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Identification of Regulatory RNA Elements Based on Structural Identification of regulatory RNA elements based on structural conservation Dem Fachbereich Biologie der Technischen Universität Darmstadt zur Erlangung des akademischen Grades eines Dr. rer. nat eingereichte Dissertation von Herr M. Sc. Johannes Braun aus Landau i. d. Pfalz 1. Referentin: Prof. Dr. Beatrix Süss 2. Referent: Prof. Dr. Viktor Stein Datum der Einreichung: 04.12.2018 Tag der mündlichen Prüfung: 24.01.2019 Darmstadt 2019 Braun, Johannes: Identification of regulatory RNA elements based on structural con- servation Darmstadt,Technische Universität Darmstadt, Jahr der Veröffentlichung der Dissertationauf TUprints: 2019 Tag der mündlichen Prüfung: 24.01.2019 Veröffentlicht unter CC BY-SA 4.0 International https://creativecommons.org/licenses/ Für meine Mädels. The posttranscriptional regulation of gene expression determines the amount of a protein produced from a specific mRNA. All stages of the mRNA life cycle are post- transcriptionally regulated. The untranslated regions (UTRs) of mRNA play an im- portant role in this process. UTRs encode cis-regulatory elements that interact with trans-acting factors such as RNA binding proteins or non-coding RNAs. These interac- tions are based on the specific recognition of sequence or structured motifs. Similar to the conservation of linear sequences, the conservation of secondary structures can be an indicative of a functional cis-regulatory element. In the first part of my doctoral thesis, I identified structurally conserved regulatory elements in 3’UTRs of mRNAs. For this, I performed reporter gene assays with bioin- formatically predicted structurally conserved RNA elements and discovered a regulatory element in the 3’UTR of the UCP3 (uncoupling protein 3) mRNA. UCP3 is a protein of the inner mitochiondrial membrane and is associated with the development of diabetes melitus type 2 (T2DM). Through sequence and structural analysis, I discovered that the element has an active conformation that consists of two short RNA stem-loops. In further experiments, I was able to confirm that the presence of both RNA stem-loops is necessary for efficient regulation. Furthermore, I showed that the reduction of reporter gene expression is caused by a reduction of the mRNA half-life. The prediction of con- served RNA structures thus provides a powerful tool for the de novo identification of cis-regulatory elements. In the second part of my doctoral thesis, I characterized the regulatory element from the 3’UTR of UCP3 in detail. First, I performed RNA affinity purification to identify proteins specifically associated with the UCP3 element by mass spectrometry. This allowed me to show that the proteins Roquin-1 and Roquin-2 bind to the RNA stem loops of the UCP3 element. Furthermore, I showed that endogenous UCP3 is regulated by Roquin. Roquin proteins bind to constitutive (CDEs) and alternative (ADEs) decay elements and induce the rapid degradation of mRNAs of genes that play an important role in the immune response. Binding studies with the Roquin-1 ROQ domain showed that Roquin binds with significantly higher affinity to the UCP3 element, when both CDEs are present. Both CDEs in the UCP3 element do not correspond to the previously suggested CDE consensus. Performing a detailed mutational analysis, I revised the CDE consensus. With my data >160 new CDE- and 19 new ADE-coding mRNAs could be identified. Furthermore, I confirmed new CDE/ADE-containing mRNAs as targets of Roquin. Interestingly, I was able to show that not only the expression of CDE- encoding mRNAs, but also regulation by Roquin is cell type dependent. In conclusion, I have extended Roquin’s role in the posttranscriptional regulation of gene expression and suggest that its role is not limited to the regulation of the immune response. Die posttranskriptionelle Regulation der Genexpression bestimmt maßgeblich die Men- ge eines Proteins, welches von einer bestimmten mRNA hergestellt wird. Alle Prozesse im Lebenszyklus einer mRNA werden posttranskriptionell reguliert. Eine besondere Rolle spielen dabei die nicht translatierten Bereiche (UTRs) der mRNA. UTRs kodieren cis- regulatorische Elemente welche mit trans-agierende Faktoren wie RNA Bindeproteinen oder nicht-kodierenden RNAs interagieren. Diese Interaktionen basieren auf der spezi- fischen Erkennung von Sequenz- oder Strukturmotifen. Ähnlich wie die Konservierung von Sequenzen, kann auch die Konservierung von Sekundärstrukturen Hinweis auf ein funktionales cis-regulatorisches Element sein. Im ersten Teil meiner Doktorarbeit identifizierte ich strukturkonservierte, regulatori- sche Elemente in 3’UTRs der mRNA. Dazu führte ich Reportergenanalysen mit bioin- formatisch vorhergesagten strukturkonservierten RNA Elementen durch und entdeckte ein regulatorisches Element im 3’UTR der UCP3 (uncoupling protein 3) mRNA. UCP3 ist ein mitochondriales Membranprotein und mit der Entstehung von Diabetes Melitus Typ 2 