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 RNPs regulate the life cycle of mRNAs in neurons, and how this can go wrong in neurologic diseases. To study the assembly of RNPs, we obtain detailed maps of protein-RNA binding sites by using transcriptomic techniques. For this purpose, we developed the nucleotide-resolution UV crosslinking and immunoprecipitation (iCLIP) (1,2), which identifies protein-RNA contacts by using a series of steps, as described in the figure above. We are further developing similar methods, as well as computational tools to interpret the high-throughput sequencing data (3-5). Thereby, we gain a comprehensive view of RNP assembly and dynamics within intact cells.
Regulation of pre-mRNA processing
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. In this context, we mainly focus on the roles of RNPs in the regulation of splicing, as well as cleavage and polyadenylation. We demonstrated that most RNPs regulate RNA processing according to positional principles, which can be visualised as RNA maps (1,6-9). We showed that these positional principles can be combined with the analysis of multivalent sequence motifs in order to detect functionally important binding sites (8,9). This approach helped us to understand combinatorial regulatory mechanisms, and to predict the likely functions of protein-RNA binding sites.
The structure of RNPs
We developed a new method, termed hybrid iCLIP (hiCLIP) (3), 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.
Regulation of repetitive and non-canonical RNA elements
One of the major surprises of our iCLIP studies was the major role that transposable elements (TEs) play as hubs for RNP assembly (10,11). We have found that TEs are a rich source for cryptic sites of RNA processing, and therefore regulatory RNPs need to assemble on thousands of TEs in order to repress their processing. By analysing the evolutionary sequence variations in TEs, we found that changes in RNP assembly drive the emergence of new tissue-specific exons. This also provided insights into the way that mutations can disrupt RNP assembly to cause diseases (10-12). Moreover, we uncovered recursive splice sites in the longest introns of human genes that are expressed in the brain, thus indicating a role for non-canonical splicing events in human transcripts (13). 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 might contribute to human diseases.
RNPs in motor neuron disease
We study how RNPs regulate gene expression in neurons and glial cells during neuronal differentiation, aging, and diseases. We focus on Amyotrophic lateral sclerosis (ALS), a disease affecting motor neurons that is often caused by mutations that perturb the function of RNPs. We monitor the assembly and dynamics of RNPs involved in ALS in order to better understand how treatments could ameliorate the effects of these mutations. We have studied three RBPs, TDP-43, MATR3 and FUS, which are target of mutations causing ALS (7,9,14,15). We now work on the function of the intrinsically disordered regions of these RBPs, since most disease-causing mutations are located in these regions. We use mouse and human pluripotent stem cells as a model system to study how mutant RNA-binding proteins affect RNP assembly and function at specific stages of motor neuron differentiation.
1. Konig et al, Nat Str Mol Biol, 2010
2. Lee et al, Mol Cell, 2018
3. Sugimoto et al, Nature, 2015
4. Sugimoto et al, Gen Biol, 2012
5. Haberman et al, Gen Biol, 2017
6. Wang et al, Plos Biol, 2010
7. Tollervey et al, Nat Neuro, 2011
8. Cereda et al, Gen Biol, 2014
9. Rot et al, Cell Rep, 2017
10. Zarnack et al, Cell, 2013
11. Sibley et al, Nat Rev Gen, 2016
12. Attig et al, eLife, 2016
13. Sibley et al, Nature, 2015
14. Soreq et al, Cell Rep, 2017
15. Tollervey et al, Gen Res, 2011