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

Approaches for Dissecting RNA-Binding Protein Networks

RNA-binding proteins (RBPs) play pivotal roles in post-transcriptional regulation, acting as master controllers of the RNA’s life. The development of a variety of global analysis tools during the last decade has tremendously enriched our understanding of the function of RBPs and their implications in environmental and cellular cues. In this chapter, we discuss the properties and functions of the RBPs in fungi, and we outline the genome-wide approaches that have been developed during the last decade to systematically identify their RNA targets. We further sketch recent approaches aimed to search for novel RBPs, revealing that many more proteins such as metabolic enzymes could have additional functions in RNA regulation. Based on a rich dataset obtained in budding yeast, we further refer to attempts to integrate RNA targets for more than 60 RBPs into molecular networks to reveal structures and motifs. Finally, we elaborate on the future tasks and challenges on our way to complete our understanding of this highly connected and interwoven protein-RNA interaction network and its importance for cell physiology

Slt2 MAPK pathway is essential for cell integrity in the presence of arsenate.

Arsenate is a common toxic metalloid found in drinking water worldwide that causes several human diseases. The biochemical action underlying cellular response to arsenate, however, is not yet completely understood. Here we used Saccharomyces cerevisiae as an eukaryotic model system to identify proteins essential for adaptation to arsenate treatment. Previous studies have demonstrated a function for Hog1 MAPK in modulating the cellular response to arsenite. Our results, however, showed that cells deficient in Hog1 did not show increased sensitivity to arsenate, suggesting that perhaps other MAPKs may be involved in the response to this particular arsenic species. Here, we found that Slt2 MAPK and several of its upstream regulators are essential in modulating the response to arsenate, and that Slt2 is phosphorylated after arsenate treatment. Furthermore, whole-genome transcriptional analysis showed that Slt2 is required for the induction of several genes in response to arsenate exposure. Many of these genes are involved in the cellular response to heat, suggesting an overlap between these two stress response pathways, and pointing toward a common response to both arsenate and heat exposure in Saccharomyces cerevisiae. Furthermore, our results support the idea that cellular exposure to arsenate results in induction of cellular signalling pathways different from those induced under arsenite treatment

Slt2 MAPK pathway is essential for cell integrity in the presence of arsenate.

Arsenate is a common toxic metalloid found in drinking water worldwide that causes several human diseases. The biochemical action underlying cellular response to arsenate, however, is not yet completely understood. Here we used Saccharomyces cerevisiae as an eukaryotic model system to identify proteins essential for adaptation to arsenate treatment. Previous studies have demonstrated a function for Hog1 MAPK in modulating the cellular response to arsenite. Our results, however, showed that cells deficient in Hog1 did not show increased sensitivity to arsenate, suggesting that perhaps other MAPKs may be involved in the response to this particular arsenic species. Here, we found that Slt2 MAPK and several of its upstream regulators are essential in modulating the response to arsenate, and that Slt2 is phosphorylated after arsenate treatment. Furthermore, whole-genome transcriptional analysis showed that Slt2 is required for the induction of several genes in response to arsenate exposure. Many of these genes are involved in the cellular response to heat, suggesting an overlap between these two stress response pathways, and pointing toward a common response to both arsenate and heat exposure in Saccharomyces cerevisiae. Furthermore, our results support the idea that cellular exposure to arsenate results in induction of cellular signalling pathways different from those induced under arsenite treatment

Conserved mRNA-binding proteomes in eukaryotic organisms

RNA-binding proteins (RBPs) are essential for post-transcriptional regulation of gene expression. Recent high-throughput screens have dramatically increased the number of experimentally identified RBPs; however, comprehensive identification of RBPs within living organisms is elusive. Here we describe the repertoire of 765 and 594 proteins that reproducibly interact with polyadenylated mRNAs in Saccharomyces cerevisiae and Caenorhabditis elegans, respectively. Furthermore, we report the differential association of mRNA-binding proteins (mRPBs) upon induction of apoptosis in C. elegans L4-stage larvae. Strikingly, most proteins composing mRBPomes, including components of early metabolic pathways and the proteasome, are evolutionarily conserved between yeast and C. elegans. We speculate, on the basis of our evidence that glycolytic enzymes bind distinct glycolytic mRNAs, that enzyme-mRNA interactions relate to an ancient mechanism for post-transcriptional coordination of metabolic pathways that perhaps was established during the transition from the early 'RNA world' to the 'protein world'

A versatile tandem RNA isolation procedure to capture in vivo formed mRNA-protein complexes

We describe a tandem RNA isolation procedure (TRIP) that enables purification of in vivo formed messenger ribonucleoprotein (mRNP) complexes. The procedure relies on the purification of polyadenylated mRNAs with oligo(dT) beads from cellular extracts, followed by the capture of specific mRNAs with 3'-biotinylated 2'-O-methylated antisense RNA oligonucleotides, which are recovered with streptavidin beads. TRIP was applied to isolate in vivo crosslinked mRNP complexes from yeast, nematodes and human cells for subsequent analysis of RNAs and bound proteins. The method provides a basis for adaptation to other types of polyadenylated RNAs, enabling the comprehensive identification of bound proteins/RNAs, and the investigation of dynamic rearrangement of mRNPs imposed by cellular or environmental cues

Functional characterization of Upf1 targets in Schizosaccharomyces pombe.

Nonsense-mediated mRNA decay (NMD) is a highly conserved mechanism of mRNA degradation. NMD eliminates mRNAs containing premature termination codons (PTCs), preventing the production of truncated proteins with possible deleterious effects. However, there is mounting evidence that NMD factors, like Upf1, Upf2 and Upf3, participate in general regulation of gene expression, affecting the expression of genes lacking PTCs. We have used the fission yeast Schizosaccharomyces pombe to identify mRNAs directly regulated by NMD. Using a combination of genetic and biochemical approaches, we have defined a population of fission yeast mRNAs specifically regulated by Upf1. We show that other components of the Upf complex, Upf2 and Upf3, are required for binding of Upf1 to its RNA targets and for the proper response of fission yeast to oxidative stress. Finally, we investigated the physiological importance of this phenomenon, and demonstrate that the Upf1-dependent downregulation of some of its direct targets is necessary for normal resistance to oxidative stress

Response to arsenate treatment in Schizosaccharomyces pombe and the role of its arsenate reductase activity.

Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast