Die 5′-Kappe ist ein Charakteristikum eukaryotischer mRNAs und nimmt im RNA-Metabolismus entscheidende Rollen ein, die sich von der Qualitätskontrolle über den Export bis hin zur Translation erstrecken. Die Modifizierung der 5′-Kappe könnte daher die Modulierung dieser Prozesse und die Erforschung verschiedener biologischer Funktionen ermöglichen. Wir präsentieren einen direkten Ansatz zur Erzeugung einer Auswahl N7-modifizierter Kappen basierend auf der hoch promiskuitiven Methyltransferase Ecm1. Wir zeigen, dass diese sowie N²-modifizierte 5′-Kappen zur Steuerung der Translation entsprechender mRNAs in vitro und in Zellen genutzt werden können. Entsprechende Modifikationen ermöglichen die anschließende Anwendung bioorthogonaler Reaktionen, z. B. die intrazelluläre Markierung einer Ziel-mRNA in lebenden Zellen. Die effiziente und vielseitige Manipulierung des N7-Atoms der mRNA-Kappe ist ein wertvoller Beitrag zum Methodenrepertoire der chemischen Biologie.

The 5′-cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5′-cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7-modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N²-modified 5′-caps, can be used to tune translation of the respective mRNAs both in vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live-cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox.

Multicolor readout is an important feature of RNA detection techniques aiming at the investigation of RNA localization. Several detection methods have been developed, however they require either transfection of cells with the probe or prior tagging of the target RNA. We report a fully genetically encodable system for simultaneous detection of two RNAs by using green and yellow fluorescence based on tetramolecular fluorescence complementation (TetFC). To obtain yellow fluorescent protein (YFP), substitution T203Y was introduced into one of the three non-fluorescent GFP fragments; this was fused to different variants of the Homo sapiens Pumilio homology domain. Using different sets of fusion proteins we were able to discriminate between two closely related target RNAs based on the fluorescence signals at the respective wavelengths.

Methyltransferases have proven useful to install functional groups site-specifically in different classes of biomolecules when analogues of their cosubstrate S-adenosyl-l-methionine (AdoMet) are available. Methyltransferases have been used to address different classes of RNA molecules selectively and site-specifically, which is indispensable for biophysical and mechanistic studies as well as labeling in the complex cellular environment. However, the AdoMet analogues are not cell-permeable, thus preventing implementation of this strategy in cells. We present a two-step enzymatic cascade for site-specific mRNA modification starting from stable methionine analogues. Our approach combines the enzymatic synthesis of AdoMet with modification of the 5′ cap by a specific RNA methyltransferase in one pot. We demonstrate that a substrate panel including alkene, alkyne, and azido functionalities can be used and further derivatized in different types of click reactions.

Methyltransferasen sind nützlich, um funktionelle Gruppen ortsspezifisch in Biomoleküle einzubauen, wenn Analoga des Kosubstrates S-Adenosyl-l-Methionin (AdoMet) verfügbar sind. Methyltransferasen wurden eingesetzt, um RNA-Moleküle selektiv und ortsspezifisch zu adressieren, was für biophysikalische und mechanistische Studien sowie die Markierung in der komplexen zellulären Umgebung unerlässlich ist. AdoMet-Analoga sind jedoch nicht zellgängig, sodass diese Strategie nicht in Zellen anwendbar ist. Wir präsentieren eine zweistufige enzymatische Kaskade zur ortsspezifischen mRNA-Modifizierung ausgehend von Methioninanaloga. Unser Ansatz kombiniert die enzymatische Synthese von AdoMet mit der Modifikation der 5′-Kappe durch eine spezifische mRNA-Methyltransferase in einem Eintopfverfahren. Wir zeigen, dass eine Reihe von Substraten, unter anderem mit Alken-, Alkin- und Azidoresten, eingesetzt werden kann und dass die weitere Derivatisierung mit Klick-Reaktionen gelingt.

Ribonucleoprotein (RNP) complexes are widespread in nature and play crucial roles in gene regulation, RNA processing, and translation. Novel technologies, such as CRISPR-mediated genome engineering, stress the potential of RNP complexes to carry out complex tasks in molecular biology. Here we report a bottom-up approach for the programmable self-assembly of RNP complexes. The building blocks for RNP complex formation are RNAs and Pumilio proteins that can bind to RNA sequence-specifically. Correct RNP assembly triggers protein complementation of a tripartite GFP, thereby resulting in up to 25-fold increased fluorescence, and is strictly dependent on the correct RNA sequences. Our results indicate that Pumilio and guide RNAs are suitable building blocks for the correct self-assembly of RNP complexes consisting of up to six different components. Self-assembling RNP complexes might prove useful for complex biotechnological applications in RNA sensing, imaging, or processing.

Degradation of cellulose represents a key step for efficient generation of biofuels such as ethanol or butanol from nonfood crops. Several strategies aim to improve the performance of cellulases in order to make the overall process more cost-efficient.

In one strategy, novel arrangements of cellulases are developed. The resulting so-called designer cellulosomes might allow to equip the fermenting hosts with sufficient cellulase activity to grow on cellulose as sole carbon source and thus enable consolidated bioprocessing.

Other strategies aim to engineer cellulases with higher thermostability and activity to make cellulose hydrolysis more efficient. This review summarizes current developments in the field of cellulase engineering and highlight achievements as well as limitations.

Over recent years, click reactions have become recognized as valuable and flexible tools to label biomacromolecules such as proteins, nucleic acids, and glycans. Some of the developed strategies can be performed not only in aqueous solution but also in the presence of cellular components, as well as on (or even in) living cells. These labeling strategies require the initial, specific modification of the target molecule with a small, reactive moiety. In the second step, a click reaction is used to covalently couple a reporter molecule to the biomolecule. Depending on the type of reporter, labeling by the click reaction can be used in many different applications, ranging from isolation to functional studies of biomacromolecules. In this minireview, we focus on labeling strategies for RNA that rely on the click reaction. We first highlight click reactions that have been used successfully to label modified RNA, and then describe different strategies to introduce the required reactive groups into target RNA. The benefits and potential limitations of the strategies are critically discussed with regard to possible future developments.