A gene needs to express itself in order to contribute to cellular functions. This requires information from the gene to be transcribed from DNA into an RNA molecule. Upon its transcription, each RNA molecule undergoes processing, such as splicing and 3’ end processing, and passes through many stages of quality control and regulation. The coordinators of these stages are ribonucleoprotein complexes (RNP), which form when multiple proteins bind to an RNA molecule.
We develop techniques that integrate biochemistry and computational biology to obtain a comprehensive map of interactions between a specific protein and its RNA partners within our cells. We developed the individual-nucleotide resolution UV crosslinking and immunoprecipitation of protein-RNA complexes (iCLIP), and a related method called hiCLIP, which reveals the higher-order conformation of RNPs.
We use these methods in collaboration with the group of Nicholas Luscombe to study how the sequence and structure of RNAs defines the composition and function of RNPs.
Cells can change their gene expression by modulating the function of RNPs. Moreover, genetic studies have identified mutations that disrupt the normal function of RNPs. These mutations often cause neurologic diseases, particularly the motor neuron disease, also referred to as amyotrophic lateral sclerosis (ALS).
We study this disease in collaboration with the group of Rickie Patani by using induced pluripotent stem cells with specific genetic mutations, and differentiating them into motor neurons. We wish to understand how these mutations affect the assembly of RNPs, thereby initiating the molecular cascade leading to disease. We study the following questions:
1) How do RNA-RNA and protein-RNA contacts define the assembly of RNPs, and thereby coordinate RNA processing and regulation?
2) How do RNPs modulate neuronal functions in response to neuronal differentiation or synaptic activity?
3) How does evolution tinker with the RNA regulatory circuits? What is the role of transposable elements and non-canonical splicing in evolution?
4) How do mutations cause disease by disrupting the function of RNPs, and what treatments could ameliorate this?
And here are some of the RNA stories that we have passed through:
Understanding the function of protein-RNA binding sites.
Techniques to identify RNA binding sites.We developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) to quantify protein-RNA interactions in the whole transcriptome. 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.
RNA maps: how does the location of RNA binding site guide its function?We integrate transcriptomic data on protein-RNA interactions and their function, which can tell us how ribonucleoproteins (RNPs) assemble at specific positions on their target transcripts and thereby regulate alternative splicing, mRNA decay or translation. For example, we used iCLIP to assess where an RBP binds its target transcripts, and RNA-seq to assess how this RBP controls pre-mRNA processing. This approach revealed that most RBPs regulate alternative splicing according to genome-wide positional principles, or RNA splicing maps. For example. 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, Julian König, Kathi Zarnack, Nejc Haberman, Jan Attig
RNA map gives first comprehensive understanding of alternative splicing
Understanding the regulation and function of cryptic splicing elements.We reviewed the studies that mapped the functional binding sites of RNA-binding proteins across the transcriptome, which have uncovered an unprecedented diversity of previously unknown non-canonical splicing events (check our review). These studies identified many cryptic events located far from the currently annotated exons and unconventional splicing mechanisms that have important roles in regulating gene expression. These non-canonical splicing events are also a major source of newly emerging transcripts during evolution, especially when they involve sequences derived from transposable elements. They are therefore under precise regulation and quality control, while mutations in these elements can disrupt gene expression and lead to diseases. Image on the right shows the cover, inspired by our review.
Alu-derived exonsBy identifying RNA binding sites of hnRNP C across the transcriptome, we have shown that hnRNP C specifically recognizes long uridine tracts, and can thereby repress splicing of alternative exons. This was evident by the ultraviolet crosslinking of the hnRNP C1/C2 tetramer, which demonstrated that hnRNP C forms higher-order complexes that bind across the repressed exons (see the paper). This uncovered a major role for hnRNP C in the repression of cryptic splicing elements(see the paper). We found 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.
Julian König, Kathi Zarnack, Mojca Tajnik, Jan Attig, Igor Ruiz de los Mozos, Federico Agostini
Regulating Alu element exonization
The guardian of the transcriptome
Recursive splicing in long intronsLong introns contain hundreds of so-called ‘cryptic sequences’ that appear very similar to exons, but are not supposed to be used. The cellular machinery faces great challenges in distinguishing true exons from these cryptic sites. We found that cells sometimes select a cryptic exon that is present deep within a long intron, but later discard it, in a process called recursive splicing (see the paper here). Normally recursive exon removes this cryptic exon, allowing it to remain invisible. However, if the recursive site is preceded by other cryptic splicing events, then the exon is not removed – creating a ‘binary switch’ or checkpoint that can distinguish correct splicing events from the newly emerging cryptic events, which could be potentially damaging. Thus, long introns on one hand enable emergence of many cryptic splicing events during evolution, whereas recursive splicing ensures that this evolutionary tinkering does not disturb the primary mRNA that needs to be made from the gene. We observed this process happening in some of the longest genes that are expressed in human brain, which are often implicated in autism or other neurodevelopmental disorders.
Chris Sibley, Warren Emmett, Lorea Blazquez, Andrea Elser
A new genetic switch uncovered in the long genes expressed in our brain
Splicing does the two-step
In a commentary, many scientists express fascination by introns.
Understanding the secondary structure of full-length mRNAs, and its role in RNP assembly.The secondary structure of mRNAs has important effects on its stability and translation. To understand the in vivo structure of full-length mRNAs, developed a technique called hiCLIP to identify the connections that hook sections of an mRNA together, which are called RNA duplexes. We were amazed to find that mRNAs form thousands of such duplexes, and often these duplexes hook together very distant parts of mRNA molecules. We found that that these duplexes interact with the double-stranded RNA binding protein Staufen 1. We also found that these RNA duplexes have less genetic variation in humans than surrounding areas of the mRNA, indicating that mutations could cause disease by disrupting the structure of mRNAs. See the paper here.
Yoichiro Sugimoto, Christina Militti, Flora Lee
Structure of genetic messenger molecules reveals key role in diseases
hiCLIP: New method finds structures of mRNA molecules
Detailed probing of RNA structure in vivo
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). Moreover, we uncovered the regulatory networks controlled by TDP-43 and FUS, two proteins can cause amyotrophic lateral sclerosis when mutated. We showed that both proteins regulate a functionally coherent set of transcripts, many of which encode proteins implicated in neurodegenerative disorders click here or here).
James Tollervey, Boris Rogelj, Rickie Patani, Michael Briese, Lilach Soreq, Claire Hall, Martina Halleger, Frederique Rau, Julian Zagalak
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.