assoziiert. Durch Sequenz- und Strukturanalyse fand ich heraus, dass das Element eine aktive Konformation mit zwei kurzen RNA Stammschleifen einnimmt. In weiteren Experimenten konnte ich bestätigen, dass die Anwesenheit beider RNA Stammschlei- fen notwendig für die Regulation ist. Darüber hinaus zeigte ich, dass die Reduktion der Reportergenexpression durch eine drastische Verringerung der mRNA Halbwertszeit ver- ursacht wird. Die Vorhersage konservierter RNA Strukturen, stellt somit ein effizientes Werkzeug zur de novo Identifikation cis-regulatorischer Elemente dar. Im zweiten Teil meiner Doktorarbeit untersuchte ich das regulatorische Element aus dem 3’UTR von UCP3 im Detail. Zunächst führte ich RNA Affinitätsaufreinigung durch um Proteine, die speziefisch mit dem UCP3 Element interagieren, mittels Massenspek- tromentrie zu identifizieren. Dadurch konnte ich zeigen, dass die Proteine Roquin-1 und Roquin-2 an die RNA Stammschleifen des UCP3 Elements binden. Darüber hinaus zeig- te ich, dass endogenes UCP3 durch Roquin reguliert wird. Roquin Proteine binden an konstitutive (CDEs) und alternative (ADEs) Abbauelemente und induzieren eine schnel- len Abbau der mRNA von Genen die eine wichtige Rolle bei der Immunantwort spielen. Bindungstudien mit der Roquin-1 ROQ Domäne zeigten, dass Roquin mit deutlich hö- herer Affinität an das UCP3 Element bindet, wenn beide CDEs vohanden sind. Beide CDEs im UCP3 Element entspechen nicht dem bisherigen Consesus für CDEs. Mit Hil- fe einer Mutationsstudie revidierte ich den CDE Konsesus. Mit meinen Daten konnten >160 neue CDE- und 19 neue ADE-kodierende mRNAs identifiziert werden. Drüber hinaus bestätigte ich neue CDE/ADE-kodierende mRNAs als Ziele von Roquin. Inter- essanterweise konnte ich zeigen, dass nicht nur die Expression CDE-kodierender mRNAs, sondern auch die Regulation durch Roquin, Zelltyp abhängig ist. Durch meine Arbeit konnte die Rolle von Roquin in der posttranskriptionellen Regulation der Genexpression erweitert werden und und gezeigt werden, dass die Regulation durch Roquin nicht auf Gene der Immunantwort beschränkt ist. Contents 1 Introduction1 1.1 Post-transcriptional Regulation . .1 1.1.1 Pre-mRNA Splicing . .4 1.1.2 Alternative pre-mRNA Splicing . .5 1.1.3 mRNA Surveillance . .6 1.1.4 mRNA Decay . .7 1.1.5 Non-coding RNAs in Gene Regulation . .8 1.1.6 RNA-binding Proteins . .9 1.1.7 The Roquin Protein Family . 12 1.2 The Role of UTRs in Gene Regulation . 14 1.2.1 Regulation by 5’UTRs . 15 1.2.2 Regulation by 3’UTRs . 17 1.2.3 AU-rich Elements . 17 1.2.4 Structured Regulatory RNA Elements . 19 1.2.5 Prediction of Regulatory RNA Elements . 20 1.3 Aim of this Study . 22 2 Results 23 2.1 Identification of Regulatory RNA Elements Based on Structural Conser- vation . 24 2.1.1 A Conserved Structure in the 3’UTR of UCP3 Reduces Gene Ex- pression . 24 2.1.2 The reduction of gene expression is not induced by miR-152 . 27 2.1.3 A 64 nt Minimal Motif is Sufficient for Gene Repression . 29 2.1.4 The Repressive Element is a Conserved Tandem Stem-Loop Struc- ture . 30 2.1.5 The UCP3 Element Reduces mRNA Half-Life . 31 2.2 Identification of Trans-acting Factors . 34 2.2.1 RNA Affinity Purification of RNA-binding Proteins . 35 2.2.2 Roquin Proteins Mediate Repression by Binding the UCP3 wt Element . 37 2.3 Redefining Roquin Binding Preferences . 38 2.3.1 The UCP3 Element Encodes Two Roquin Binding Sites . 39 2.3.2 In vivo Mutational Analysis of the UCP3 Tandem CDE . 42 I Contents 2.4 Identification of New High Affinity Targets of Roquin . 46 2.4.1 Computational Prediction of CDEs . 47 2.4.2 In vivo Verification of New Roquin Target Genes . 49 2.4.3 Knockdown of Roquin Proteins Increases mRNA Abundance of CDE-encoding mRNAs . 51 2.4.4 Computational Prediction of ADEs . 53 3 Discussion 55 3.1 Identification of Regulatory RNA Elements Based on Structural Conser- vation . 55 3.1.1 Prediction of Structurally Conserved RNA Elements . 56 3.1.2 Screening for Functional RNA Elements . 57 3.1.3 A Repressive RNA Element in the 3’UTR of UCP3 ........ 58 3.1.4 The UCP3 Repressive Element is not a miR Binding Site . 59 3.2 The Tandem CDE in the UCP3 3’UTR . 60 3.2.1 Structure-Sequence-Function Analysis of the Tandem CDE . 62 3.2.2 Identification of New Roquin Target Genes . 63 3.2.3 ADEs are Self-sufficient Roquin Binding Sites . 66 3.2.4 Determinants of Roquin-mediated Regulation . 67 3.3 Summary and Outlook . 68 4 Material and Methods 70 4.1 Materials . 70 4.2 Methods . 70 4.2.1 Cell Culture and Transfection . 70 4.2.2 Plasmid Construction . 71 4.2.3 Hybridization and Phosphorylation of Oligonucleotides . 73 4.2.4 Polymerase Chain Reaction (PCR) . 74 4.2.5 Genomic Integration . 74 4.2.6 Flow Cytometry . 75 4.2.7 Luciferase Reporter Assay . 75 4.2.8 RNA Extraction and Reverse Transcription . 76 4.2.9 Quantitative RT-PCR . 78 4.2.10 mRNA Decay Assay . 78 4.2.11 Western Blot Analysis . 79 4.2.12 In Vitro Transcription . 80 4.2.13 RNA Affinity Purification . 80 4.2.14 Mass Spectrometry .
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