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Articles by Gregor Rot in JoVE
JoVE General
iCLIP - transkriptom hela Kartläggning av protein-RNA interaktioner med individuella Nukleotid Upplösning
1Laboratory of Molecular Biology, Medical Research Council - MRC, 2European Bioinformatics Institute, EMBL Heidelberg, 3Computer and Information Science, University of Ljubljana, 4Wellcome Trust Genome Campus, Wellcome Trust Sanger Institute
Den rumsliga arrangemang av RNA-bindande proteiner på en utskrift är en viktig faktor för post-transkriptionell reglering. Därför utvecklade vi individuella nukleotida crosslinking upplösning UV och immunoprecipitation (iCLIP) som ger en exakt genomet hela kartläggning av bindningar på ett RNA-bindande protein.
Other articles by Gregor Rot on PubMed
DictyExpress: a Dictyostelium Discoideum Gene Expression Database with an Explorative Data Analysis Web-based Interface
Bioinformatics often leverages on recent advancements in computer science to support biologists in their scientific discovery process. Such efforts include the development of easy-to-use web interfaces to biomedical databases. Recent advancements in interactive web technologies require us to rethink the standard submit-and-wait paradigm, and craft bioinformatics web applications that share analytical and interactive power with their desktop relatives, while retaining simplicity and availability.
Conserved Developmental Transcriptomes in Evolutionarily Divergent Species
Evolutionarily divergent organisms often share developmental anatomies despite vast differences between their genome sequences. The social amoebae Dictyostelium discoideum and Dictyostelium purpureum have similar developmental morphologies although their genomes are as divergent as those of man and jawed fish.
ICLIP Reveals the Function of HnRNP Particles in Splicing at Individual Nucleotide Resolution
In the nucleus of eukaryotic cells, nascent transcripts are associated with heterogeneous nuclear ribonucleoprotein (hnRNP) particles that are nucleated by hnRNP C. Despite their abundance, however, it remained unclear whether these particles control pre-mRNA processing. Here, we developed individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) to study the role of hnRNP C in splicing regulation. iCLIP data show that hnRNP C recognizes uridine tracts with a defined long-range spacing consistent with hnRNP particle organization. hnRNP particles assemble on both introns and exons but remain generally excluded from splice sites. Integration of transcriptome-wide iCLIP data and alternative splicing profiles into an 'RNA map' indicates how the positioning of hnRNP particles determines their effect on the inclusion of alternative exons. The ability of high-resolution iCLIP data to provide insights into the mechanism of this regulation holds promise for studies of other higher-order ribonucleoprotein complexes.
ICLIP Predicts the Dual Splicing Effects of TIA-RNA Interactions
The regulation of alternative splicing involves interactions between RNA-binding proteins and pre-mRNA positions close to the splice sites. T-cell intracellular antigen 1 (TIA1) and TIA1-like 1 (TIAL1) locally enhance exon inclusion by recruiting U1 snRNP to 5' splice sites. However, effects of TIA proteins on splicing of distal exons have not yet been explored. We used UV-crosslinking and immunoprecipitation (iCLIP) to find that TIA1 and TIAL1 bind at the same positions on human RNAs. Binding downstream of 5' splice sites was used to predict the effects of TIA proteins in enhancing inclusion of proximal exons and silencing inclusion of distal exons. The predictions were validated in an unbiased manner using splice-junction microarrays, RT-PCR, and minigene constructs, which showed that TIA proteins maintain splicing fidelity and regulate alternative splicing by binding exclusively downstream of 5' splice sites. Surprisingly, TIA binding at 5' splice sites silenced distal cassette and variable-length exons without binding in proximity to the regulated alternative 3' splice sites. Using transcriptome-wide high-resolution mapping of TIA-RNA interactions we evaluated the distal splicing effects of TIA proteins. These data are consistent with a model where TIA proteins shorten the time available for definition of an alternative exon by enhancing recognition of the preceding 5' splice site. Thus, our findings indicate that changes in splicing kinetics could mediate the distal regulation of alternative splicing.
Characterizing the RNA Targets and Position-dependent Splicing Regulation by TDP-43
TDP-43 is a predominantly nuclear RNA-binding protein that forms inclusion bodies in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The mRNA targets of TDP-43 in the human brain and its role in RNA processing are largely unknown. Using individual nucleotide-resolution ultraviolet cross-linking and immunoprecipitation (iCLIP), we found that TDP-43 preferentially bound long clusters of UG-rich sequences in vivo. Analysis of RNA binding by TDP-43 in brains from subjects with FTLD revealed that the greatest increases in binding were to the MALAT1 and NEAT1 noncoding RNAs. We also found that binding of TDP-43 to pre-mRNAs influenced alternative splicing in a similar position-dependent manner to Nova proteins. In addition, we identified unusually long clusters of TDP-43 binding at deep intronic positions downstream of silenced exons. A substantial proportion of alternative mRNA isoforms regulated by TDP-43 encode proteins that regulate neuronal development or have been implicated in neurological diseases, highlighting the importance of TDP-43 for the regulation of splicing in the brain.
SNPsyn: Detection and Exploration of SNP-SNP Interactions
SNPsyn (http://snpsyn.biolab.si) is an interactive software tool for the discovery of synergistic pairs of single nucleotide polymorphisms (SNPs) from large genome-wide case-control association studies (GWAS) data on complex diseases. Synergy among SNPs is estimated using an information-theoretic approach called interaction analysis. SNPsyn is both a stand-alone C++/Flash application and a web server. The computationally intensive part is implemented in C++ and can run in parallel on a dedicated cluster or grid. The graphical user interface is written in Adobe Flash Builder 4 and can run in most web browsers or as a stand-alone application. The SNPsyn web server hosts the Flash application, receives GWAS data submissions, invokes the interaction analysis and serves result files. The user can explore details on identified synergistic pairs of SNPs, perform gene set enrichment analysis and interact with the constructed SNP synergy network.
Analysis of Alternative Splicing Associated with Aging and Neurodegeneration in the Human Brain
Age is the most important risk factor for neurodegeneration; however, the effects of aging and neurodegeneration on gene expression in the human brain have most often been studied separately. Here, we analyzed changes in transcript levels and alternative splicing in the temporal cortex of individuals of different ages who were cognitively normal, affected by frontotemporal lobar degeneration (FTLD), or affected by Alzheimer's disease (AD). We identified age-related splicing changes in cognitively normal individuals and found that these were present also in 95% of individuals with FTLD or AD, independent of their age. These changes were consistent with increased polypyrimidine tract binding protein (PTB)-dependent splicing activity. We also identified disease-specific splicing changes that were present in individuals with FTLD or AD, but not in cognitively normal individuals. These changes were consistent with the decreased neuro-oncological ventral antigen (NOVA)-dependent splicing regulation, and the decreased nuclear abundance of NOVA proteins. As expected, a dramatic down-regulation of neuronal genes was associated with disease, whereas a modest down-regulation of glial and neuronal genes was associated with aging. Whereas our data indicated that the age-related splicing changes are regulated independently of transcript-level changes, these two regulatory mechanisms affected expression of genes with similar functions, including metabolism and DNA repair. In conclusion, the alternative splicing changes identified in this study provide a new link between aging and neurodegeneration.
