Project 1 hosted by the Rajewsky lab at the MDC, Berlin, Germany
Single cell circRNA expression analysis by total RNA sequencing
Objectives: From our single molecule imaging data (unpublished) we have noticed that CDR1as, one of the most well-known circRNAs, has variable expression across cells. At any point in time, some cells are devoid of CDR1as molecules while others express many. This observation makes it interesting to investigate circRNA expression in single cells. We have recently successfully set up“Drop-Seq” in our lab, a microfluidics based method that allows sequencing of substantial parts of the individual transcriptome of thousands of cells in little time. For this purpose, ribosomal RNA must be either depleted or omitted. Several strategies will be tried to achieve this task. Moreover, circRNAs in the brain usually have highly specific expression patterns. Quantifying circRNAs expression in neuronal cells from primary tissues would therefore benefit from single cell analysis (WP4).
Expected Results: Development of a method for total RNA sequencing in single cells that allows circRNA quantification. If successful, this would open up many exciting possibilities, which are important beyond circRNAs. For example, many lincRNAs are not polyadenylated and can only be quantified via total RNA sequencing. The developed methods would be for obvious reasons of merit to the entire ETN consortium and could also be commercialized.
Secondments are planned with the Preibisch lab and qpa.
Project 2 hosted by the Rajewsky lab at the MDC, Berlin, Germany
Clustering analysis solutions for circRNA single cell sequencing
Objectives: Either by circRNAs-specific microarrays which we are currently developing in collaboration with qpa or by success of the ESR1 project, it will be clear that extraordinarily large numbers of samples will need to be clustered to identify circRNA biomarker candidates. This problem is also in general one of the most important upcoming problems in genomics because single sequencing data already generate tens of thousands of samples in as little as 1 hour (Drop-seq) and can easily be scaled up to sequence practically unlimited numbers of cells. On the other hand, available clustering algorithms in biology are not able to deal with these numbers- they compare each sample to all other samples and therefore scale O (N**2) with the number N of samples. In practice, it is already very difficult for a normale compute-cluster in a top life science institute to perform the clustering of the 45,000 cells published by Macosko et al. Here the aim would be to read the literature on other fields where similar problems have been encountered and heuristic based methods have been implemented and tested. For example, in astronomy millions of digital spectra are recorded and need to be clustered to give raise to “maps” of stellar objects. An experienced PostDoc in the Lab, Dr Karaiskos, is an expert in theoretical physics and would guide the student through this project. The student would then focus on implementing heuristic solutions and testing them on our data.
Expected Results: Implementation of heuristic solutions to the problem of clustering large numbers of data points from single cell sequencing analyses. The results would be important beyond circRNAs for transcript analysis in general. Again, the developed methods would be of merit to the entire consortium and might be commercialized by licensing.
Secondments are planned with Qiagen Aarhus.
Project 3 hosted by the Rajewsky lab at the MDC, Berlin, Germany
Surface-based circRNA detection for analytics and diagnostics
Objectives: Establishment of surface-based semi-quantitative assays, well plate- and microarray-based, for the detection of disease related circRNA expression patterns, novel biomarkers and comparison between circRNA and linear isoforms, allow analysing diverse clinically relevant specimens in neuronal disease (tissue biopsies, blood etc.)
Expected Results: Proof-of-Concept for surface-based assays using well-established read-out systems and evaluation software for semi-quantitative circRNA profiling with specific plates/chips for the detection of ALS/TLE/SD/BD related expression patterns from total RNA extracts. Systematic comparison to sequencing data.
Secondments are planned with qpa.
Project 4 hosted by the Preibisch lab at the MDC, Berlin, Germany
In-toto single molecule circRNAs imaging & characterization
Objectives: 1. Establish single-molecule (sm) circRNA imaging; 2. Automated imaging of sm-circRNA expression and and statistical analysis describing its regulation 3. sm-circRNA imaging of large cleared samples using lightsheet microscopy and big-data analysis.
Expected Results: Methods to robustly detect single circRNA molecules using automated microscopy and software methods capable of acquiring and analyzing large amounts of image data automatically. Imaging of specific transcript candidates (Irimia & Rajewsky) characterizing regulation of circRNA expression by comparative model-based analysis. Development of methods for fixation and clearing of mouse brain tissue, adaption of sm-circRNA imaging using lightsheet microscopy, and development of software infrastructure to efficiently handle the datasets.
