Prof. Dr. Albrecht Bindereif
Our current research interests focus on alternative mRNA splicing, RNA-binding proteins, and noncoding RNAs in the human system. Specifically, we study pathways and networks of RNA-mediated gene regulation, including circular RNAs and RNA transfer via extracellular vesicles, combining RNA biochemistry, genomewide approaches, and RNA bioinformatics.
Circular RNAs in humansCircular RNAs (circRNAs) were recently discovered as a novel and abundant class of noncoding RNAs in higher eukaryotes. They are generated from pre-mRNAs by a special mode of alternative splicing, circularization of one or several adjacent exons. CircRNAs are expressed in a cell-type- and tissue-specific manner, evolutionarily conserved, and mostly cytoplasmic. For a few specific cases, miRNA sponge and gene-regulatory roles have been demonstrated, but their functional spectrum is largely unexplored.
We are interested in their biogenesis, specifically on the protein factors and sequence elements that determine circular versus linear splicing. We have obtained first evidence for the canonical spliceosome functioning in circRNA processing. Our projects also focus on circRNA-protein interactions, and we have carried out an initial analysis of circRNA-protein complexes (circRNPs) in mammalian cells, focussing on a multifunctional RNA-binding protein, IMP3 (see also above): This protein turned out to be a common protein component of a subclass of circRNPs.
Engineering designer circRNAs as protein sponges and antisense vectorsWe established methodology to design and produce synthetic circRNAs in preparative quantities, both of small (up to 100 nts) and large sizes (100 nts to 1 kb), functioning as specific protein sponges and as antisense vectors. Protein sponges are designed to inactivate specific RNA-binding proteins, for example when upregulated in cancer tissue; in addition, antisense-circRNAs are designed to block mRNA function on translation initiation or stability levels. The overall aim is to use such designer circRNAs for interfering with gene-regulatory networks, opening up new therapeutic strategies for human diseases.
RNA recognition and function of multidomain RNA-binding proteinsHow multidomain RNA-binding proteins recognize their specific target sequences, based on a combinatorial code, represents a fundamental unsolved question and has not been studied systematically so far. We focus on a prototypical multidomain RNA-binding protein, IMP3 (also called IGF2BP3), which contains six RNA-binding domains (RBDs): four KH and two RRM domains. We have established an integrative systematic strategy, combining single-domain-resolved SELEX-seq, motif-spacing analyses, in vivo iCLIP, functional validation assays, and structural biology. This approach identifies the RNA-binding specificity and RNP topology of IMP3, involving all six RBDs and a cluster of up to five distinct and appropriately spaced CA-rich and GGC-core RNA elements, covering a >100 nucleotide-long target RNA region. Our generally applicable approach explains both specificity and flexibility of IMP3-RNA recognition, allows the prediction of IMP3 targets, and provides a paradigm for studying the function of multidomain RNA-binding proteins in gene regulation.
RNAs in extracellular vesiclesCircular RNAs are particularly abundant in human platelets (thrombocytes), which play important roles in haemostasis, wound healing, inflammation, and cancer metastasis. We demonstrated that there is a selective export of circRNAs in extracellular vesicles (both exosomes and microvesicles) released from activated platelets, suggesting specific sorting mechanisms.
What are the RNA sequence and factor determinants for such specific release within vesicles? What are the biological functions of vesicle-mediated transfer of RNAs, including circRNAs to target cells? These studies should also extend the potential of circRNAs as prognostic and diagnostic biomarkers.
Alternative splicing in the human system, splice defects and diseaseThe human genome project has revealed that the complexity of gene expression in the human system is not determined by the gene number, which is -compared with other organisms- relatively low. Instead, posttranscriptional processes such as alternative splicing play an important role and multiply the diversity of the proteome. Most human protein-coding genes undergo alternative RNA processing and yield functionally multiple proteins of diverse functions, regulated in a cell type-, tissue- and development-specific manner. Furthermore, splicing defects caused by mutations represent an important disease mechanism in the human system. Alternative splicing is determined by the combinatorial control of a relatively small set of splicing regulator proteins.
To investigate molecular mechanisms of alternative splicing, we apply genomewide approaches, combining RNA-Seq with SELEX (Systematic Evolution of Ligands by EXponential Enrichment) and iCLIP strategies (individual CrossLinking-Immunoprecipitation) to analyze in vitro and in vivo RNA-binding characteristics. The overall goal is to functionally characterize RNA-binding proteins and to define regulatory networks between factors and targets, which is also of relevance to molecular medicine.