TY - JOUR A1 - Denk, S. A1 - Schmidt, S. A1 - Schurr, Y. A1 - Schwarz, G. A1 - Schote, F. A1 - Diefenbacher, M. A1 - Armendariz, C. A1 - Dejure, F. A1 - Eilers, M. A1 - Wiegering, Armin T1 - CIP2A regulates MYC translation (via its 5′UTR) in colorectal cancer JF - International Journal of Colorectal Disease N2 - Background Deregulated expression of MYC is a driver of colorectal carcinogenesis, suggesting that decreasing MYC expression may have significant therapeutic value. CIP2A is an oncogenic factor that regulates MYC expression. CIP2A is overexpressed in colorectal cancer (CRC), and its expression levels are an independent marker for long-term outcome of CRC. Previous studies suggested that CIP2A controls MYC protein expression on a post-transcriptional level. Methods To determine the mechanism by which CIP2A regulates MYC in CRC, we dissected MYC translation and stability dependent on CIP2A in CRC cell lines. Results Knockdown of CIP2A reduced MYC protein levels without influencing MYC stability in CRC cell lines. Interfering with proteasomal degradation of MYC by usage of FBXW7-deficient cells or treatment with the proteasome inhibitor MG132 did not rescue the effect of CIP2A depletion on MYC protein levels. Whereas CIP2A knockdown had marginal influence on global protein synthesis, we could demonstrate that, by using different reporter constructs and cells expressing MYC mRNA with or without flanking UTR, CIP2A regulates MYC translation. This interaction is mainly conducted by the MYC 5′UTR. Conclusions Thus, instead of targeting MYC protein stability as reported for other tissue types before, CIP2A specifically regulates MYC mRNA translation in CRC but has only slight effects on global mRNA translation. In conclusion, we propose as novel mechanism that CIP2A regulates MYC on a translational level rather than affecting MYC protein stability in CRC. KW - CIP2A KW - MYC KW - translation KW - colon cancer Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-280092 VL - 36 IS - 5 ER - TY - JOUR A1 - Libre, Camille A1 - Seissler, Tanja A1 - Guerrero, Santiago A1 - Batisse, Julien A1 - Verriez, Cédric A1 - Stupfler, Benjamin A1 - Gilmer, Orian A1 - Cabrera-Rodriguez, Romina A1 - Weber, Melanie M. A1 - Valenzuela-Fernandez, Agustin A1 - Cimarelli, Andrea A1 - Etienne, Lucie A1 - Marquet, Roland A1 - Paillart, Jean-Christophe T1 - A conserved uORF regulates APOBEC3G translation and is targeted by HIV-1 Vif protein to repress the antiviral factor JF - Biomedicines N2 - The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular restriction factors APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutation during reverse transcription. Vif counteracts A3G at several levels (transcription, translation, and protein degradation) that altogether reduce the levels of A3G in cells and prevent its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5′ untranslated region (5′-UTR) of A3G mRNA for this process. A3G translation occurs through a combination of leaky scanning and translation re-initiation and the presence of an intact uORF decreases the extent of global A3G translation under normal conditions. Interestingly, the uORF is also absolutely required for Vif-mediated translation inhibition and redirection of A3G mRNA into stress granules. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific feature to repress its translation. KW - HIV-1 KW - APOBEC3G KW - Vif KW - mRNA KW - translation KW - uORF Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-252147 SN - 2227-9059 VL - 10 IS - 1 ER - TY - JOUR A1 - Chithelen, Janice A1 - Franke, Hannah A1 - Länder, Nora A1 - Grafen, Anika A1 - Schneider-Schaulies, Jürgen T1 - The sphingolipid inhibitors ceranib-2 and SKI-II reduce measles virus replication in primary human lymphocytes: effects on mTORC1 downstream signaling JF - Frontiers in Physiology N2 - The bioactive sphingolipids ceramide and sphingosine-1-phosphate (S1P) are involved in the regulation of cell homeostasis and activity ranging from apoptosis to proliferation. We recently described that the two compounds ceranib-2 (inhibiting acid ceramidase) and SKI-II [inhibiting the sphingosine kinases 1 and − 2 (SphK1/2)] reduce mTORC1 activity and measles virus (MV) replication in human primary peripheral blood lymphocytes (PBL) by about one log step. We now further investigated