Genotoxic stress causes the accumulation of the splicing regulator Sam68 in nuclear foci of transcriptionally active chromatin

DNA-damaging agents cause a multifaceted cellular stress response. Cells set in motion either repair mechanisms or programmed cell death pathways, depending on the extent of the damage and on their ability to withstand it. The RNA-binding protein (RBP) Sam68, which is up-regulated in prostate carcinoma, promotes prostate cancer cell survival to genotoxic stress. Herein, we have investigated the function of Sam68 in this cellular response. Mitoxantrone (MTX), a topoisomerase II inhibitor, induced relocalization of Sam68 from the nucleoplasm to nuclear granules, together with several other RBPs involved in alternative splicing, such as TIA-1, hnRNP A1 and the SR proteins SC35 and ASF/SF2. Sam68 accumulation in nuclear stress granules was independent of signal transduction pathways activated by DNA damage. Using BrU labelling and immunofluorescence, we demonstrate that MTX-induced nuclear stress granules are transcriptionally active foci where Sam68 and the phosphorylated form of RNA polymerase II accumulate. Finally, we show that MTX-induced relocalization of Sam68 correlates with changes in alternative splicing of its mRNA target CD44, and that MTX-induced CD44 splicing depends on Sam68 expression. These results strongly suggest that Sam68 is part of a RNA-mediated stress response of the cell that modulates alternative splicing in response to DNA damage

Transcription of Satellite III non-coding RNAs is a general stress response in human cells

In heat-shocked human cells, heat shock factor 1 activates transcription of tandem arrays of repetitive Satellite III (SatIII) DNA in pericentromeric heterochromatin. Satellite III RNAs remain associated with sites of transcription in nuclear stress bodies (nSBs). Here we use real-time RT-PCR to study the expression of these genomic regions. Transcription is highly asymmetrical and most of the transcripts contain the G-rich strand of the repeat. A low level of G-rich RNAs is detectable in unstressed cells and a 104-fold induction occurs after heat shock. G-rich RNAs are induced by a wide range of stress treatments including heavy metals, UV-C, oxidative and hyper-osmotic stress. Differences exist among stressing agents both for the kinetics and the extent of induction (>100- to 80.000-fold). In all cases, G-rich transcripts are associated with nSBs. On the contrary, C-rich transcripts are almost undetectable in unstressed cells and modestly increase after stress. Production of SatIII RNAs after hyper-osmotic stress depends on the Tonicity Element Binding Protein indicating that activation of the arrays is triggered by different transcription factors. This is the first example of a non-coding RNA whose transcription is controlled by different transcription factors under different growth conditions

Non-coding murine centromeric transcripts associate with and potentiate Aurora B kinase

Non-coding RNAs are emerging as key players in many fundamental biological processes, including specification of higher-order chromatin structure. We examined the implication of RNA transcribed from mouse centromeric minor satellite repeats in the formation and function of centromere-associated complexes. Here we show that the levels of minor satellite RNA vary during cell-cycle progression, peaking in G2/M phase, concomitant with accumulation of proteins of the chromosomal passenger complex near the centromere. Consistent with this, we describe that murine minor satellite RNA are components of CENP-A-associated centromeric fractions and associate with proteins of the chromosomal passenger complex Aurora B and Survivin at the onset of mitosis. Interactions of endogenous Aurora B with CENP-A and Survivin are sensitive to RNaseA. Likewise, the kinase activity of Aurora B requires an RNA component. More importantly, Aurora B kinase activity can be potentiated by minor satellite RNA. In addition, decreased Aurora B activity after RNA depletion can be specifically rescued by restitution of these transcripts. Together, our data provide new functional evidence for minor satellite transcripts as key partners and regulators of the mitotic kinase Aurora B

Cytokine-induced killer cells express CD39, CD38, CD203a, CD73 ectoenzymes and P1 adenosinergic receptors

Cytokine-induced killer (CIK) cells, a heterogeneous T cell population obtained by in vitro differentiation of peripheral blood mononuclear cells (PBMC), represent a promising immunological approach in cancer. Numerous studies have explored the role of CD38, CD39, CD203a/PC-1, and CD73 in generating extracellular adenosine (ADO) and thus in shaping the tumor niche in favor of proliferation. The findings shown here reveal that CIK cells are able to produce extracellular ADO via traditional (CD39/CD73) and/or alternative (CD38/CD203a/CD73 or CD203a/CD73) pathways. Transcriptome analysis showed the mRNA expression of these molecules and their modulation during PBMC to CIK differentiation. When PBMC from normal subjects or cancer bearing patients were differentiated into CIK cells under normoxic conditions, CD38 and CD39 were greatly up-regulated while the number of CD203a, and CD73 positive cells underwent minor changes. Since hypoxic conditions are often found in tumors, we asked whether CD39, CD38, CD203a, and CD73 expressed by CIK cells were modulated by hypoxia. PBMC isolated from cancer patients and differentiated into CIK cells in hypoxic conditions did not show relevant changes in CD38, CD39, CD73, CD203a, and CD26. CIK cells also expressed A1, A2A, and A2B ADO receptors and they only underwent minor changes as a consequence of hypoxia. The present study sheds light on a previously unknown functional aspect of CIK cells, opening the possibility of pharmacologically modulated ADO-generating ectoezymes to improve CIK cells performance