Secondments are planned with Bioneer, and the Irimia and Pasterkamp labs.
Project 5 hosted by the Bindereif lab at the Justus-Liebig University, Giessen, Germany
Identification and characterization of circRNA-protein complexes (circRNPs)
Objectives: 1. Biochemical identification of circRNA-protein complexes (circRNPs) in human cells. CircRNPs will be biochemically identified from a human cell line (HeLa) as well as from two primary cells (human platelets; HUVEC cells). In each case, lysates will be prepared, followed by sucrose gradient centrifugation, fractionation, and RT-PCR analysis for circular and linear splice isoforms. In addition, special conditions will be applied to fractionate polysome-associated RNAs, to systematically search for potentially translated circRNAs. Protein components of circRNPs will be determined by two means: First, gradient centrifugation and affinity antisense selections will be combined, followed by mass spectrometry of protein factors associated with specific circRNAs (in collaboration with H. Urlaub, MPI Gö̈ttingen) and validation by immunoprecipitation. Second, we have already established a procedure to identify classes of circRNPs associated with a certain protein component (manuscript in preparation): This is based on specific immunoprecipitation from cell lysates, followed by RNase R treatment (to enrich for circRNAs), RNA-Seq analysis, bioinformatic circRNA identification, and validation. 2. Functional analysis of protein components of human circRNPs. To address the functional role of protein factors identified (see part 1) in biogenesis they will be RNAi-downregulated in HeLa cells, and the effects of knockdowns on processing (linear versus circular isoforms; RT-PCR) and cellular localization (cell fractionation and RT-PCR; FISH assays) will be characterized. 3. Are certain circRNAs translated into peptides or proteins? We will use the approach described above under #1 (HeLa and HUVEC cells, human activated platelets) to enrich for translationally active mRNAs and circRNAs, followed by RNA-Seq analysis of polysome fractions of different size classes. This will yield candidate circRNAs to be further analyzed for translation activity on the protein level.
Expected Results: 1. Characterization of selected circRNPs from several human cell types in terms of size and protein components. 2. Insights into circRNP functions, in particular the functional contributions of specific protein components to cellular biogenesis and processing. 3. Assessment of translational potential of human circRNAs.
Secondments are planned with the Rajewsky, Kjems and Bozzoni labs as well as qpa.
Project 6 hosted by the Bindereif lab at the Justus-Liebig University, Giessen, Germany
Extracellular-vesicle-mediated transfer of human circRNAs
Objectives: 1. Identification of circRNAs in human platelets and platelet-derived extracellular vesicles. Platelets play central roles during blood coagulation and during cardiovascular disease; in addition, they are currently discussed as novel diagnostic tumor markers. CircRNAs are relatively abundant in platelets, compared with various hematopoetic cell types. Under activation conditions, platelets release extracellular vesicles (exosomes, microvesicles). Using platelet preparations we will establish circRNA catalogs from platelets as well as from platelet-derived vesicles. 2. Functional analysis of circRNA transfer via platelet-derived vesicles. Since extracellular vesicles may transmit macromolecules including RNA to target cells in the endothelium, we plan to characterize to what extent circRNAs may participate in signal transduction. CircRNA transfer from platelets and platelet-derived vesicles to primary HUVEC cells will be characterized in vitro with the aim to quantitatively study the transfer and to reveal novel biological functions of circRNAs (such as during shear stress of HUVEC cells). 3. Validation of circRNAs as biomarkers in cardiovascular disease. CircRNAs from human serum and from serum-derived exosome fractions will be analyzed and screened during cardiovascular disease states (in collaboration with Cardiology, Charité, Berlin), with the aim to derive diagnostic circRNA biomarkers.
Expected Results: 1. Establishment of validated circRNA catalogs from human platelets and platelet–derived vesicles. 2. Characterization of circRNA transfer from platelets/vesicles to endothelial cells. 3. Evaluation of circRNAs as potential new biomarkers for cardiovascular disease.
Secondments are planned with the Rajewsky and Kjems labs as well as Exosomics.
