Published in Methods (San Diego, Calif.)
RNA molecules have the intrinsic ability to fold into complex structures that are important in regulating many biological processes, including transcription, translation, processing and degradation. However, our knowledge for RNA structures remains very limited. Previously, we developed icSHAPE, a high-throughput method to probe single-stranded RNA...
Published in Methods (San Diego, Calif.)
Precise knowledge on the binding sites of an RNA-binding protein (RBP) is key to understanding the complex post-transcriptional regulation of gene expression. This information can be obtained from individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiments. Here, we present a complete data analysis workflow to relia...
Published in Methods (San Diego, Calif.)
Several protein-RNA cross linking protocols have been established in recent years to delineate the molecular interaction of an RNA Binding Protein (RBP) and its target RNAs. However, functional dissection of the role of the RBP binding sites in modulating the post-transcriptional fate of the target RNA remains challenging. CRISPR/Cas9 genome editin...
Published in Methods (San Diego, Calif.)
To understand the regulation of gene expression it is critical to determine how proteins interact with and discriminate between different RNAs. In this review, we discuss experimental techniques that utilize high throughput approaches to characterize the interactions of proteins with large numbers of RNAs in vitro. We describe the underlying princi...
Published in Methods (San Diego, Calif.)
mRNA binding proteins (RBPs) play a major role in post-transcriptional control of gene expression. To understand the complex regulatory processes regulating a specific mRNA during its life-time, a comprehensive view of the bound RBPs is essential. Here, we describe a method for transcript-specific isolation of endogenous ribonucleoprotein complexes...
Published in Methods (San Diego, Calif.)
Mammalian Native Elongating Transcript sequencing (mNET-seq) is a recently developed technique that generates genome-wide profiles of nascent transcripts associated with RNA polymerase II (Pol II) elongation complexes. The ternary transcription complexes formed by Pol II, DNA template and nascent RNA are first isolated, without crosslinking, by imm...
Published in Methods (San Diego, Calif.)
Given their central role in translation, splicing, localization and stability of transcripts, RNA binding proteins (RBPs) are key regulators of several cellular processes. While experimental efforts have been put to study how RBPs bind to transcripts, very little is known about the RNA contributions to the interaction. Here, we review the most comm...
Published in Methods (San Diego, Calif.)
Understanding which proteins and RNAs directly interact is crucial for revealing cellular mechanisms of gene regulation. Efficient methods allowing to detect RNA-protein interactions and dissect the underlying molecular origin for RNA-binding protein (RBP) specificity are in high demand. The recently developed recombination-Y3H screening (rec-Y3H) ...
Published in Methods (San Diego, Calif.)
Post-transcriptional regulation makes an important contribution to adjusting the transcriptome to environmental changes in plants. RNA-binding proteins are key players that interact specifically with mRNAs to co-ordinate their fate. While the regulatory interactions between proteins and RNA are well understood in animals, until recently little info...
Published in Methods (San Diego, Calif.)
Post-transcriptional regulation of gene expression in cells is facilitated by formation of RNA-protein complexes (RNPs). While many methods to study eukaryotic (m)RNPs rely on purification of polyadenylated RNA, other important regulatory RNA classes or bacterial mRNA could not be investigated at the same depth. To overcome this limitation, we deve...
