Publications by authors named "Julia E Weigand"

42 Publications

A Novel, Universally Active C-terminal Protein Degradation Signal Generated by Alternative Splicing.

J Mol Biol 2021 Apr 23;433(8):166890. Epub 2021 Feb 23.

Department of Biology, Technical University of Darmstadt, Darmstadt 64287, Germany. Electronic address:

Proteome integrity is crucial for cellular homeostasis and adaptation to stress conditions such as hypoxia. One mechanism for rapid adaptation of the proteome in response to changing environmental signals is alternative splicing. In addition to generating different protein isoforms, alternative splicing is also capable of controlling total protein levels by the regulated synthesis of non-productive mRNA isoforms. The hypoxia-induced isoform E of the tumor suppressor MAX is produced by retention and translation of the last intron. This leads to an alternative C-terminus that harbors a potent C-degron, the isoE degron. Strikingly, the isoE degron represents a universal protein degradation signal that is not only functional in mammalian cells, but also in yeast and even in bacteria. Essential for efficient protein decay is a conserved (F/W)xxW motif. Degradation of isoE tagged proteins is mediated by the proteasome in eukaryotes and Lon protease in bacteria. Thus, the isoE degron is a broadly applicable and highly efficient tool in protein analyses.
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http://dx.doi.org/10.1016/j.jmb.2021.166890DOI Listing
April 2021

H, C and N chemical shift assignment of the stem-loop 5a from the 5'-UTR of SARS-CoV-2.

Biomol NMR Assign 2021 04 23;15(1):203-211. Epub 2021 Jan 23.

Institute for Molecular Biosciences, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Str. 9, 60438, Frankfurt/M., Germany.

The SARS-CoV-2 (SCoV-2) virus is the causative agent of the ongoing COVID-19 pandemic. It contains a positive sense single-stranded RNA genome and belongs to the genus of Betacoronaviruses. The 5'- and 3'-genomic ends of the 30 kb SCoV-2 genome are potential antiviral drug targets. Major parts of these sequences are highly conserved among Betacoronaviruses and contain cis-acting RNA elements that affect RNA translation and replication. The 31 nucleotide (nt) long highly conserved stem-loop 5a (SL5a) is located within the 5'-untranslated region (5'-UTR) important for viral replication. SL5a features a U-rich asymmetric bulge and is capped with a 5'-UUUCGU-3' hexaloop, which is also found in stem-loop 5b (SL5b). We herein report the extensive H, C and N resonance assignment of SL5a as basis for in-depth structural studies by solution NMR spectroscopy.
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http://dx.doi.org/10.1007/s12104-021-10007-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822759PMC
April 2021

H, C, and N backbone chemical shift assignments of the C-terminal dimerization domain of SARS-CoV-2 nucleocapsid protein.

Biomol NMR Assign 2021 04 3;15(1):129-135. Epub 2020 Dec 3.

Institute for Molecular Biosciences, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Str. 9, 60438, Frankfurt/M, Germany.

The current outbreak of the highly infectious COVID-19 respiratory disease is caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). To fight the pandemic, the search for promising viral drug targets has become a cross-border common goal of the international biomedical research community. Within the international Covid19-NMR consortium, scientists support drug development against SARS-CoV-2 by providing publicly available NMR data on viral proteins and RNAs. The coronavirus nucleocapsid protein (N protein) is an RNA-binding protein involved in viral transcription and replication. Its primary function is the packaging of the viral RNA genome. The highly conserved architecture of the coronavirus N protein consists of an N-terminal RNA-binding domain (NTD), followed by an intrinsically disordered Serine/Arginine (SR)-rich linker and a C-terminal dimerization domain (CTD). Besides its involvement in oligomerization, the CTD of the N protein (N-CTD) is also able to bind to nucleic acids by itself, independent of the NTD. Here, we report the near-complete NMR backbone chemical shift assignments of the SARS-CoV-2 N-CTD to provide the basis for downstream applications, in particular site-resolved drug binding studies.
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http://dx.doi.org/10.1007/s12104-020-09995-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711055PMC
April 2021

Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy.

Nucleic Acids Res 2020 12;48(22):12415-12435

Institute for Organic Chemistry and Chemical Biology, Max-von-Laue-Strasse 7, 60438 Frankfurt/M., Germany.

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.
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http://dx.doi.org/10.1093/nar/gkaa1013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736788PMC
December 2020

H, C, and N backbone chemical shift assignments of the nucleic acid-binding domain of SARS-CoV-2 non-structural protein 3e.

Biomol NMR Assign 2020 10 8;14(2):329-333. Epub 2020 Aug 8.

Institute for Molecular Biosciences, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany.

