Genomic views of the RNA world
Posttranscriptional regulation of gene expression plays an important role in development and tissue identity but is also implicated in the aetiology of diseases such as cancer. Regulation is achieved by the cooperative action of RNA-binding proteins (RBPs) that recognise RNA sequences in the pre-mRNA and modulate the activity of cellular machineries such as the ribosome and the spliceosome. The information in the RNA sequence and how it is interpreted by RBPs is commonly referred to as the mRNP code (messenger ribonucleoprotein code). Our vision is to crack this mRNP code and to obtain mechanistic insights into the regulation of splicing and translation.
Understanding the mRNP code requires profound knowledge about the interplay of cis-regulatory RNA elements, trans-acting RBPs and large cellular machineries, like the spliceosome. To build this knowledge, our group has achieved three major goals:
(1) In order to study the intrinsic RNA binding activity of RBPs, we established in vitro iCLIP experiments, in which recombinant RBPs are incubated with long transcripts. This approach allowed us to study how the core splicing factor U2AF2 recognises RNA sequences. By measuring its affinity at hundreds of binding sites and comparing the in vitro and in vivo binding landscapes, we found that trans-acting RBPs extensively regulate U2AF2 binding in vivo. The most prevalent regulatory mechanisms include enhanced recruitment to 3' splice sites and clearance of introns. Using machine learning, we identified and experimentally validated novel trans-acting RBPs (including FUBP1, CELF6 and PCBP1) that modulate U2AF2 binding and affect splicing outcomes. (Sutandy et al., Genome Res, 2018)
(2) We developed a high-throughput screen of randomly mutated minigenes to decode the cis-regulatory landscape that determines alternative splicing of the proto-oncogene MST1R (RON). Mathematical modelling of splicing kinetics enabled us to identify more than 1,000 mutations affecting RON exon 11 skipping, which corresponds to the pathological isoform RON∆165. Importantly, the effects correlate well with RON alternative splicing in cancer patients bearing the same mutations. (Braun et al., Nat Commun, 2018)
(3) Using a broad spectrum of proteomics and functional genomics approaches, we investigated the RNA binding behaviour as well as the protein interaction partners and ubiquitylation targets of the RNA-binding E3 ubiquitin ligase MKRN1. This identified its role as a new sensor of ribosome-associated quality control (RQC). Based on these results, we are currently investigating the molecular mechanism of MKRN1 action and its physiological relevance for quality maintenance. (Hildebrandt et al., bioRxiv, 2019)
The key technologies established in our laboratory, including iCLIP, in vitro iCLIP and high-throughput screens, form the basis for several ongoing research projects in the lab, including mRNP assembly at splice sites, alternative splicing in paediatric leukaemia,the evolution of human splicing patterns, translational regulation, the impact of RNA modifications on translation, intrinsically disordered protein regions and phase separation.