Fast and unbiased purification of RNA-protein complexes after UV cross-linking.

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.

TriPepSVM: de novo prediction of RNA-binding proteins based on short amino acid motifs.

In recent years, hundreds of novel RNA-binding proteins (RBPs) have been identified, leading to the discovery of novel RNA-binding domains. Furthermore, unstructured or disordered low-complexity regions of RBPs have been identified to play an important role in interactions with nucleic acids. However, these advances in understanding RBPs are limited mainly to eukaryotic species and we only have limited tools to faithfully predict RNA-binders in bacteria. Here, we describe a support vector machine-based method, called TriPepSVM, for the prediction of RNA-binding proteins. TriPepSVM applies string kernels to directly handle protein sequences using tri-peptide frequencies. Testing the method in human and bacteria, we find that several RBP-enriched tri-peptides occur more often in structurally disordered regions of RBPs. TriPepSVM outperforms existing applications, which consider classical structural features of RNA-binding or homology, in the task of RBP prediction in both human and bacteria. Finally, we predict 66 novel RBPs in Salmonella Typhimurium and validate the bacterial proteins ClpX, DnaJ and UbiG to associate with RNA in vivo.

The RNA-bound proteome of MRSA reveals post-transcriptional roles for helix-turn-helix DNA-binding and Rossmann-fold proteins.

RNA-binding proteins play key roles in controlling gene expression in many organisms, but relatively few have been identified and characterised in detail in Gram-positive bacteria. Here, we globally analyse RNA-binding proteins in methicillin-resistant Staphylococcus aureus (MRSA) using two complementary biochemical approaches. We identify hundreds of putative RNA-binding proteins, many containing unconventional RNA-binding domains such as Rossmann-fold domains. Remarkably, more than half of the proteins containing helix-turn-helix (HTH) domains, which are frequently found in prokaryotic transcription factors, bind RNA in vivo. In particular, the CcpA transcription factor, a master regulator of carbon metabolism, uses its HTH domain to bind hundreds of RNAs near intrinsic transcription terminators in vivo. We propose that CcpA, besides acting as a transcription factor, post-transcriptionally regulates the stability of many RNAs.

Organic phase separation opens up new opportunities to interrogate the RNA-binding proteome.

Protein-RNA interactions regulate all aspects of RNA metabolism and are crucial to the function of catalytic ribonucleoproteins. Until recently, the available technologies to capture RNA-bound proteins have been biased toward poly(A) RNA-binding proteins (RBPs) or involve molecular labeling, limiting their application. With the advent of organic-aqueous phase separation-based methods, we now have technologies that efficiently enrich the complete suite of RBPs and enable quantification of RBP dynamics. These flexible approaches to study RBPs and their bound RNA open up new research avenues for systems-level interrogation of protein-RNA interactions.

Purification of cross-linked RNA-protein complexes by phenol-toluol extraction.

Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium.