Abstract
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 developed Phenol Toluol extraction (PTex), a novel and unbiased method for the purification of UV cross-linked RNPs in living cells. PTex is a fast (2-3 h) and simple protocol. The purification principle is solely based on physicochemical properties of cross-linked RNPs, enabling us to interrogate RNA-protein interactions system-wide and beyond poly(A) RNA from a variety of species and source material. Here, we are presenting an introduction of the underlying separation principles and give a detailed discussion of the individual steps as well as incorporation of PTex in high-throughput pipelines.
Highlights
Cellular gene expression is regulated at different levels
MRNA is interacting with RNA-binding proteins (RBPs) to form ribonucleoprotein complexes (RNPs), forming a complex network of (m)RNPs in which post-transcriptional regulation is facilitated [3, 4]
3.1.1 Phenol-toluol ratios For a start, we focused on understanding the influence of the different chemical compounds on the clRBPs partitioning during the extraction
Summary
Cellular gene expression is regulated at different levels. Post-transcriptional regulation comprising mRNA localisation, degradation, translation as well as miRNA-mediated or non-coding RNA-mediated regulation has become a major focus of research in the past years [1, 2]. MRNA is interacting with RNA-binding proteins (RBPs) to form ribonucleoprotein complexes (RNPs), forming a complex network of (m)RNPs in which post-transcriptional regulation is facilitated [3, 4]. In 2012, the RNA interactome capture (RIC) approach was presented; after UV cross-linking of RBPs to RNA in vivo, eukaryotic mRNA is selected using oligo(dT) magnetic beads. The co-purified RBPs are stringently washed using denaturing conditions and identified by mass spectrometry [5, 6]. This resulted in mapping of mRNA-bound proteomes in diverse cell lines and to the identification of hundreds of novel RBPs [2, 7]. Investigating other RNA classes (transcripts which are products of RNA polymerase I or III) or mRNA from bacteria and archea cannot be accomplished with this method
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