We developed individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) followed by high-throughput sequencing to study protein-RNA interactions. We developed algorithms and the iCount pipeline for mapping iCLIP sequence reads to the human genome, quality-control filtering, removal of PCR duplicates and quantification of binding using random barcodes, generation of cross-link maps, identification of significant clusters of cross-links and analysis of enriched pentanucleotides. We then studied the positioning of hnRNP C particles and their role in alternative splicing.
COBISS.SI-ID: 7800916
We used iCLIP and the computational pipeline iCount to study the role of TIA1 and TIAL1 proteins in alternative splicing. Studies of splicing regulation generally focus on RNA elements located in the proximity of alternative exons. For the two mentioned proteins we have shown that observing the recognition of distal regulatory sites in the preceding 5’ splice sites may play an important role in fully understanding the regulation of alternative splicing.
COBISS.SI-ID: 8022356
Based on extensive bioinformatics analyses of iCLIP data, we have found that TDP-43 preferentially binds long clusters of UG-rich sequences and that MALAT1 and NEAT1 are the main targets in subjects with FTLD. 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.
COBISS.SI-ID: 8278100
Comparing samples from healthy individuals ranging from 16 to 102 years old with samples from diseased individuals, we uncovered a striking similarity in the changes in gene expression patterns associated with aging and the neurodegenerative diseases. We evaluated changes in alternative splicing in great detail, which showed that changes associated with aging largely affected genes associated with cellular metabolism, while disease-specific changes were associated with genes involved in neuron-specific function. Finally, we found changes in the expression of several genes coding for RNA binding proteins, which are likely responsible for at least part of the observed alterations in splicing.
COBISS.SI-ID: 8549204
We propose that a CREB-like (cAMP response element-binding) protein BzpF integrates signaling through the cAMP-dependent protein kinase A (PKA), which plays a critical role in late development of Dictyostelium. We show that bzpF- mutants produce extremely compromised and ustable spores, and fail to express 205 genes, many of which encode cellulose-binding and sugar-binding proteins. Using extensive computational analyses of promoter sequences and data from protein-binding microarray (PBM) experiments we predicted putative BzpF targets. We experimentally confirmed that BzpF is necessary to activate the transcription of at least 15 PKA-regulated, late-developmental target genes whose promoters contain BzpF binding sites.
COBISS.SI-ID: 8548180