Project 7 hosted by the Bozzoni lab at the Sapienza University, Rome, Italy
circRNA function in muscle differentiation and in dystrophies
Objectives: In a screening for circRNA expression during in vitro differentiation of murine and human myoblasts Uniroma has characterized several species whose deregulation affects the correct conversion of precursor cells into mature myofibers. Among them a specific circRNA was identified that displays protein coding ability. The objective is: a) study circRNA function in muscle differentiation and in DMD and analyze whether circRNAs deregulated in Duchenne Muscular Dystrophy contribute to its pathogenesis; b) set up conditions for circRNA pull down and purification of interacting factors (RNA and protein); c) identify cis and trans acting factors required for translation of circRNA species and characterize the machinery required for proper translation of these uncapped and non-adenylated species; d) define whether the control of circRNA translation responds to specific signalling of either physiological (proliferation, differentiation) or pathological (stress, cell damage, apoptosis) nature. e) Identify the machinery responsible for circRNA turnover and the regulatory elements involved in the control of this process. A high throughput screening for RNAi against cellular endonucleases or RNA modifiers will be carried out in order to identify components involved in the control of circRNA (relating to WP1).
Expected Results: 1. Identify circRNAs with a relevant role in muscle differentiation and altered in DMD conditions. 2. Identify circRNP composition and the mode of action of the circRNAs under study.3. Identify the translation machinery required for circRNA translation.4. Identify components required for circRNA turnover.
Secondments are planned with the Kjems and Rajewsky labs and Qiagen Aarhus.
Project 8 hosted by the Bozzoni lab at the Sapienza University, Rome, Italy
circRNAs in neurodegenerative disorders
Objectives: Specific circRNAs have been recently characterized in human iPS-derived motoneurons which are deregulated in ALS motoneurons carrying mutations in the RNA binding protein FUS. The major objectives for ESR7 will be: a) to clarify whether FUS controls the biogenesis of circRNAs with a relevant function in motoneuron activity ; b) to determine whether deregulation of MN-specific circRNAs impact on ALS pathogenesis; c) to define circRNP composition and to get proteins and/or RNA interactors that might possibly help to identify the function of these molecules. Specific emphasis will be devoted to study the subcellular localization of MN-specific circRNAs in in vitro differentiated MN through in situ hybridization (ISH) and to analyse whether their localization is affected in FUS mutant cells. Along this direction ESR7 will exploit the PLA (proximal ligation assay) technology in order to specifically detect circRNA molecules, by using combinations of oligos complementary to the flanking regions of the back-splicing junctions.
Expected Results: 1. Identify circRNAs whose biogenesis is controlled by the RNA-binding protein FUS and test whether FUS mutations associated to ALS affect circRNA expression.2. Identify species that are altered in ALS MNs and test whether they impact on MN survival. 3. Identify protein/RNA interactors of MN-specific circRNAs and derive regulatory/functional processes in which they participate.
Secondments are planned Pasterkamp and Kadener labs as well as Bioneer.
Project 9 hosted by the Irimia lab at the CRG, Barcelona, Spain
Regulatory and functional interactions between alternative splicing, circRNAs and other unusual forms of splicing
Objectives: To investigate the extent of cross-talk between alternative splicing, circRNAs and other forms of unconventional splicing-related phenomena, and the potential regulatory and functional implications of this cross-talk.
Expected Results: 1. Computational pipeline to quantify circRNA formation and alternative splicing from RNA-seq data. 2. Catalog of exons and introns from the human genome involved in circRNA formation and regulated alternative splicing. 3. List of splicing factors involve in the co-regulation of both circRNA biogenesis and alternative splicing 4. Atlas of unconventional splicing-related phenomena in the human genome. 5. Integrated database for alternative splicing and circRNAs and other unconventional splicing-related phenomena.
Secondments are planned with Qiagen Aarhus and the Bindereif and Rajewsky labs.
Project 10 hosted by the Kadener lab at the Hebrew University, Jerusalem, Israel
Unraveling Molecular and Physiological functions of circRNAs
Objectives: Regulation of RNA metabolism is highly important in neurons and the brain contains the higher number and concentration of circRNAs. Interestingly, we have observed that downregulation of four different circRNAs leads to specific behavioral phenotypes. This project aims to unravel the mechanism of action of those specific circRNAs at the molecular level. In particular we hypothesize that some circRNAs might affect general RNA metabolism, in particular alternative splicing. We plan to determine general patterns of gene expression in our collection of 150 strains lacking specific circRNAs in the brain and determine their effect at the gene expression levels (including splicing) as well as on the behavioral level. To investigate the molecular of these physiological effects we will determine the composition of specific circRNPs by precipitation followed by mass spectrometry as well as the mechanism of translation of the subset which produce protein.
