Laboratory of Molecular Neuroscience
The primary task of the nervous system is to process and store information, and we study how this is achieved at the level of RNA molecules. The nervous system contains many types of neurons and glia, which are highly polarized, with diverse shapes and distinct cellular compartments. Messenger RNA (mRNA) carries the genetic information from DNA to the machinery in our cells that makes the proteins. Often, specific mRNAs need to be localized to distal neuronal compartments, such as the axon growth cone or the dendrites, before they can be translated into proteins. As an mRNA travels through the different cellular compartments, it passes through several regulatory stages. These stages are controlled by RNA-binding proteins (RBPs) and non-coding RNAs (ncRNAs), which assemble on the mRNA into a regulatory ribonucleoprotein complex (RNP). In recent years, we showed how the position of RBP-RNA interaction determines its effects on alternative splicing, characterised the function of several RBPs that are implicated in neurologic diseases, and revealed a mechanism that controls the emergence of new exons from transposable elements.
We study the structure and function of regulatory RNPs in the brain. Each RNP in the cell has a unique structure, which depends on the sequence-specific interactions between mRNA, RBPs and ncRNAs. Moreover, the RNP structure is highly dynamic, since its composition can change in response to cellular signals, such as the signals that may initiate neurodegenerative diseases. To fully understand the dynamic nature of RNPs, we study them within intact cells using innovative genomic techniques. We developed iCLIP, which we now use to study RNPs in brain tissue or in pluripotent stem cells that are differentiated into specific neuronal or glial cell types. We integrate genomic, biochemical and computational techniques to uncover the importance of RNP structure for neuronal biology and neurologic diseases. Specifically, we aim to:
1) Determine how the structure of regulatory RNPs instructs their function in brain development and disease.
2) Understand how regulatory RNPs respond to cellular signals, in particular the signals that affect neurons during the initial stages of neurodegenerative diseases, with the primary focus on motor neuron disease.
3) Define how mutations can either drive evolution of mammalian brain, or cause neurologic diseases, by modifying the regulatory RNPs.
As an RNA passes through the cellular regulatory stages, it is like a character from Mozart's Magic Flute, passing through the ordeals of space and time. And here are some of the RNA stories that we have passed through:
New technologies for studies of protein-RNA interactions.
We developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) to quantify protein-RNA interactions in the whole transcriptome, thereby fully exploiting the power of high-throughput sequencing. We review the progress made in the last years in the technologies for studies of protein-RNA interactions. You can download the manuscript here. We also performed a comparative analysis of iCLIP and CLIP, click here.
RNA Analysis Unearths Invaluable Insights
Published iCLIP data
All published iCLIP sequencing data are available both as raw format (fastq file), as well as processed format on the public server iCount.
Question-answer forum on the iCLIP method
Two journals dedicated an issue in 2014 to RNA-binding proteins. An issue of Genome Biology is dedicated to RBPome, and an issue of Methods is dedicated to protein-RNA interactions, both including manuscripts on CLIP and related experimental and computational methods.
Introns and evolution
In collaboration with the Nick Luscombe lab, we have discovered a major new role for the RNA-binding protein hnRNP C. We have originally shown that hnRNP C specifically recognizes long uridine tracts, and can thereby repress splicing of exons. This was evident by the ultraviolet crosslinking of the hnRNP C1/C2 tetramer, which suggested that the repressed exons are incorporated into the hnRNP particles (see the paper).
We later showed (see the paper) that hnRNP C controls the emergence of new exons from Alu elements, which are retrotransposable elements that are specific for primate genomes, and constitute 10% of human genome. hnRNP C represses recognition of cryptic splice sites in Alu elements by displacing the splicing factor U2AF65 from uridine tracts. Loss of hnRNP C leads to formation of thousands of harmful exons, and mutations disrupting hnRNP C binding cause human diseases. Since the repressive function of hnRNP C prevents the damaging effects of immediate Alu exonization, it enables mutations to gradually create Alu-derived exons. This represents an elegant molecular mechanism that could mediate incremental evolution of new cellular functions.
Kathi Zarnack (dry lab), Julian König (wet lab), Mojca Tajnik, Iñigo Martincorena
Nature Reviews Genetics on "Regulating Alu element exonization"
LMB news on "The guardian of the transcriptome"
Sixty years of genome biology
In a recent commentary, many researchers found the discovery of introns our biggest inspiration, especially since this uncovered the unforeseen evolutionary potential of our genome.
Understanding how RNPs regulate pre-mRNA processing.
We use an integrative genomic approach to uncover how RBP regulate their target transcripts. We use methods such as iCLIP to assess where an RBP binds its target transcripts, and integrate this with methods such as RNA-seq that assess how this RBP controls pre-mRNA processing. This approach revealse that most RBPs regulate alternative splicing according to genome-wide positional principles, or RNA splicing maps. For instance, by integrating TIA iCLIP with splicing analysis upon TIA knockdown, we were able to derive nucleotide-resolution RNA splicing maps of TIA proteins. Moreover, we developed software (RNAmotifs) that can derive RNA splicing maps by analysis of multivalent RNA motifs that are often bound by RBPs.
Zhen Wang, Matteo Cereda, Gregor Rot, Melis Kayikci, Kathi Zarnack
RNA map gives first comprehensive understanding of alternative splicing
Understanding the role of RNPs in brain function and disease.
Alternative splicing can produce several mRNA isoforms from a gene, and these isoforms can change in the human brain during aging or neurodegeneration (click here). In collaboration with the Chris Shaw lab (KCL), we uncovered the regulatory networks controlled by TDP-43 and FUS. Mutations in these two RBPs can cause amyotrophic lateral sclerosis, therefore it is important to understand their functions in the brain. We showed that TDP-43 binds to long clusters of UG-rich RNA motifs to recognise specific sites on pre-mRNAs and thereby regulate splicing. Moreover, TDP-43 increases its interactions with specific non-coding RNAs in diseased brain, click here. Surprisingly, FUS and TDP-43 rarely regulate splicing the same exons. FUS has little specificity for RNA sequence or structure, and binds across the whole pre-mRNA, with enriched binding to introns flanking the regulated exons. Nevertheless, both proteins regulate a functionally coherent set of transcripts, many of which encode proteins implicated in neurodegenerative disorders (click here). For a review of this field, click here
James Tollervey, Boris Rogelj, Rickie PataniTomaz Curk, Michael Briese
CLIPs of TDP-43 Provide a Glimpse Into Pathology, Alzheimer Research Forum
FUS and Friends: Two Studies Probe FUS’ RNA Partners
New Link Revealed Between Alzheimer's Disease and Healthy Aging, Science Daily
An issue of Frontiers in Neuroscience dedicated to mRNA life cycle in the brain includes review articles covering the many fascinating functions of mRNA regulation in the brain.