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A key prerequisite to understand how gene regulatory processes are controlled by the interplay of RNA-binding proteins and ribonucleoprotein complexes with RNAs is the generation of comprehensive high-resolution maps of protein–RNA interactions. Recent advances in next-generation sequencing technology accelerated the development of various crosslinking and immunoprecipitation (CLIP) approaches to...
The ability to observe protein dynamics in living cells is critical for the mechanistic understanding of highly flexible biological processes such as pre-mRNA splicing by the spliceosome. Splicing relies on intricate RNA and protein networks that are repeatedly rearranged during spliceosome assembly. Here we describe a method based on fluorescence microscopy that has been used by our and other laboratories...
Reconstitution of protein complexes has been a valuable tool to test molecular functions and to interpret in vivo observations. In recent years, a large number of RNA–protein complexes has been identified to regulate gene expression and to be important for a range of cellular functions. In contrast to protein complexes, in vitro analyses of RNA–protein complexes are hampered by the fact that recombinant...
The correct processing and faithful decoding of mRNAs during gene expression depends on the interaction with RNA-binding proteins (RBPs). The association of RBPs with pre-mRNAs starts during transcription by RNA polymerase II and undergoes constant remodeling during pre-mRNA processing and later steps of genes expression. Recently developed high throughput methods enabled to define RBP binding sites...
Associations between proteins and RNA–RNA duplexes are important in post-transcriptional regulation of gene expression. The CLASH (Cross-linking, Ligation and Sequencing of Hybrids) technique captures RNA–RNA interactions by physically joining two RNA molecules associated with a protein complex into a single chimeric RNA molecule. These events are relatively rare and considerable effort is needed...
The majority of human genes undergo alternative splicing to generate multiple isoforms with distinct functions. This process is generally controlled by cis-acting splicing regulatory elements (SREs) that recruit trans-acting factors to promote or inhibit the use of nearby splice sites. The growing interest in understanding the regulatory rules of splicing necessitates the systematic identification...
RNA-binding proteins (RBPs) are key players in the post-transcriptional regulation of gene expression. Precise knowledge about their binding sites is therefore critical to unravel their molecular function and to understand their role in development and disease. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) identifies protein–RNA crosslink sites on a genome-wide scale...
Development of high-throughput approaches to map the RNA interaction sites of individual RNA binding proteins (RBPs) transcriptome-wide is rapidly transforming our understanding of post-transcriptional gene regulatory mechanisms. Here we describe a ribonucleoprotein (RNP) footprinting approach we recently developed for identifying occupancy sites of both individual RBPs and multi-subunit RNP complexes...
In the past few years, RNA molecules have been revealed to be at the center of numerous biological processes. Long considered as passive molecules transferring genetic information from DNA to proteins, it is now well established that RNA molecules play important regulatory roles. Associated with that, the number of identified RNA binding proteins (RBPs) has increased considerably and mutations in...
Protein–RNA interactions play fundamental roles in many biological processes, such as regulation of gene expression, RNA splicing, and protein synthesis. The understanding of these processes improves as new structures of protein–RNA complexes are solved and the molecular details of interactions analyzed. However, experimental determination of protein–RNA complex structures by high-resolution methods...
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