Expected Results: As a result of the proposed research we will: 1. Identify circRNAs that when modulate affect brain gene expression and behavior. 2. Determine their mechanism of action by identifiying the proteins bound to it, the role of these interaction for the circRNA activity as well as the effect on gene expression. 3. Determine mechanism of translation of ribosome-associated cirRNAs.
Secondments are planned with the Rajewsky and Irimia labs.
Project 11 hosted by the Kadener lab at the Hebrew University, Jerusalem, Israel
Relationship between neurodegeneration and circRNAs using Drosophila
Objectives: It has been demonstrated demonstrated that circRNAs accumulate with age, suggesting that their function could be related to age-related diseases. Furthermore, we found a number of genes that are involved in distinct neurodegenerative disorders (ATXN1, 2, 3, 7, and 10 as well as APP and PSEN1) host brain circRNAs and that several circRNAs are misregulated in the brain of Parkinson’s Disease patients as well as in a fly model of ALS. This project aims to address the role of circRNAs in the development of ALS utilizing Drosophila models of the disease. We will utilize two models: Overexpression of wild-type human TDP-43 (hTDP-43 model) and overexpression of a RNA molecule containing 288 GGGGCC repeats (C9orf72 model). With the help of these models we will perform a screening to determine downregulation of which circRNAs affect the development and progress of ALS-like symptoms in these flies. Moreover, we will determine how RNA splicing and translation are affected in these models, hoping to extend our findings to mammalian systems. As miss-RNA localization is a key landmark of ALS, we will determine putative roles of circRNA in RNA foci formation as well as determine the subcellular localization of ALS-relevant circRNAs.
Expected Results: As a result of the proposed research we will: 1. Identify circRNAs with altered expression in ALS fly models; 2. Determine alterations in RNA metabolism and translation in the fly ALS models; 3. Characterize the mechanism of action of ALS-relevant circRNAs.
Secondments are planned with the Bozzoni lab and Bioneer.
Project 12 hosted by the Kjems lab at the Aarhus University, Aarhus, Denmark
Function of differentially regulated circRNAs in mammalian brain development
Objectives: To elucidate the putative functions of tissue, developmental or disease specific circRNA candidates we will map them to sub-cellular regions in neurons by in situ hybridization (ISH) and in fixed brain sections from healthy and diseased brains from animals and humans. To obtain information about intracellular dynamics of circRNA, short fluorescent aptamer tags will be cloned into selected RNA candidates allowing real-time visualization in isolated neurons at UMC Pasterkamp. CircRNA depletion using RNAi or vector-based overexpression will be conducted to disclose downstream effect of circRNA expression/depletion. In vivo expression cassettes for circRNA overexpression or short-hairpin cassettes for RNA depletion will also be introduced to fetal mouse brain by in utero electroporation at UMC to observe the effect on the brain, e.g. by functional characterization of study synapse development, axon growth and guidance in vivo. Finally, selected candidates will be studied by genome targeting techniques (CRISPR/Cas) of splice sites or intronic motives important for circularization. Rescue expression of full-length and truncated RNA circles will be conducted to ensure that any obtained phenotype is circRNA associated and to determine the essential sequence elements within the circRNA involved. This approach will initially be exploited in ex vivo setups where known and expected effects based on the above depletion/overexpression analyses will be scrutinized.
Expected Results: 1. List of circRNA for which expression has an influence on neuronal development. 2. Cell lines with absent circRNA expression for functional studies. 3. Identification of proteins interacting with circRNA 4. Integration of selected circRNA in functional pathways.
Secondments are planned with the Pasterkamp and Bozzoni labs and Bioneer.
Project 13 hosted by Qiagen, Aarhus, Denmark
Brain tissue and biofluid associated circRNAs as biomarkers and causative agent in aging and neurological diseases
Objectives: Next generation sequencing (NGS) profiling of RNA in brain tissue and blood (exosomes) from humans and animal models suffering from neurodegenerative diseases including ALS, TLE, SD, BD, and PD as well as healthy controls will be performed to establish circRNA expression profiles. For a subset of samples circRNA profiles will be performed in the synaptosome, cytoplasm, and nuclear fractions. Exosomics will advise in standardization of exosome purification. We aim to recover circRNA from formalin-fixed, paraffin-embedded (FFPE) tissue which, if successful, will potentially give us access to 159 SD and 141 BD patients and 20 normal subjects with associated medical records. Samples will be sequenced at high read depth and analysed by pipelines developed at MDC and Qiagen. Differentially expressed circRNA candidates will be bioinformatically analyzed for miRNA and protein binding sites to identify potential “sponging” functions and compared to publicly available HITS-CLIP datasets for validation.