The ongoing pandemic caused by the Betacoronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) demonstrates the urgent need of coordinated and rapid research towards inhibitors of the COVID-19 lung disease. The covid19-nmr consortium seeks to support drug development by providing publicly accessible NMR data on the viral RNA elements and proteins. The SARS-CoV-2 genome encodes for approximately 30 proteins, among them are the 16 so-called non-structural proteins (Nsps) of the replication/transcription complex. The 217-kDa large Nsp3 spans one polypeptide chain, but comprises multiple independent, yet functionally related domains including the viral papain-like protease. The Nsp3e sub-moiety contains a putative nucleic acid-binding domain (NAB) with so far unknown function and consensus target sequences, which are conceived to be both viral and host RNAs and DNAs, as well as protein-protein interactions. Its NMR-suitable size renders it an attractive object to study, both for understanding the SARS-CoV-2 architecture and drugability besides the classical virus' proteases. We here report the near-complete NMR backbone chemical shifts of the putative Nsp3e NAB that reveal the secondary structure and compactness of the domain, and provide a basis for NMR-based investigations towards understanding and interfering with RNA- and small-molecule-binding by Nsp3e.
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http://dx.doi.org/10.1007/s12104-020-09971-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414254PMC
October 2020

Structural basis for the recognition of transiently structured AU-rich elements by Roquin.

Nucleic Acids Res 2020 07;48(13):7385-7403

Institute for Molecular Biosciences, Goethe University Frankfurt and Center for Biomolecular Magnetic Resonance (BMRZ), 60438 Frankfurt, Germany.

Adenylate/uridylate-rich elements (AREs) are the most common cis-regulatory elements in the 3'-untranslated region (UTR) of mRNAs, where they fine-tune turnover by mediating mRNA decay. They increase plasticity and efficacy of mRNA regulation and are recognized by several ARE-specific RNA-binding proteins (RBPs). Typically, AREs are short linear motifs with a high content of complementary A and U nucleotides and often occur in multiple copies. Although thermodynamically rather unstable, the high AU-content might enable transient secondary structure formation and modify mRNA regulation by RBPs. We have recently suggested that the immunoregulatory RBP Roquin recognizes folded AREs as constitutive decay elements (CDEs), resulting in shape-specific ARE-mediated mRNA degradation. However, the structural evidence for a CDE-like recognition of AREs by Roquin is still lacking. We here present structures of CDE-like folded AREs, both in their free and protein-bound form. Moreover, the AREs in the UCP3 3'-UTR are additionally bound by the canonical ARE-binding protein AUF1 in their linear form, adopting an alternative binding-interface compared to the recognition of their CDE structure by Roquin. Strikingly, our findings thus suggest that AREs can be recognized in multiple ways, allowing control over mRNA regulation by adapting distinct conformational states, thus providing differential accessibility to regulatory RBPs.
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http://dx.doi.org/10.1093/nar/gkaa465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7367199PMC
July 2020

Muscleblind-like 2 controls the hypoxia response of cancer cells.

RNA 2020 05 3;26(5):648-663. Epub 2020 Mar 3.

Department of Biology, Technical University of Darmstadt, Darmstadt, 64287, Germany.

Hypoxia is a hallmark of solid cancers, supporting proliferation, angiogenesis, and escape from apoptosis. There is still limited understanding of how cancer cells adapt to hypoxic conditions and survive. We analyzed transcriptome changes of human lung and breast cancer cells under chronic hypoxia. Hypoxia induced highly concordant changes in transcript abundance, but divergent splicing responses, underlining the cell type-specificity of alternative splicing programs. While RNA-binding proteins were predominantly reduced, hypoxia specifically induced muscleblind-like protein 2 (MBNL2). Strikingly, MBNL2 induction was critical for hypoxia adaptation by controlling the transcript abundance of hypoxia response genes, such as vascular endothelial growth factor A ( MBNL2 depletion reduced the proliferation and migration of cancer cells, demonstrating an important role of MBNL2 as cancer driver. Hypoxia control is specific for MBNL2 and not shared by its paralog MBNL1. Thus, our study revealed MBNL2 as central mediator of cancer cell responses to hypoxia, regulating the expression and alternative splicing of hypoxia-induced genes.
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http://dx.doi.org/10.1261/rna.073353.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7161353PMC
May 2020

A combined computational pipeline to detect circular RNAs in human cancer cells under hypoxic stress.

J Mol Cell Biol 2019 10;11(10):829-844

Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt am Main, Germany.

Hypoxia is associated with several diseases, including cancer. Cells that are deprived of adequate oxygen supply trigger transcriptional and post-transcriptional responses, which control cellular pathways such as angiogenesis, proliferation, and metabolic adaptation. Circular RNAs (circRNAs) are a novel class of mainly non-coding RNAs, which have been implicated in multiple cancers and attract increasing attention as potential biomarkers. Here, we characterize the circRNA signatures of three different cancer cell lines from cervical (HeLa), breast (MCF-7), and lung (A549) cancer under hypoxia. In order to reliably detect circRNAs, we integrate available tools with custom approaches for quantification and statistical analysis. Using this consolidated computational pipeline, we identify ~12000 circRNAs in the three cancer cell lines. Their molecular characteristics point to an involvement of complementary RNA sequences as well as trans-acting factors in circRNA biogenesis, such as the RNA-binding protein HNRNPC. Notably, we detect a number of circRNAs that are more abundant than their linear counterparts. In addition, 64 circRNAs significantly change in abundance upon hypoxia, in most cases in a cell type-specific manner. In summary, we present a comparative circRNA profiling in human cancer cell lines, which promises novel insights into the biogenesis and function of circRNAs under hypoxic stress.
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http://dx.doi.org/10.1093/jmcb/mjz094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6884703PMC
October 2019

Structure guided fluorescence labeling reveals a two-step binding mechanism of neomycin to its RNA aptamer.

