Project

microRNA, miR-CLIP, psoralen, crosslinker

Lead
MicroRNAs (miRNAs) sind kleine, wichtige RNAs, die die Genexpression regulieren.
Lay summary
In menschlichen Zellen binden sie an andere RNAs, um ihre Funktion auszuüben. Einige miRNAs regulieren Mechanismen, die in verschiedenen Krankheiten eine Rolle spielen, z.B. in HCV infektionen. Um zu verstehen welche miRNAs für die in der Zelle ablaufenden Mechanismen von Bedeutung sind, müssen wir deren Bindungspartner („Zielstrukturen“) identifizieren. Seit ihrer Entdeckung ist dies eines der Hauptprobleme auf diesem Arbeitsgebiet. Der sicherste Weg um die Bindungspartner zu identifizieren ist, durch eine chemische Reaktion die miRNA an Ihre Zielstruktur zu binden. Bei diesem Ansatz, wird die miRNA aktiviert, nachdem sie ihren Bindungspartner gefunden hat. Dafür werden chemische Reaktionen für diese kleinen RNAs benötigt. Im 2015 haben wir eine neue Methode veröffentlicht um die RNA-Zielstrukturen zu finden und haben den Wert dieser Technik durch die Identifizierung der Bindungspartner von zwei miRNAs gezeigt. Die Methode funktioniert gut, jedoch ist eine weitere Optimierung nötig. Das übergeordnete Ziel dieses Forschungsvorhabens ist die Entwicklung neuer chemischer Werkzeuge um die RNA-Zielstrukturen von miRNAs in der Zelle zu identifizieren. Wir werden verschiedene auf Konjugation basierende Ansätze untersuchen, jede in verschiedenen Varianten mit Ihren jeweiligen Stärken und Schwächen.  Ein Ziel ist es, effiziente Reagenzien der zweiten Generation  für alle miRNAs, unabhängig von Ihrer Sequenz, zu entwicklen. In einem Teil der Arbeit werden wir den der ersten Generation von Reagenzien zu Grunde liegenden Quervernetzungsmechanismus in vereinfachten Testsystemen detailliert untersuchen. Im zweiten Teil werden wir neue Methoden zur miRNA-Bindungspartner-Identifizierung entwickeln.

Direct link to Lay Summary

Last update: 02.11.2016

Active participation

Self-organised

Title	Year

Number	Title	Start	Funding scheme

MicroRNAs (miRNAs) are small RNAs that regulate gene expression post-transcriptionally, either fine-tuning expression or acting as phenotypic “switches. Some miRNAs play important roles in diseases and are under clinical investigation as drugs or as drug targets, such as miR-34a and miR-103/107, respectively, for cancer and insulin disorders, respectively. Understanding miRNA function requires that their binding partners be identified. MiRNAs bind target messenger RNAs (mRNAs) mostly in their 3’UTRs, using approx. 7 nucleotides (nt) at their 5’ ends (“seed”). This is considered the canonical mechanism of regulation: it leads to translational repression and/or mRNA decay. A miRNA may bind hundreds to thousands of RNAs in a given cell. Accounts of non-canonical miRNA-RNA interactions, e.g. involving the 3’ends of miRNAs are increasing but base-pairing patterns for these interactions are poorly understood. Thus, there is a need for new methods to identify non-canonical binding interactions, particularly for miRNAs that contribute to programs important for gene expression, or for miRNAs with an important role in pathological mechanisms. Although computational predictions have proven crucial in revealing principles of miRNA-dependent mRNA regulation, the gold standard for the identification of miRNA targets is experimental approaches, in which miRNA binding partners are captured covalently in cells and then validated. These wet-bench-based approaches are still in stages of infancy. The overall objective of this proposal is to provide new chemical tools to capture covalently RNA targets of miRNAs in cells. Identification of a miRNA targetome (all targets) is done by two general capture-based approaches, each with its variants, each with strengths and weak points. In one, all expressed miRNAs and their targets are captured under native conditions in a selected cell type genome-wide fashion; in the other, targets of a single, selected miRNA are captured under non-native conditions. The principal basis for this application was our development of miR-CLIP (miRNA cross-linking and immunoprecipitation), a protocol comprising the design and synthesis of psoralen- and biotin-labeled miRNA probes which mimic native miRNAs in cells and cross-link to their “targetome” when transfected into cells. Whereas a miR-CLIP probe for miR-106a cross-linked efficiently to its target mRNAs in HeLa cells, many other miRNAs were much less efficient cross-linkers, suggesting that the psoralen was not optimally positioned in the probe and that probe sequence was important. The main objective of this proposal is to design and develop a second generation of miR-CLIP probes so that efficient reagents can be obtained for all miRNAs, independent of their sequence. This will provide a resource that permits the targetome of any selected (or in the long term, all) miRNA to be determined, in any transfectable cells. For miR-CLIP probes to be of general use, it is critical that cross-linking occurs from the nucleotide bearing the psoralen to the complementary base in a target. In Part 1 of the proposal we will investigate the cross-linking mechanism for 1st generation miR-CLIP reagents using in vitro and reporter assays. In Part 2 we will investigate alternative modes of conjugating psoralen cross-linkers. In Part 3 we will investigate the use of miRNA-mRNA ligation as a new form of cross-linking. The best performing miR-CLIP reagents will be used to capture the targetomes of ongoing miRNAs of interest in our laboratory.