dependent CAR induction and the subsequent induction of T3-eliminating enzymes might be of physiological significance for the fasting-incluced reduction in energy expenditure by fatty acids as natural PPARa ligands. The synergism of the PPAR alpha agonist WY14643 and phenobarbital in inducing thyroid hormone breakdown might serve as a paradigm for the synergistic disruption of endocrine control by other combinations of xenobiotics. (C) 2009 Elsevier Inc. All rights reserved.

Lithium toxicity in yeast is due to the inhibition of RNA processing enzymes

Hal2p is an enzyme that converts pAp (adenosine 3',5' bisphosphate), a product of sulfate assimilation, into 5' AMP and Pi. Overexpression of Hal2p confers lithium resistance in yeast, and its activity is inhibited by submillimolar amounts of Li+in vitro. Here we report that pAp accumulation in HAL2 mutants inhibits the 5'3' exoribonucleases Xrn1p and Rat1p. Li+ treatment of a wild-type yeast strain also inhibits the exonucleases, as a result of pAp accumulation due to inhibition of Hal2p; 5' processing of the 5.8S rRNA and snoRNAs, degradation of pre-rRNA spacer fragments and mRNA turnover are inhibited. Lithium also inhibits the activity of RNase MRP by a mechanism which is not mediated by pAp. A mutation in the RNase MRP RNA confers Li+ hypersensitivity and is synthetically lethal with mutations in either HAL2 or XRN1. We propose that Li+ toxicity in yeast is due to synthetic lethality evoked between Xrn1p and RNase MRP. Similar mechanisms may contribute to the effects of Li+ on development and in human neurobiology.

c-Cbl facilitates endocytosis and lysosomal degradation of cystic fibrosis transmembrane conductance regulator in human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl− channel expressed in the apical membrane of fluid-transporting epithelia. The apical membrane density of CFTR channels is determined, in part, by endocytosis and the postendocytic sorting of CFTR for lysosomal degradation or recycling to the plasma membrane. Although previous studies suggested that ubiquitination plays a role in the postendocytic sorting of CFTR, the specific ubiquitin ligases are unknown. c-Cbl is a multifunctional molecule with ubiquitin ligase activity and a protein adaptor function. c-Cbl co-immunoprecipitated with CFTR in primary differentiated human bronchial epithelial cells and in cultured human airway cells. Small interfering RNA-mediated silencing of c-Cbl increased CFTR expression in the plasma membrane by inhibiting CFTR endocytosis and increased CFTR-mediated Cl− currents. Silencing c-Cbl did not change the expression of the ubiquitinated fraction of plasma membrane CFTR. Moreover, the c-Cbl mutant with impaired ubiquitin ligase activity (FLAG-70Z-Cbl) did not affect the plasma membrane expression or the endocytosis of CFTR. In contrast, the c-Cbl mutant with the truncated C-terminal region (FLAG-Cbl-480), responsible for protein adaptor function, had a dominant interfering effect on the endocytosis and plasma membrane expression of CFTR. Moreover, CFTR and c-Cbl co-localized and co-immunoprecipitated in early endosomes, and silencing c-Cbl reduced the amount of ubiquitinated CFTR in early endosomes. In summary, our data demonstrate that in human airway epithelial cells, c-Cbl regulates CFTR by two mechanisms: first by acting as an adaptor protein and facilitating CFTR endocytosis by a ubiquitin-independent mechanism, and second by ubiquitinating CFTR in early endosomes and thereby facilitating the lysosomal degradation of CFTR.

RNA interference inhibits yellow fever virus replication in vitro and in vivo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Transcriptoma (RNA-seq) de laranja doce Valência (Citrus sinensis L. Osbeck) e Kumquat (Fortunella spp.) infectadas por Xanthomonas citri subsp. citri

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR

Isolated total RNA and protein are preserved after thawing for more than twenty-four hours

OBJECTIVE: The preservation of biological samples at a low temperature is important for later biochemical and/or histological analyses. However, the molecular viability of thawed samples has not been studied sufficiently in depth. The present study was undertaken to evaluate the viability of intact tissues, tissue homogenates, and isolated total RNA after defrosting for more than twenty-four hours. METHODS: The molecular viability of the thawed samples (n = 82) was assessed using the A260/A280 ratio, the RNA concentration, the RNA integrity, the level of intact mRNA determined by reverse transcriptase polymerase chain reaction, the protein level determined by Western blotting, and an examination of the histological structure. RESULTS: The integrity of the total RNA was not preserved in the thawed intact tissue, but the RNA integrity and level of mRNA were perfectly preserved in isolated defrosted samples of total RNA. Additionally, the level of beta-actin protein was preserved in both thawed intact tissue and homogenates. CONCLUSION: Isolated total RNA does not undergo degradation due to thawing for at least 24 hours, and it is recommended to isolate the total RNA as soon as possible after tissue collection. Moreover, the protein level is preserved in defrosted tissues.

