Hi! You reached the research group of Jernej Ule, based at The Francis Crick Institute and the Department of Molecular Neuroscience at the UCL Institute of Neurology.

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    Why RNA?

    A gene needs to express itself in order to contribute to cellular functions. This requires information from DNA to be transcribed into an RNA molecule. RNAs are never naked in the cells; as soon as an RNA is transcribed, it becomes coated by proteins. The RNA molecule then passes through many stages of processing, quality control, transport and regulation. These stages are coordinated by protein-RNA complexes, which form when the proteins wrap around the RNA. Often these complexes involve more than one RNA, and dozens, if not hundreds of proteins.

    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 protein-RNA complexes. 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 protein-RNA complexes.

    Cells can change their gene expression by modulating the function of protein-RNA complexes. Moreover, genetic studies have identified mutations that disrupt the normal function of protein-RNA complexes. 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 protein-RNA complexes, 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 protein-RNA complexes, and thereby coordinate RNA processing and regulation?

    2) How does evolution tinker with the RNA regulatory circuits? What is the role of transposable elements and non-canonical splicing in evolution?

    3) How do protein-RNA complexes modulate the functions of neurons or glial cells during brain development, aging or neurodegenerative diseases?

    4) How do mutations cause disease by disrupting the function of protein-RNA complexes, and what treatments could ameliorate this?

    And here are some of the RNA stories that we have passed through:

    Understanding protein-RNA complexes.

    Techniques to identify RNA binding sites.

    Methods for nucleotide-resolution studies of protein-RNA interactions We developed individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) to quantify protein-RNA interactions in the whole transcriptome. We We described the details of the iCLIP method, presented it in a video, and reviewed the transcriptomic techniques for studies of protein-RNA interactions. We also demonstrated that the cDNAs in iCLIP truncate at crosslink sites, and developed computational methods based on cDNAs-starts for analysis of iCLIP data.

    RNA maps: how does the location of RNA binding site guide its function?

    Image of the decision-making process of the splicesome as it scans the pre-mRNA for exons, and the role of RNA-binding proteins in modulating this process. We integrate transcriptomic data on protein-RNA interactions and their function, which can tell us how ribonucleoproteins (protein-RNA complexes) 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.

    Understanding the regulation and function of cryptic splicing elements.

    NRG cover 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 is from the cover of the journal.

    Alu-derived exons

    By identifying RNA binding sites of hnRNP C across the transcriptome with iCLIP), we have shown that hnRNP C binds to long uridine tracts, and thereby regulates splicing of alternative exons.

    We have found that hnRNP C can strongly repress inclusion of exons that are derived from Alu elements, or so-called Alu-exons. Alu retrotransposable elements are specific for primate genomes, and they have probably played an important role in the evolution of primates, since they constitute 10% of the human genome. hnRNP C represses recognition of cryptic splice sites in Alu elements by displacing the splicing factor U2AF65 from the uridine tracts. Loss of hnRNP C leads to formation of thousands of harmful exons, and mutations within uridine tracts in Alu elements can cause many human diseases.

    Co-evolution of positive and repressive regulatory forces in Alu elements. We have shown that the positive and negative regulatory forces are tightly coupled in the evolution) of Alu-exons. In species where mutations made the splice sites of Alu-exons stronger, the uridine tracts are longer, which allows hnRNP C to act as a counteracting force. This allows the Alu-exons to remain in a harmless cryptic state over long evolutionary periods, during which they accumulate additional mutations. We hypothesise that the repressive function of hnRNP C prevents the damaging effects of immediate Alu exonization, and the length of uridine tracts represents a ‘molecular rheostat’. After Alu-exons accumulate lots of mutations, the uridine tracts gradually shorten, and as a result the Alu-exons start escaping from repression, and contributing to new cellular functions. We hypothesise that the uridine tracts in Alu elements can buy the time needed for mutations to make beneficial changes, rather than disruptive ones, during the evolution of a species.
    Artistic rendering of ultraviolet crosslinking of the hnRNP C1/C2 tetramer interacting with pre-mRNA.

    Recursive splicing in long introns

    recursive splicing Long 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.

    Understanding the secondary structure of mRNAs.

    Lines between the two arms of RNA duplexes 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.

    Understanding the mechanisms of protein-RNA complexes in brain aging and neurodegeneration.

    protein-RNA complexes play many important roles in the brain by regulating gene expression in many different ways, including alternative splicing, RNA transport and translation. We study the regulatory networks controlled by TDP-43 and FUS), two RNA-binding proteins that can cause amyotrophic lateral sclerosis when mutated. We showed that both proteins regulate alternative splicing of a functionally coherent set of transcripts, many of which encode proteins implicated in neurodegenerative disorders.

    We have examined changes in gene expression in human postmortem brain samples to compare the effects of healthy aging with two neurodegenerative diseases: Alzheimer's disease and frontotemporal lobar degeneration (FTLD). We observed widespread changes in alternative splicing: most were specific to diseased samples, but some were common to aging and disease. Especially the changes in glial-specific genes appeared to be shared, with the decrease in oligodendrocyte-specific genes being most apparent. Therefore, we further analysed gene expression in three large cohorts of samples to examine changes in ten brain regions upon aging. Stratifying the gene expression by cell type, we found that astrocytes and oligodendrocytes diminish their regional identity upon aging. We also developed a machine learning method to analyse high-resolution images of brain sections, and applied it to a more limited number of samples. This indicates that the number of oligodendrocytes decreases, which less change is seen in the total number of neurons upon aging. However, the neurons with largest cell bodies appear to also decline in their number. On the other hand, we find dramatic increase in the expression of microglia and endothelial-specific genes in all brain regions upon aging. We hope that these findings will be of use for further studies of the cellular phase of aging and the Alzheimer's Disease.