whether mTORC1 downstream signaling and viral protein expression may be affected by ceranib-2 and/or SKI-II. Western blot analyses showed that in uninfected cells the phosphorylation of the eukaryotic initiation factor 4E (eIF4E) was reduced by both inhibitors. Interestingly, MV infection led to an increase of rpS6 protein levels and phosphorylation of eIF4E. Treatment with both inhibitors reduced the rpS6 protein expression, and in addition, SKI-II reduced rpS6 phosphorylation. The phosphorylation of eIF4E was slightly reduced by both inhibitors. In addition, SKI-II led to reduced levels of IKK in MV-infected cells. Both inhibitors reduced the expression of viral proteins and the titers of newly synthesized MV by approximately one log step. As expected, SKI-II and rapamycin reduced also the virally encoded GFP expression; however, ceranib-2 astonishingly led to increased levels of GFP fluorescence. Our findings suggest that the inhibitors ceranib-2 and SKI-II act via differential mechanisms on MV replication. The observed effects on mTORC1 downstream signaling, predominantly the reduction of rpS6 levels by both inhibitors, may affect the translational capacity of the cells and contribute to the antiviral effect in human primary PBL. KW - acid ceramidase inhibitor ceranib-2 KW - sphingosine kinase inhibitor SKI-II KW - mTORC1 KW - translation KW - measles virus Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265988 SN - 1664-042X VL - 13 ER - TY - JOUR A1 - Wencker, Freya D. R A1 - Marincola, Gabriella A1 - Schoenfelder, Sonja M. K. A1 - Maaß, Sandra A1 - Becher, Dörte A1 - Ziebuhr, Wilma T1 - Another layer of complexity in Staphylococcus aureus methionine biosynthesis control: unusual RNase III-driven T-box riboswitch cleavage determines met operon mRNA stability and decay JF - Nucleic Acids Research N2 - In Staphylococcus aureus, de novo methionine biosynthesis is regulated by a unique hierarchical pathway involving stringent-response controlled CodY repression in combination with a T-box riboswitch and RNA decay. The T-box riboswitch residing in the 5′ untranslated region (met leader RNA) of the S. aureus metICFE-mdh operon controls downstream gene transcription upon interaction with uncharged methionyl-tRNA. met leader and metICFE-mdh (m)RNAs undergo RNase-mediated degradation in a process whose molecular details are poorly understood. Here we determined the secondary structure of the met leader RNA and found the element to harbor, beyond other conserved T-box riboswitch structural features, a terminator helix which is target for RNase III endoribonucleolytic cleavage. As the terminator is a thermodynamically highly stable structure, it also forms posttranscriptionally in met leader/ metICFE-mdh read-through transcripts. Cleavage by RNase III releases the met leader from metICFE-mdh mRNA and initiates RNase J-mediated degradation of the mRNA from the 5′-end. Of note, metICFE-mdh mRNA stability varies over the length of the transcript with a longer lifespan towards the 3′-end. The obtained data suggest that coordinated RNA decay represents another checkpoint in a complex regulatory network that adjusts costly methionine biosynthesis to current metabolic requirements. KW - allelic replacement KW - expression KW - translation KW - mechanism KW - acid KW - endoribonuclease KW - antitermination KW - transcription KW - proteins KW - geometry Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259029 VL - 49 IS - 4 ER - TY - JOUR A1 - Whisnant, Adam W. A1 - Jürges, Christopher S. A1 - Hennig, Thomas A1 - Wyler, Emanuel A1 - Prusty, Bhupesh A1 - Rutkowski, Andrzej J. A1 - L'hernault, Anne A1 - Djakovic, Lara A1 - Göbel, Margarete A1 - Döring, Kristina A1 - Menegatti, Jennifer A1 - Antrobus, Robin A1 - Matheson, Nicholas J. A1 - Künzig, Florian W. H. A1 - Mastrobuoni, Guido A1 - Bielow, Chris A1 - Kempa, Stefan A1 - Liang, Chunguang A1 - Dandekar, Thomas A1 - Zimmer, Ralf A1 - Landthaler, Markus A1 - Grässer, Friedrich A1 - Lehner, Paul J. A1 - Friedel, Caroline C. A1 - Erhard, Florian A1 - Dölken, Lars T1 - Integrative functional genomics decodes herpes simplex virus 1 JF - Nature Communications N2 - The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution. Here, using computational integration of multi-omics data, the authors provide a detailed transcriptome and