Nucleic Acids Res 2019 01;47(1):15-28

Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt am Main, Max-von-Laue-Strasse 7, 60438 Frankfurt, Germany.

The ability of the cytidine analog Çmf to act as a position specific reporter of RNA-dynamics was spectroscopically evaluated. Çmf-labeled single- and double-stranded RNAs differ in their fluorescence lifetimes, quantum yields and anisotropies. These observables were also influenced by the nucleobases flanking Çmf. This conformation and position specificity allowed to investigate the binding dynamics and mechanism of neomycin to its aptamer N1 by independently incorporating Çmf at four different positions within the aptamer. Remarkably fast binding kinetics of neomycin binding was observed with stopped-flow measurements, which could be satisfactorily explained with a two-step binding. Conformational selection was identified as the dominant mechanism.
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http://dx.doi.org/10.1093/nar/gky1110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326822PMC
January 2019

Influence of Mg on the conformational flexibility of a tetracycline aptamer.

RNA 2019 01 18;25(1):158-167. Epub 2018 Oct 18.

Institute of Physical and Theoretical Chemistry and Center of Biomolecular Magnetic Resonance, Goethe University Frankfurt, 60438 Frankfurt am Main, Germany.

The tetracycline-binding RNA aptamer (TC-aptamer) is a synthetic riboswitch that binds the antibiotic tetracycline (TC) with exceptionally high affinity. Although a crystal structure exists of the TC-bound state, little is known about the conformational dynamics and changes upon ligand binding. In this study, pulsed electron paramagnetic resonance techniques for measuring distances (PELDOR) in combination with rigid nitroxide spin labels (Çm spin label) were used to investigate the conformational flexibility of the TC-aptamer in the presence and absence of TC at different Mg concentrations. TC was found to be the essential factor for stabilizing the tertiary structure at intermediate Mg concentrations. At higher Mg concentrations, Mg alone is sufficient to stabilize the tertiary structure. In addition, the orientation of the two spin-labeled RNA helices with respect to each other was analyzed with orientation-selective PELDOR and compared to the crystal structure. These results demonstrate for the first time the unique value of the Çm spin label in combination with PELDOR to provide information about conformational flexibilities and orientations of secondary structure elements of biologically relevant RNAs.
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http://dx.doi.org/10.1261/rna.068684.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298572PMC
January 2019

Identification of new high affinity targets for Roquin based on structural conservation.

Nucleic Acids Res 2018 12;46(22):12109-12125

Department of Biology, Technische Universität Darmstadt, Darmstadt 64287, Germany.

Post-transcriptional gene regulation controls the amount of protein produced from a specific mRNA by altering both its decay and translation rates. Such regulation is primarily achieved by the interaction of trans-acting factors with cis-regulatory elements in the untranslated regions (UTRs) of mRNAs. These interactions are guided either by sequence- or structure-based recognition. Similar to sequence conservation, the evolutionary conservation of a UTR's structure thus reflects its functional importance. We used such structural conservation to identify previously unknown cis-regulatory elements. Using the RNA folding program Dynalign, we scanned all UTRs of humans and mice for conserved structures. Characterizing a subset of putative conserved structures revealed a binding site of the RNA-binding protein Roquin. Detailed functional characterization in vivo enabled us to redefine the binding preferences of Roquin and identify new target genes. Many of these new targets are unrelated to the established role of Roquin in inflammation and immune responses and thus highlight additional, unstudied cellular functions of this important repressor. Moreover, the expression of several Roquin targets is highly cell-type-specific. In consequence, these targets are difficult to detect using methods dependent on mRNA abundance, yet easily detectable with our unbiased strategy.
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http://dx.doi.org/10.1093/nar/gky908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294493PMC
December 2018

A small, portable RNA device for the control of exon skipping in mammalian cells.

Nucleic Acids Res 2018 05;46(8):e48

Department of Biology, Technical University Darmstadt, Schnittspahnstr. 10, 64287 Darmstadt, Germany.