Footprints of a trypanosomatid RNA world: pre-small subunit rRNA processing by spliced leader addition trans-splicing

The addition of a capped mini-exon [spliced leader (SL)] through trans-splicing is essential for the maturation of RNA polymerase (pol) II-transcribed polycistronic pre-mRNAs in all members of the Trypanosomatidae family. This process is an inter-molecular splicing reaction that follows the same basic rules of cis-splicing reactions. In this study, we demonstrated that mini-exons were added to precursor ribosomal RNA (pre-rRNA) are transcribed by RNA pol I, including the 5' external transcribed spacer (ETS) region. Additionally, we detected the SL-5' ETS molecule using three distinct methods and located the acceptor site between two known 5' ETS rRNA processing sites (A' and A1) in four different trypanosomatids. Moreover, we detected a polyadenylated 5' ETS upstream of the trans-splicing acceptor site, which also occurs in pre-mRNA trans-splicing. After treatment with an indirect trans-splicing inhibitor (sinefungin), we observed SL-5' ETS decay. However, treatment with 5-fluorouracil (a precursor of RNA synthesis that inhibits the degradation of pre-rRNA) led to the accumulation of SL-5' ETS, suggesting that the molecule may play a role in rRNA degradation. The detection of trans-splicing in these molecules may indicate broad RNA-joining properties, regardless of the polymerase used for transcription.

RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

Abstract Background The development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old) FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR) for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp. Results Biologically useful RNA (minimum RNA integrity number, RIN, 1.4) was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old") samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp. Conclusion All protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of extracted RNA, although it produced similar quality RNA to other protocols. If a chosen protocol fails to extract biologically useful RNA from a given sample in a first attempt, another attempt and then another protocol should be tried before excluding the case from molecular analysis.

Dynamics of small interfering RNA - FRET based integrity measurements of siRNA in the cuvette & inside cells

RNAi ist ein bedeutendes Werkzeug zur Funktionsanalyse von Genen und hat großes Potential für den Einsatz in der Therapie. Obwohl effiziente Knockdowns in der Zellkultur erzielt werden, erweist sich eine in vivo Anwendung als schwierig. Die großen Hürden sind dabei der Transport der siRNA ins Zielgewebe und deren voranschreitende Degradierung.rnMarkierte siRNA kann sowohl zur eigenen Integritätsmessung als auch zur Lokalisierung verwendet werden. Zwei Farbstoffe an den jeweiligen 3’- bzw. -5’-Enden des Sense- bzw. Antisense-Stranges erzeugen ein robustes FRET-System (Hirsch et al. 2012). Das Verhältnis von FRET- zu Donor-Signal, das R/G-Ratio, dient zur sensitiven Klassifizierung des Integritätslevels einer siRNA Probe (Järve et al. 2007; Hirsch et al. 2011; Kim et al. 2010). Mit diesem System kann eine Degradierung von weniger als 5 % in der Küvette und in Zellen nachgewiesen werden.rnDie vorliegende Arbeit beschäftigt sich mit der Evaluierung von potentiellen FRET Farbstoffpaaren hinsichtlich deren Eignung für in vitro und in vivo Anwendung. Verschiedenste FRET-Paare, die das gesamte sichtbare Spektrum abdecken, wurden evaluiert und ermöglichen nun die Auswahl eines geeigneten Paares für die jeweilige Anwendung oder Kombination mit anderen Farbstoffen.rnMit Hilfe von Alexa555/Atto647N siRNA wurde ein erfolgreicher Einschluss von siRNA in Liposomen beobachtet. Eine anschließende Evaluierung der RNase-Protektion ergab für Liposomen, Nanohydrogele und kationische Peptide hervorragende protektive Eigenschaften. Basierend auf den Ergebnisse können diese und andere Transportsysteme nun für eine zelluläre Aufnahme optimiert werden.rnAtto488/Atto590 zeigte die besten Eigenschaften für Echtzeit-Integritätsmessungen in der Lebendzellmikroskopie. Verringerte Bleicheigenschaften und minimaler spektraler “Cross-Talk” ermöglichten es, transfizierte Zellen über einen Zeitraum von bis zu 8 Stunden zu beobachten. Mittels Atto488/Atto590 siRNA wurde die Einschleusung und Freisetzung in Zellen in Echtzeit untersucht. Dabei konnten Freisetzung und Verteilung in einzelnen Zellen beobachtet und analysiert werden. rnAuf eine anfängliche Phase mit hoher Freisetzungsrate folgte eine Phase mit geringerer Rate für den restlichen Beobachtungszeitraum. Die durchschnittliche Verweildauer im Zytosol betrug 24 und 58 Minuten, wobei zwischen lang- und kurzanhaltenden Ereignissen unterschieden werden konnte. Obwohl ein Import von siRNA in den Zellkern beobachtet wurde, konnte kein Schema bzw. genauer Zeitpunkt, in Bezug auf den Transfektionszeitraum für diese Ereignisse bestimmt werden. Die beobachteten Freisetzungsprozesse fanden sporadisch statt und Änderungen in der zellulären Verteilung geschahen innerhalb von wenigen Minuten. Einmal freigesetzte siRNA verschwand mit der Zeit wieder aus dem Zytosol und es blieben nur kleine Aggregate von siRNA mit immer noch geringer Integrität zurück.rn