translatome of herpes simplex virus 1 (HSV-1), including previously unidentified ORFs and N-terminal extensions. The study also provides a HSV-1 genome browser and should be a valuable resource for further research. KW - infected-cell protein KW - messenger RNA KW - binding protein KW - type 1 KW - identification KW - ICP27 KW - translation KW - expression KW - sequence KW - domain Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-229884 VL - 11 ER - TY - JOUR A1 - Dugar, Gaurav A1 - Svensson, Sarah L. A1 - Bischler, Thorsten A1 - Waldchen, Sina A1 - Reinhardt, Richard A1 - Sauer, Markus A1 - Sharma, Cynthia M. T1 - The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni JF - Nature Communications N2 - The widespread CsrA/RsmA protein regulators repress translation by binding GGA motifs in bacterial mRNAs. CsrA activity is primarily controlled through sequestration by multiple small regulatory RNAs. Here we investigate CsrA activity control in the absence of antagonizing small RNAs by examining the CsrA regulon in the human pathogen Campylobacter jejuni. We use genome-wide co-immunoprecipitation combined with RNA sequencing to show that CsrA primarily binds flagellar mRNAs and identify the major flagellin mRNA (flaA) as the main CsrA target. The flaA mRNA is translationally repressed by CsrA, but it can also titrate CsrA activity. Together with the main C. jejuni CsrA antagonist, the FliW protein, flaA mRNA controls CsrA-mediated post-transcriptional regulation of other flagellar genes. RNA-FISH reveals that flaA mRNA is expressed and localized at the poles of elongating cells. Polar flaA mRNA localization is translation dependent and is post-transcriptionally regulated by the CsrA-FliW network. Overall, our results suggest a role for CsrA-FliW in spatiotemporal control of flagella assembly and localization of a dual-function mRNA. KW - bacterial genetics KW - cell signalling KW - translation KW - Campylobacter jejuni Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-173201 VL - 7 ER - TY - JOUR A1 - Preising, Christina A1 - Schneider, Reinhard A1 - Bucher, Michael A1 - Gekle, Michael A1 - Sauvant, Christoph T1 - Regulation of expression of renal organic anion transporters OAT1 and OAT3 in a model of ischemia/reperfusion injury JF - Cellular Physiology and Biochemistry N2 - Background: Recently, we gained evidence that impairment of rOat1 and rOat3 expression induced by ischemic acute kidney injury (AKI) is mediated by COX metabolites and this suppression might be critically involved in renal damage. Methods: (i) Basolateral organic anion uptake into proximal tubular cells after model ischemia and reperfusion (I/R) was investigated by fluorescein uptake. The putative promoter sequences from hOAT1 (SLC22A6) and hOAT3 (SCL22A8) were cloned into a reporter plasmid, transfected into HEK cells and (ii) transcriptional activity was determined after model ischemia and reperfusion as a SEAP reporter gen assay. Inhibitors or antagonists were applied with the beginning of reperfusion. Results: By using inhibitors of PKA (H89) and PLC (U73122), antagonists of E prostanoid receptor type 2 (AH6809) and type 4 (L161,982), we gained evidence that I/R induced down regulation of organic anion transport is mediated by COX1 metabolites via E prostanoid receptor type 4. The latter signaling was confirmed by application of butaprost (EP2 agonist) or TCS2510 (EP4 agonist) to control cells. In brief, the latter signaling was verified for the transcriptional activity in the reporter gen assay established. Therein, selective inhibitors for COX1 (SC58125) and COX2 (SC560) were also applied. Conclusion: Our data show (a) that COX1 metabolites are involved in the regulation of renal organic anion transport(ers) after I/R via the EP4 receptor and (b) that this is due to transcriptional regulation of the respective transporters. As the promoter sequences cloned were of human origin and expressed in a human renal epithelial cell line we (c) hypothesize that the regulatory mechanisms described after I/R is meaningful for humans as well. KW - opossum kidney cells KW - prostaglandin e2 KW - reperfusion KW - transport experiments KW - translation KW - reporter gen assay KW - cloning of putative human promoter sequence KW - regulation of expression KW - OAT1 KW - OAT3 KW - OK cells KW - ischemic acute