Splicing is an essential and highly regulated process in mammalian cells. We developed a synthetic riboswitch that efficiently controls alternative splicing of a cassette exon in response to the small molecule ligand tetracycline. The riboswitch was designed to control the accessibility of the 3' splice site by placing the latter inside the closing stem of a conformationally controlled tetracycline aptamer. In the presence of tetracycline, the cassette exon is skipped, whereas it is included in the ligand's absence. The design allows for an easy, context-independent integration of the regulatory device into any gene of interest. Portability of the device was shown through its functionality in four different systems: a synthetic minigene, a reporter gene and two endogenous genes. Furthermore, riboswitch functionality to control cellular signaling cascades was demonstrated by using it to specifically induce cell death through the conditionally controlled expression of CD20, which is a target in cancer therapy.
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http://dx.doi.org/10.1093/nar/gky062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5934650PMC
May 2018

Auto- and cross-regulation of the hnRNPs D and DL.

RNA 2018 03 20;24(3):324-331. Epub 2017 Dec 20.

Department of Biology, Technische Universität Darmstadt, 64287 Darmstadt, Germany.

HnRNP D, better known as AUF1, is an extensively studied protein that controls a variety of cellular pathways. Consequently, its expression has to be tightly regulated to prevent the onset of pathologies. In contrast, the cellular functions and regulation of its ubiquitously expressed paralog hnRNP DL are barely explored. Here, we present an intricate crosstalk between these two proteins. Both hnRNP D and DL are able to control their own expression by alternative splicing of cassette exons in their 3'UTRs. Exon inclusion produces mRNAs degraded by nonsense-mediated decay. Moreover, hnRNP D and DL control the expression of one another by the same mechanism. Thus, we identified two novel ways of how hnRNP D expression is controlled. The tight interconnection of expression control directly links hnRNP DL to hnRNP D-related diseases and emphasizes the importance of a systematic analysis of its cellular functions.
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http://dx.doi.org/10.1261/rna.063420.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5824352PMC
March 2018

Design and implementation of a synthetic pre-miR switch for controlling miRNA biogenesis in mammals.

Nucleic Acids Res 2017 Dec;45(22):e181

Department of Biology, Technical University Darmstadt, Darmstadt 64287, Germany.

Synthetic RNA-based systems have increasingly been used for the regulation of eukaryotic gene expression. Due to their structural properties, riboregulators provide a convenient basis for the development of ligand-dependent controllable systems. Here, we demonstrate reversible conditional control of miRNA biogenesis with an aptamer domain as a sensing unit connected to a natural miRNA precursor for the first time. For the design of the pre-miR switch, we replaced the natural terminal loop with the TetR aptamer. Thus, the TetR aptamer was positioned close to the Dicer cleavage sites, which allowed sterical control over pre-miR processing by Dicer. Our design proved to be highly versatile, allowing us to regulate the biogenesis of three structurally different miRNAs: miR-126, -34a and -199a. Dicer cleavage was inhibited up to 143-fold via co-expression of the TetR protein, yet could be completely restored upon addition of doxycycline. Moreover, we showed the functionality of the pre-miR switches for gene regulation through the interaction of the respective miRNA with its specific target sequence. Our designed device is capable of robust and reversible control of miRNA abundance. Thus, we offer a novel investigational tool for functional miRNA analysis.
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http://dx.doi.org/10.1093/nar/gkx858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5727447PMC
December 2017

sST2 translation is regulated by FGF2 via an hnRNP A1-mediated IRES-dependent mechanism.

Biochim Biophys Acta 2016 Jul 8;1859(7):848-59. Epub 2016 May 8.

Institute of Biochemistry I, Faculty of Medicine, Goethe-University Frankfurt, 60590 Frankfurt, Germany. Electronic address:

Translation is an energy-intensive process and tightly regulated. Generally, translation is initiated in a cap-dependent manner. Under stress conditions, typically found within the tumor microenvironment in association with e.g. nutrient deprivation or hypoxia, cap-dependent translation decreases, and alternative modes of translation initiation become more important. Specifically, internal ribosome entry sites (IRES) facilitate translation of specific mRNAs under otherwise translation-inhibitory conditions. This mechanism is controlled by IRES trans-acting factors (ITAF), i.e. by RNA-binding proteins, which interact with and determine the activity of selected IRESs. We aimed at characterizing the translational regulation of the IL-33 decoy receptor sST2, which was enhanced by fibroblast growth factor 2 (FGF2). We identified and verified an IRES within the 5'UTR of sST2. Furthermore, we found that MEK/ERK signaling contributes to FGF2-induced, sST2-IRES activation and translation. Determination of the sST2-5'UTR structure by in-line probing followed by deletion analyses identified 23 nucleotides within the sST2-5'UTR to be required for optimal IRES activity. Finally, we show that the RNA-binding protein heterogeneous ribonucleoprotein A1 (hnRNP A1) binds to the sST2-5'UTR, acts as an ITAF, and thus controls the activity of the sST2-IRES and consequently sST2 translation. Specifically, FGF2 enhances nuclear-cytoplasmic translocation of hnRNP A1, which requires intact MEK/ERK activity. In summary, we provide evidence that the sST2-5'UTR contains an IRES element, which is activated by a MEK/ERK-dependent increase in cytoplasmic localization of hnRNP A1 in response to FGF2, enhancing the translation of sST2.
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http://dx.doi.org/10.1016/j.bbagrm.2016.05.005DOI Listing
July 2016

What a Difference an OH Makes: Conformational Dynamics as the Basis for the Ligand Specificity of the Neomycin-Sensing Riboswitch.

