The Journey of an mRNA
In multicellular organisms, most genes are transcribed into a pre-mRNA that needs to be cut into pieces before a mature mRNA is made. Some of these pieces (introns) are discarded, whereas others (exons) are put together into the mRNA. The choice of the pieces that end up as exons is regulated via alternative splicing and 3' end processing. Cells can produce multiple mRNA isoforms from a single gene, which increases the variety of proteins made from a finite number of genes. This helps the cells to follow multiple developmental pathways, since different cell types can produce different mRNA isoforms from the same gene. Cells can also control where and when protein is made from an mRNA molecule. In neurons, synaptic activity can induce local translation of mRNAs, which leads to long-term synaptic plasticity, and thereby enables us to retain memories.
During its life cycle, the mRNA interacts with a variety of RNA-binding proteins (RBPs) and non-coding RNAs. Together with these interacting partners, each mRNA assembles into a dynamic ribonucleoprotein complex (RNP), which changes in its composition as the mRNA moves within the cell. The structure of the RNP controls the alternative pre-mRNA processing, mRNA localisation, translation and degradation. Defects at any of these stages can lead to human diseases. Each mRNA assembles into a regulatory RNP with a unique structure and dynamics, and therefore systems biology approaches are required to understand the assembly and function of these regulatory RNPs.
The goal of our research group is to reveal how protein-RNA and RNA-RNA interactions regulate the life cycle of mRNAs during neuronal development, and how this can go wrong in the process leading to neurodegenerative diseases. We employ experimental and computational techniques to determine the structure and function of ribonucleoprotein complexes (RNPs). We developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) to investigate protein-RNA interactions in intact cells or tissues. iCLIP enables to map all RNA binding sites an RBP, which revealed intricate interplay between RBPs and their target mRNAs: even though RBPs control the life cycle of an mRNAs, it is the sequence, position and structure of each RNA-binding site that guides the effect of the RBPs. Thus, by mapping the interactions between RBPs, non-coding RNAs, pre-mRNAs and mRNAs, we can understand the combinatorial action of these diverse components of RNPs. To study how RNPs contribute to motor neuron disease, we model the disease using induced pluripotent stem cells.
Regulation of pre-mRNA processing
Many different RBPs interact with nascent transcripts to regulate splicing, as well as cleavage and polyadenylation. We study the functions of RBPs that regulate both alternative splicing and alternative polyadenylation in order to understand if these two aspects of RNA processing are co-ordinately regulated. We particularly focus on RBPs such as TDP-43 and FUS that are target of mutations causing motor neuron disease. We study the function of the low-complexity regions in these RBPs, which are poorly understood, even though most disease-causing mutations are located in these regions.
Non-coding RNA elements as a source of new gene functions
With iCLIP and related methods, we can rapidly characterize the functions of non-coding elements of human genome that interact with RBPs, and are important for regulation of RNA processing. We study conserved cryptic processing sites, such as recursive splice sites, as well as the highly variable sites derived from transposable elements or microsattelites. We wish to understand how variation in these elements across species, individuals and somatic tissues leads to changes in RNPs assembly and RNA regulation to facilitate evolutionary exploration of new gene functions. Moreover, we study how polymorphisms in these elements contribute to human diseases.
RNA-protein interactions in disease
We use induced pluripotent stem cells as a model system to study RNA-dependent mechanisms involved in neuronal development and disease. We study iPSCs derived from patients with neurodegenerative conditions, in particular ALS. This enables us to examine how defects in protein-RNA complexes emerge at a specific stage of iPSC differentiation.
The structure of RNPs
We developed a new method, termed hybrid iCLIP (hiCLIP), which identifies RNA-RNA hybrids that are bound by double-strand RNA-binding proteins (dsRBPs). This identifies both the RNA-RNA hybrids that form between different regions of the same mRNA, as well as interactions between different RNAs, such as long-noncoding RNAs and mRNAs. We particularly wish to understand the importance of RNP structure for translational regulation.