Expected Results: 1. Biomarkers for ALS, TLE, SD, BD and PD in human. 2. Biomarkers for TLE, SD and BD in mice and PD in rats. 3. Protocoal for FFPE circRNA isolation.
Secondments are planned with the Rajewksy and Kjems labs and Exosomics.
Project 14 hosted by the Pasterkamp lab at the UMC, Utrecht, Netherlands
Function of circRNA in neural circuit development
Objectives: Functional characterization of circRNAs in axon guidance and other aspects of neural circuit development. A large number of circRNAs has been detected in the embryonic brain. Interestingly, many of these circRNAs derive from neuron-specific genes or from genes with crucial roles in the developing or adult nervous system. An important class of molecules contributing to neural circuit development are axon guidance proteins. Several classes of axon guidance proteins have been identified, including semaphorins. More than 25 semaphorins have been identified several of which are known to give rise to circRNAs. To elucidate the role of circRNAs derived from semaphorin genes or other axon guidance genes we will first perform FISH and RT-PCR on different regions and primary neuron cultures from mice. To unveil the functional role of candidates, circRNA expression will be lowered in specific brain regions by targeting the unique circularization site by RNAi and in utero electroporation. Possible defects in axon guidance or other aspects of neural circuit development will be visualized by combining tissue clearing (3DISCO) and ultramicroscope lightsheet imaging in our in house microscopy facility (MIND; headed by Pasterkamp). To uncover the downstream effects of circRNA knockdown we will perform RNA-seq on electroporated brain regions and perform ChIRP-MS approach (collaboration with Kjems). Regulated RNAs and/or candidate interactors will be confirmed and manipulated using the knockdown, electroporation and microscopy approaches listed above. This project is related to WP2 and aims to decipher novel biological functions for circRNAs in different cell types.
Expected Results:1. Localization of axon guidance gene-derived circRNAs. 2. Function of axon guidance gene-derived circRNAs. 3. Mechanism-of-action of axon guidance gene-derived circRNAs. 4. High resolution expression map axon guidance circRNAs. 5. Defects in neural circuit development due to circRNA manipulation. 6. Novel targets for circRNAs.
Secondments are planned with the Kjems lab and InterRNA.
Project 15 hosted by the Pasterkamp lab at the UMC, Utrecht, Netherlands
To characterize the functional role of circRNAs in ALS and TLE
Objectives: We have recently performed profiling of 1) human brain samples from temporal lobe epilepsy (TLE) patients, and 2) IPSC-derived motor neuron cultured generated from healthy control and ALS patients (ATXN2 mutations). These RNA seq studies have provided a wealth of information on RNA changes in neurological disease, such as changes in circRNAs or microexons. To elucidate the role of candidate circRNAs we will first analyse their expression in patient material (brain/spinal cord) using FISH, qPCR, and NB analysis. Next we will manipulate the expression of circRNAs in IPSC-generated human neuron/glia cultures or cerebral organoids. We will either use lentiviral RNAi or CRISPR/CAS. Different neuronal subtypes will be generated (motor, cortical, sensory neurons) in the BCRM IPSC facility (headed by Pasterkamp) and analysed using biochemical methods, immunolabeling, calcium imaging and electrophysiology. To uncover the downstream effects of circRNA knockdown we will perform RNA-seq on IPSC cultures/cerebral organoids and perform ChIRP-MS approach (collaboration with Kjems). Regulated RNAs and/or candidate interactors will be confirmed in patient brain material/IPSC neurons, and manipulated using the knockdown, electroporation and microscopy approaches listed above. This project aims to dissect the functional role and mechanism-of-action of circRNAs in neurological disease.
Expected Results: 1. High resolution expression map circRNAs in ALS and TLE. 2. circRNA manipulation to influence neurological disease. 3. Novel targets for disease-associated circRNAs.
Secondments are planned with the Kjems lab and InterRNA.