Angew Chem Int Ed Engl 2016 Jan 11;55(4):1527-30. Epub 2015 Dec 11.

Institut für Molekulare Biowissenschaften and Zentrum für Biomolekulare Magnetische Resonanz (BMRZ), Goethe-Universität Frankfurt, Max-von-Laue Str. 9, 60438, Frankfurt/M, Deutschland.

To ensure appropriate metabolic regulation, riboswitches must discriminate efficiently between their target ligands and chemically similar molecules that are also present in the cell. A remarkable example of efficient ligand discrimination is a synthetic neomycin-sensing riboswitch. Paromomycin, which differs from neomycin only by the substitution of a single amino group with a hydroxy group, also binds but does not flip the riboswitch. Interestingly, the solution structures of the two riboswitch-ligand complexes are virtually identical. In this work, we demonstrate that the local loss of key intermolecular interactions at the substitution site is translated through a defined network of intramolecular interactions into global changes in RNA conformational dynamics. The remarkable specificity of this riboswitch is thus based on structural dynamics rather than static structural differences. In this respect, the neomycin riboswitch is a model for many of its natural counterparts.
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http://dx.doi.org/10.1002/anie.201507365DOI Listing
January 2016

A Universal Aptamer Chimera for the Delivery of Functional microRNA-126.

Nucleic Acid Ther 2015 Jun 6;25(3):141-51. Epub 2015 Apr 6.

1Institute for Cardiovascular Regeneration, Center of Molecular Medicine, Goethe University Frankfurt, Frankfurt, Germany.

microRNAs (miRs) regulate vascular diseases such as atherosclerosis and cancer. miR-126 is important for endothelial cell signaling and promotes angiogenesis, protects against atherosclerosis, and reduces breast cancer cell growth and metastasis. The overexpression of miR-126, therefore, may be an attractive therapeutic strategy for the treatment of cardiovascular disease or cancer. Here we report a novel strategy to deliver miR-126 to endothelial and breast cancer cells. We tested three different strategies to deliver miR-126 by linking the miR to an aptamer for the ubiquitously expressed transferrin receptor (transferrin receptor aptamer, TRA). Linking the precursor of miR-126 (pre-miR-126) to the TRA by annealing of a complementary stick led to efficient uptake and processing of miR-126, resulting in the delivery of 1.6×10(6)±0.3×10(6) copies miR-126-3p per ng RNA in human endothelial cells and 7.4×10(5)±2×10(5) copies miR-126-3p per ng in MCF7 breast cancer cells. The functionality of the active TRA-miR-126 chimera was further demonstrated by showing that the chimera represses the known miR-126 target VCAM-1 and improved endothelial cell sprouting in a spheroid assay. Moreover, the TRA-miR-126 chimera reduced proliferation and paracrine endothelial cell recruitment of breast cancer cells to a similar extent as miR-126-3p mimics introduced by conventional liposome-based transfection. Together, this data demonstrates that pre-miR-126 can be delivered by a non-specific aptamer to exert biological functions in two different cell models. The use of the TRA-miR-126 chimera or the combination of the delivery strategy with other endothelial or tumor specific aptamers may provide an interesting therapeutic option to treat vascular disease or cancers.
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http://dx.doi.org/10.1089/nat.2014.0501DOI Listing
June 2015

Tetracycline determines the conformation of its aptamer at physiological magnesium concentrations.

Biophys J 2014 Dec;107(12):2962-2971

Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt am Main, Frankfurt, Germany. Electronic address:

Synthetic riboswitches are versatile tools for the study and manipulation of biological systems. Yet, the underlying mechanisms governing its structural properties and regulation under physiological conditions are poorly studied. We performed spectroscopic and calorimetric experiments to explore the folding kinetics and thermodynamics of the tetracycline-binding aptamer, which can be employed as synthetic riboswitch, in the range of physiological magnesium concentrations. The dissociation constant of the ligand-aptamer complex was found to strongly depend on the magnesium concentration. At physiological magnesium concentrations, tetracycline induces a significant conformational shift from a compact, but heterogeneous intermediate state toward the completely formed set of tertiary interactions defining the regulation-competent structure. Thus, the switching functionality of the tetracycline-binding aptamer appears to include both a conformational rearrangement toward the regulation-competent structure and its thermodynamic stabilization.
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http://dx.doi.org/10.1016/j.bpj.2014.11.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4269781PMC
December 2014

Hypoxia reduces MAX expression in endothelial cells by unproductive splicing.

FEBS Lett 2014 Dec 15;588(24):4784-90. Epub 2014 Nov 15.