kidney injury model KW - HEK cells KW - ischemia KW - down regulation KW - nitric oxide KW - cellular physiology KW - cortical OAT1 KW - blood flow Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144504 VL - 37 IS - 1 ER - TY - JOUR A1 - Minnerup, Jens A1 - Sutherland, Brad A. A1 - Buchan, Alastair M. A1 - Kleinschnitz, Christoph T1 - Neuroprotection for Stroke: Current Status and Future Perspectives JF - International Journal of Molecular Science N2 - Neuroprotection aims to prevent salvageable neurons from dying. Despite showing efficacy in experimental stroke studies, the concept of neuroprotection has failed in clinical trials. Reasons for the translational difficulties include a lack of methodological agreement between preclinical and clinical studies and the heterogeneity of stroke in humans compared to homogeneous strokes in animal models. Even when the international recommendations for preclinical stroke research, the Stroke Academic Industry Roundtable (STAIR) criteria, were followed, we have still seen limited success in the clinic, examples being NXY-059 and haematopoietic growth factors which fulfilled nearly all the STAIR criteria. However, there are a number of neuroprotective treatments under investigation in clinical trials such as hypothermia and ebselen. Moreover, promising neuroprotective treatments based on a deeper understanding of the complex pathophysiology of ischemic stroke such as inhibitors of NADPH oxidases and PSD-95 are currently evaluated in preclinical studies. Further concepts to improve translation include the investigation of neuroprotectants in multicenter preclinical Phase III-type studies, improved animal models, and close alignment between clinical trial and preclinical methodologies. Future successful translation will require both new concepts for preclinical testing and innovative approaches based on mechanistic insights into the ischemic cascade. KW - free radical scavenger KW - ischemic cascade KW - acute ischemic stroke KW - trial KW - focal cerebral-ischemia KW - interleukin-1 receptor antagonist KW - colony-stimulating factor KW - tissue-plasminogen activator KW - traumatic brain injury KW - placebo-controlled KW - alias pilot trial KW - damage cool aid KW - neuroprotection KW - ischemic stroke KW - translation KW - STAIR Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-134730 VL - 13 IS - 9 ER - TY - JOUR A1 - Zukher, Inna A1 - Novikova, Maria A1 - Tikhonov, Anton A1 - Nesterchuk, Mikhail V. A1 - Osterman, Ilya A. A1 - Djordjevic, Marko A1 - Sergiev, Petr V. A1 - Sharma, Cynthia M. A1 - Severinov, Konstantin T1 - Ribosome-controlled transcription termination is essential for the production of antibiotic microcin C JF - Nucleic Acids Research N2 - Microcin C (McC) is a peptide-nucleotide antibiotic produced by Escherichia coli cells harboring a plasmid-borne operon mccABCDE. The heptapeptide MccA is converted into McC by adenylation catalyzed by the MccB enzyme. Since MccA is a substrate for MccB, a mechanism that regulates the MccA/MccB ratio likely exists. Here, we show that transcription from a promoter located upstream of mccA directs the synthesis of two transcripts: a short highly abundant transcript containing the mccA ORF and a longer minor transcript containing mccA and downstream ORFs. The short transcript is generated when RNA polymerase terminates transcription at an intrinsic terminator located in the intergenic region between the mccA and mccB genes. The function of this terminator is strongly attenuated by upstream mcc sequences. Attenuation is relieved and transcription termination is induced when ribosome binds to the mccA ORF. Ribosome binding also makes the mccA RNA exceptionally stable. Together, these two effects-ribosome induced transcription termination and stabilization of the message-account for very high abundance of the mccA transcript that is essential for McC production. The general scheme appears to be evolutionary conserved as ribosome-induced transcription termination also occurs in a homologous operon from Helicobacter pylori. KW - escherichia coli KW - messenger-RNA decay KW - translation KW - expression KW - synthetase KW - enterobacteria KW - inhibitors KW - maturation KW - target KW - stability Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-114839 SN - 0305-1048 VL - 42 IS - 19 ER -