SWI/SNF complexes in hematological malignancies: biological implications and therapeutic opportunities.

Hematological malignancies are a highly heterogeneous group of diseases with varied molecular and phenotypical characteristics. SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complexes play significant roles in the regulation of gene expression, being essential for processes such as cell maintenance and differentiation in hematopoietic stem cells. Furthermore, alterations in SWI/SNF complex subunits, especially in ARID1A/1B/2, SMARCA2/4, and BCL7A, are highly recurrent across a wide variety of lymphoid and myeloid malignancies.

Biochemical approach for isolation of polyadenylated RNAs with bound proteins from yeast.

characterization of RNA-protein interactions is the key for understanding RNA regulatory mechanisms. Herein, we describe a protocol for detection of proteins interacting with polyadenylated RNAs in the yeast Proteins are crosslinked to nucleic acids by ultraviolet (UV) irradiation of cells, and poly(A)-containing RNAs with bound proteins are isolated from cell lysates using oligo[dT] beads. RBPs can be detected by immunoblot analysis or with mass spectrometry to define the mRNA-binding proteome (mRBPome) and its changes under stress.

Oxidative stress induces coordinated remodeling of RNA-enzyme interactions.

RNA-binding proteins (RBPs) are key post-transcriptional regulators that play a substantial role during stress adaptation. Recent proteome-wide surveys have uncovered a large number of new and "unconventional" RBPs such as metabolic enzymes, yet little is known about the reconfiguration of the RNA-binding proteome (RBPome) and RNA-enzyme interactions in response to cellular stress. Here, we applied RNA-interactome capture to monitor the dynamics of the mRBPome upon mild oxidative stress in the yeast .

Synthetase of the methyl donor S-adenosylmethionine from nitrogen-fixing α-rhizobia can bind functionally diverse RNA species.

Function of bacterial small non-coding RNAs (sRNAs) and overall RNA metabolism is largely shaped by a vast diversity of RNA-protein interactions. However, in non-model bacteria with defined non-coding transcriptomes the sRNA interactome remains almost unexplored. We used affinity chromatography to capture proteins associated with MS2-tagged -sRNAs that regulate nutrient uptake (AbcR2 and NfeR1) and cell cycle (EcpR1) mRNAs by antisense-based translational inhibition in the nitrogen-fixing α-rhizobia .

Tandem RNA isolation reveals functional rearrangement of RNA-binding proteins on 3'UTRs in cisplatin treated cells.

Post-transcriptional control of gene expression is mediated via RNA-binding proteins (RBPs) that interact with mRNAs in a combinatorial fashion. While recent global RNA interactome capture experiments expanded the repertoire of cellular RBPs quiet dramatically, little is known about the assembly of RBPs on particular mRNAs; and how these associations change and control the fate of the mRNA in drug-treatment conditions. Here we introduce a novel biochemical approach, termed tobramycin-based tandem RNA isolation procedure (tobTRIP), to quantify proteins associated with the 3'UTRs of cyclin-dependent kinase inhibitor 1B () mRNAs .

MINA-1 and WAGO-4 are part of regulatory network coordinating germ cell death and RNAi in C. elegans.

Post-transcriptional control of mRNAs by RNA-binding proteins (RBPs) has a prominent role in the regulation of gene expression. RBPs interact with mRNAs to control their biogenesis, splicing, transport, localization, translation, and stability. Defects in such regulation can lead to a wide range of human diseases from neurological disorders to cancer.

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes.

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts.

Identification of Small RNA-Protein Partners in Plant Symbiotic Bacteria.

The identification of the protein partners of bacterial small noncoding RNAs (sRNAs) is essential to understand the mechanistic principles and functions of riboregulation in prokaryotic cells. Here, we describe an optimized affinity chromatography protocol that enables purification of in vivo formed sRNA-protein complexes in Sinorhizobium meliloti, a genetically tractable nitrogen-fixing plant symbiotic bacterium. The procedure requires the tagging of the desired sRNA with the MS2 aptamer, which is affinity-captured by the MS2-MBP protein conjugated to an amylose resin.

Generation of ribosome imprinted polymers for sensitive detection of translational responses.

Whilst the profiling of the transcriptome and proteome even of single-cells becomes feasible, the analysis of the translatome, which refers to all messenger RNAs (mRNAs) engaged with ribosomes for protein synthesis, is still an elaborate procedure requiring millions of cells. Herein, we report the generation and use of "smart materials", namely molecularly imprinted polymers (MIPs) to facilitate the isolation of ribosomes and translated mRNAs from merely 1,000 cells. In particular, we show that a hydrogel-based ribosome imprinted polymer could recover ribosomes and associated mRNAs from human, simian and mice cellular extracts, but did not selectively enrich yeast ribosomes, thereby demonstrating selectivity.

Post-transcriptional control of executioner caspases by RNA-binding proteins.

Caspases are key components of apoptotic pathways. Regulation of caspases occurs at several levels, including transcription, proteolytic processing, inhibition of enzymatic function, and protein degradation. In contrast, little is known about the extent of post-transcriptional control of caspases.

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.

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.

DNA damage, poly(ADP-Ribose) polymerase activation, and phosphorylated histone H2AX expression during postnatal retina development in C57BL/6 mouse.

Purpose: The purpose of this study was to investigate the incidence of DNA damage during postnatal development of the retina and the relationship between DNA damage and cell death.

Methods: DNA damage in the developing postnatal retina of C57BL/6 mice was assessed by determining the amounts of 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is indicative of DNA oxidation and related to the formation of DNA single-strand breaks (SSBs), and phosphorylated histone H2AX (γ-H2AX), a marker of DNA double-strand breaks (DSBs). Poly(ADP-ribose) polymerase (PARP) activation was measured by ELISA and Western blotting.

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.

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.

The RNA binding protein Csx1 promotes sexual differentiation in Schizosaccharomyces pombe.

Sexual differentiation is a highly regulated process in the fission yeast Schizosaccharomyces pombe and is triggered by nutrient depletion, mainly nitrogen source. One of the key regulatory proteins in fission yeast sexual differentiation is the transcription factor Ste11. Ste11 regulates the transcription of many genes required for the initial steps of conjugation and meiosis, and its deficiency leads to sterility.

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.