Department of Biology, Technical University Darmstadt, Schnittspahnstr. 10, 64287 Darmstadt, Germany. Electronic address:

The MYC-MAX-MXD network is involved in the regulation of cell differentiation and proliferation. Hypoxia affects the expression levels of several members of this network, but changes specific to MAX expression have so far not been shown. We found that in endothelial cells, hypoxia induces alternative splicing of MAX, thereby increasing the expression of two MAX isoforms that differ from the wild type in their 3' end. Isoform C is degraded by nonsense-mediated decay and isoform E encodes a highly unstable protein. The instability of isoform E is conferred by 36 isoform-specific amino acids, which have the capacity to destabilize heterologous proteins. Both splicing events are therefore unproductive and serve the purpose to downregulate the wild type protein.
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http://dx.doi.org/10.1016/j.febslet.2014.11.011DOI Listing
December 2014

Sequence elements distal to the ligand binding pocket modulate the efficiency of a synthetic riboswitch.

Chembiochem 2014 Jul 20;15(11):1627-37. Epub 2014 Jun 20.

Department of Biology, Technical University Darmstadt, Schnittspahnstrasse 10, 64287 Darmstadt (Germany).

Synthetic riboswitches can serve as sophisticated genetic control devices in synthetic biology, regulating gene expression through direct RNA-ligand interactions. We analyzed a synthetic neomycin riboswitch, which folds into a stem loop structure with an internal loop important for ligand binding and regulation. It is closed by a terminal hexaloop containing a U-turn and a looped-out adenine. We investigated the relationship between sequence, structure, and biological activity in the terminal loop by saturating mutagenesis, ITC, and NMR. Mutants corresponding to the canonical U-turn fold retained biological activity. An improvement of stacking interactions in the U-turn led to an RNA element with slightly enhanced regulatory activity. For the first position of the U-turn motif and the looped out base, sequence-activity relationships that could not initially be explained on the basis of the structure of the aptamer-ligand complex were observed. However, NMR studies of these mutants revealed subtle relationships between structure and dynamics of the aptamer in its free or bound state and biological activity.
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http://dx.doi.org/10.1002/cbic.201402067DOI Listing
July 2014

Building a stable RNA U-turn with a protonated cytidine.

RNA 2014 Aug 20;20(8):1163-72. Epub 2014 Jun 20.

Institute of Molecular Biosciences, Johann-Wolfgang-Goethe-University Frankfurt/M., 60438 Frankfurt, Germany Center for Biomolecular Magnetic Resonance (BMRZ), Johann-Wolfgang-Goethe-University Frankfurt/M., 60438 Frankfurt, Germany.

The U-turn is a classical three-dimensional RNA folding motif first identified in the anticodon and T-loops of tRNAs. It also occurs frequently as a building block in other functional RNA structures in many different sequence and structural contexts. U-turns induce sharp changes in the direction of the RNA backbone and often conform to the 3-nt consensus sequence 5'-UNR-3' (N = any nucleotide, R = purine). The canonical U-turn motif is stabilized by a hydrogen bond between the N3 imino group of the U residue and the 3' phosphate group of the R residue as well as a hydrogen bond between the 2'-hydroxyl group of the uridine and the N7 nitrogen of the R residue. Here, we demonstrate that a protonated cytidine can functionally and structurally replace the uridine at the first position of the canonical U-turn motif in the apical loop of the neomycin riboswitch. Using NMR spectroscopy, we directly show that the N3 imino group of the protonated cytidine forms a hydrogen bond with the backbone phosphate 3' from the third nucleotide of the U-turn analogously to the imino group of the uridine in the canonical motif. In addition, we compare the stability of the hydrogen bonds in the mutant U-turn motif to the wild type and describe the NMR signature of the C+-phosphate interaction. Our results have implications for the prediction of RNA structural motifs and suggest simple approaches for the experimental identification of hydrogen bonds between protonated C-imino groups and the phosphate backbone.
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http://dx.doi.org/10.1261/rna.043083.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4105743PMC
August 2014

Development of β -lactamase as a tool for monitoring conditional gene expression by a tetracycline-riboswitch in Methanosarcina acetivorans.

Archaea 2014 20;2014:725610. Epub 2014 Jan 20.

AG Molekulare Mikrobiologie und Bioenergetik, Institut für Molekulare Biowissenschaften, Johann Wolfgang Goethe-Universität Frankfurt, 60438 Frankfurt am Main, Germany ; Institut für Mikrobiologie, Technische Universität Dresden, 01062 Dresden, Germany.

The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, like β -galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developed β -lactamase from Escherichia coli (encoded by bla) in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeon Methanosarcina acetivorans. By using a signal peptide of a putative flagellin from M. acetivorans and different catabolic promoters, we could demonstrate growth substrate-dependent secretion of β -lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracycline-responsive riboswitch (tc-RS) was created to characterize its influence on translation initiation in M. acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation of bla expression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solely cis-active, that is, factor-independent system for controlled gene expression in Archaea.
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http://dx.doi.org/10.1155/2014/725610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3942078PMC
November 2014

Monitoring the expression level of coding and non-coding RNAs using a TetR inducing aptamer tag.

Bioorg Med Chem 2013 Oct 31;21(20):6233-8. Epub 2013 Jul 31.

Institut für Molekulare Biowissenschaften, Johann Wolfgang Goethe-Universität Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt/M., Germany.

RNA aptamers have been widely used as regulators for conditional gene expression. The TetR binding aptamer can activate tetracycline repressor TetR controlled gene expression with high efficiency. Here we demonstrate that the aptamer can also activate TetR controlled gene expression when expressed in the context of a natural transcripts. The aptamer was inserted into the untranslated regions of mRNAs as well as into small non-coding RNAs and was expressed both from a plasmid and from an endogenous locus. Our data suggest that the aptamer is a valuable tool to easily monitor the expression level of different RNAs, and it therefore represents a powerful tool for the construction of complex synthetic gene networks.
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http://dx.doi.org/10.1016/j.bmc.2013.07.035DOI Listing
October 2013

Regulation of miR-17-92a cluster processing by the microRNA binding protein SND1.

FEBS Lett 2013 Aug 13;587(15):2405-11. Epub 2013 Jun 13.

Institute of Cardiovascular Regeneration, Center for Molecular Medicine, Frankfurt, Germany.

MicroRNAs are small non-coding RNAs that regulate gene expression. Although all seven members of the miR-17-92a cluster originate from one primary transcript they are differentially expressed suggesting the presence of posttranscriptional regulation. By RNA pulldown and mass spectrometry we identified SND1, a known regulator of edited RNAs, interacting with pre-miR-92a and all mature miR-17-92a members. Hypoxic conditions lead to an elevation of the pri-miR-17-92a transcript and significantly increased levels of the precursors whereas the mature miRs were not significantly changed. SND1 silencing resolved this block in processing and induced an increase in mature miRs. Together, SND1 might be the missing link between hypoxia and the differential regulation of miRNA processing.
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http://dx.doi.org/10.1016/j.febslet.2013.06.008DOI Listing
August 2013

Hypoxia-induced alternative splicing in endothelial cells.

PLoS One 2012 2;7(8):e42697. Epub 2012 Aug 2.

Institute for Cardiovascular Regeneration, Center of Molecular Medicine, Johann Wolfgang Goethe University Frankfurt, Frankfurt am Main, Germany.

Background: Adaptation to low oxygen by changing gene expression is vitally important for cell survival and tissue development. The sprouting of new blood vessels, initiated from endothelial cells, restores the oxygen supply of ischemic tissues. In contrast to the transcriptional response induced by hypoxia, which is mainly mediated by members of the HIF family, there are only few studies investigating alternative splicing events. Therefore, we performed an exon array for the genome-wide analysis of hypoxia-related changes of alternative splicing in endothelial cells.

Methodology/principal Findings: Human umbilical vein endothelial cells (HUVECs) were incubated under hypoxic conditions (1% O(2)) for 48 h. Genome-wide transcript and exon expression levels were assessed using the Affymetrix GeneChip Human Exon 1.0 ST Array. We found altered expression of 294 genes after hypoxia treatment. Upregulated genes are highly enriched in glucose metabolism and angiogenesis related processes, whereas downregulated genes are mainly connected to cell cycle and DNA repair. Thus, gene expression patterns recapitulate known adaptations to low oxygen supply. Alternative splicing events, until now not related to hypoxia, are shown for nine genes: six which are implicated in angiogenesis-mediated cytoskeleton remodeling (cask, itsn1, larp6, sptan1, tpm1 and robo1); one, which is involved in the synthesis of membrane-anchors (pign) and two universal regulators of gene expression (cugbp1 and max).

Conclusions/significance: For the first time, this study investigates changes in splicing in the physiological response to hypoxia on a genome-wide scale. Nine alternative splicing events, until now not related to hypoxia, are reported, considerably expanding the information on splicing changes due to low oxygen supply. Therefore, this study provides further knowledge on hypoxia induced gene expression changes and presents new starting points to study the hypoxia adaptation of endothelial cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0042697PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3411717PMC
January 2013

Aptamer-regulated expression of essential genes in yeast.

Methods Mol Biol 2012 ;824:381-91

Department of Molecular Biosciences, Johann Wolfgang Goethe-University Frankfurt, Frankfurt, Germany.

Conditional gene expression systems are important tools for the functional analysis of essential genes. Tetracycline (tc)-binding aptamers can be exploited as artificial riboswitches for the efficient control of gene expression by inserting them into the 5' untranslated region of an mRNA. The ligand-bound form of those mRNAs inhibits gene expression by interfering with translation initiation. In contrast to previous tc-dependent regulatory systems, where tc inhibits or activates transcription upon binding to the repressor protein TetR, the tc-binding aptamer system inhibits translation of the respective mRNA. We describe here a simple and powerful PCR-based strategy which allows easy tagging of any target gene in yeast using a tc aptamer-containing insertion cassette. The expression window can be adjusted with different promoters and protein synthesis is rapidly switched off.
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http://dx.doi.org/10.1007/978-1-61779-433-9_20DOI Listing
April 2012

RNA-based networks: using RNA aptamers and ribozymes as synthetic genetic devices.

Methods Mol Biol 2012 ;813:157-68

Institute of Molecular Biosciences, Johann Wolfgang Goethe-University Frankfurt, Frankfurt, Germany.

Within the last few years, a set of synthetic riboswitches has been engineered, which expands the toolbox of genetic regulatory devices. Small molecule binding aptamers have been used for the design of such riboswitches by insertion into untranslated regions of mRNAs, exploiting the fact that upon ligand binding the RNA structure interferes either with translation initiation or pre-mRNA splicing in yeast. In combination with self-cleaving ribozymes, aptamers have been used to modulate RNA stability. In this chapter, we discuss the applicability of different aptamers, ways to identify novel genetic devices, the pros and cons of various insertion sites and the application of allosteric ribozymes. Our expertise help to apply synthetic riboswitches to engineer complex genetic circuits.
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http://dx.doi.org/10.1007/978-1-61779-412-4_9DOI Listing
March 2012

Conformational dynamics of the tetracycline-binding aptamer.

Nucleic Acids Res 2012 Feb 3;40(4):1807-17. Epub 2011 Nov 3.

Institut für Physikalische und Theoretische Chemie, Goethe-Universität Frankfurt, Max-von-Laue-Straße 7, Germany.

The conformational dynamics induced by ligand binding to the tetracycline-binding aptamer is monitored via stopped-flow fluorescence spectroscopy and time-correlated single photon counting experiments. The fluorescence of the ligand is sensitive to changes within the tertiary structure of the aptamer during and after the binding process. In addition to the wild-type aptamer, the mutants A9G, A13U and A50U are examined, where bases important for regulation are changed to inhibit the aptamer's function. Our results suggest a very fast two-step-mechanism for the binding of the ligand to the aptamer that can be interpreted as a binding step followed by a reorganization of the aptamer to accommodate the ligand. Binding to the two direct contact points A13 and A50 was found to occur in the first binding step. The exchange of the structurally important base A9 for guanine induces an enormous deceleration of the overall binding process, which is mainly rooted in an enhancement of the back reaction of the first binding step by several orders of magnitude. This indicates a significant loss of tertiary structure of the aptamer in the absence of the base A9, and underlines the importance of pre-organization on the overall binding process of the tetracycline-binding aptamer.
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http://dx.doi.org/10.1093/nar/gkr835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3287181PMC
February 2012

Mechanistic insights into an engineered riboswitch: a switching element which confers riboswitch activity.

Nucleic Acids Res 2011 Apr 11;39(8):3363-72. Epub 2010 Dec 11.

RNA Biochemistry, Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt/M, Germany.

While many different RNA aptamers have been identified that bind to a plethora of small molecules only very few are capable of acting as engineered riboswitches. Even for aptamers binding the same ligand large differences in their regulatory potential were observed. We address here the molecular basis for these differences by using a set of unrelated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher degree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is necessary but not sufficient for regulation. NMR data showed that a destabilized, open ground state accompanied by extensive structural changes upon ligand binding is important for regulation. In contrast, inactive aptamers are already pre-formed in the absence of the ligand. By a combination of genetic, biochemical and structural analyses, we identified a switching element responsible for destabilizing the ligand free state without compromising the bound form. Our results explain for the first time the molecular mechanism of an engineered riboswitch.
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http://dx.doi.org/10.1093/nar/gkq946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082870PMC
April 2011

Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR.

RNA 2011 Jan 19;17(1):182-8. Epub 2010 Nov 19.

Fachbereich Physik, Universität Osnabrück, 49069 Osnabrück, Germany.

RNA aptamers are in vitro-selected binding domains that recognize their respective ligand with high affinity and specificity. They are characterized by complex three-dimensional conformations providing preformed binding pockets that undergo conformational changes upon ligand binding. Small molecule-binding aptamers have been exploited as synthetic riboswitches for conditional gene expression in various organisms. In the present study, double electron-electron resonance (DEER) spectroscopy combined with site-directed spin labeling was used to elucidate the conformational transition of a tetracycline aptamer upon ligand binding. Different sites were selected for post-synthetic introduction of either the (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate by reaction with a 4-thiouridine modified RNA or of 4-isocyanato-2,6-tetramethylpiperidyl-N-oxid spin label by reaction with 2'-aminouridine modified RNA. The results of the DEER experiments indicate the presence of a thermodynamic equilibrium between two aptamer conformations in the free state and capture of one conformation upon tetracycline binding.
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http://dx.doi.org/10.1261/rna.2222811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3004059PMC
January 2011