A transcriptomic analysis of human centromeric and pericentric sequences in normal and tumor cells

Although there is now evidence that the expression of centromeric (CT) and pericentric (PCT) sequences are key players in major genomic functions, their transcriptional status in human cells is still poorly known. The main reason for this lack of data is the complexity and high level of polymorphism of these repeated sequences, which hampers straightforward analyses by available transcriptomic approaches. Here a transcriptomic macro-array dedicated to the analysis of CT and PCT expression is developed and validated in heat-shocked (HS) HeLa cells. For the first time, the expression status of CT and PCT sequences is analyzed in a series of normal and cancer human cells and tissues demonstrating that they are repressed in all normal tissues except in the testis, where PCT transcripts are found. Moreover, PCT sequences are specifically expressed in HS cells in a Heat-Shock Factor 1 (HSF1)-dependent fashion, and we show here that another independent pathway, involving DNA hypo-methylation, can also trigger their expression. Interestingly, CT and PCT were found illegitimately expressed in somatic cancer samples, whereas PCT were repressed in testis cancer, suggesting that the expression of CT and PCT sequences may represent a good indicator of epigenetic deregulations occurring in response to environmental changes or in cell transformation

Human sat III and Drosophila hsrω transcripts: a common paradigm for regulation of nuclear RNA processing in stressed cells

Exposure of cells to stressful conditions elicits a highly conserved defense mechanism termed the heat shock response, resulting in the production of specialized proteins which protect the cells against the deleterious effects of stress. The heat shock response involves not only a widespread inhibition of the ongoing transcription and activation of heat shock genes, but also important changes in post-transcriptional processing. In particular, a blockade in splicing and other post-transcriptional processing has been described following stress in different organisms, together with an altered spatial distribution of the proteins involved in these activities. However, the specific mechanisms that regulate these activities under conditions of stress are little understood. Non-coding RNA molecules are increasingly known to be involved in the regulation of various activities in the cell, ranging from chromatin structure to splicing and RNA degradation. In this review, we consider two non-coding RNAs, the hsrω transcripts in Drosophila and the sat III transcripts in human cells, that seem to be involved in the dynamics of RNA-processing factors in normal and/or stressed cells, and thus provide new paradigms for understanding transcriptional and post-transcriptional regulations in normal and stressed cells

Dead-box proteins: a family affair—active and passive players in RNP-remodeling

DEAD-box proteins are characterized by nine conserved motifs. According to these criteria, several hundreds of these proteins can be identified in databases. Many different DEAD-box proteins can be found in eukaryotes, whereas prokaryotes have small numbers of different DEAD-box proteins. DEAD-box proteins play important roles in RNA metabolism, and they are very specific and cannot mutually be replaced. In vitro, many DEAD-box proteins have been shown to have RNA-dependent ATPase and ATP-dependent RNA helicase activities. From the genetic and biochemical data obtained mainly in yeast, it has become clear that these proteins play important roles in remodeling RNP complexes in a temporally controlled fashion. Here, I shall give a general overview of the DEAD-box protein family

The Genetic Signatures of Noncoding RNAs

The majority of the genome in animals and plants is transcribed in a developmentally regulated manner to produce large numbers of non–protein-coding RNAs (ncRNAs), whose incidence increases with developmental complexity. There is growing evidence that these transcripts are functional, particularly in the regulation of epigenetic processes, leading to the suggestion that they compose a hitherto hidden layer of genomic programming in humans and other complex organisms. However, to date, very few have been identified in genetic screens. Here I show that this is explicable by an historic emphasis, both phenotypically and technically, on mutations in protein-coding sequences, and by presumptions about the nature of regulatory mutations. Most variations in regulatory sequences produce relatively subtle phenotypic changes, in contrast to mutations in protein-coding sequences that frequently cause catastrophic component failure. Until recently, most mapping projects have focused on protein-coding sequences, and the limited number of identified regulatory mutations have been interpreted as affecting conventional cis-acting promoter and enhancer elements, although these regions are often themselves transcribed. Moreover, ncRNA-directed regulatory circuits underpin most, if not all, complex genetic phenomena in eukaryotes, including RNA interference-related processes such as transcriptional and post-transcriptional gene silencing, position effect variegation, hybrid dysgenesis, chromosome dosage compensation, parental imprinting and allelic exclusion, paramutation, and possibly transvection and transinduction. The next frontier is the identification and functional characterization of the myriad sequence variations that influence quantitative traits, disease susceptibility, and other complex characteristics, which are being shown by genome-wide association studies to lie mostly in noncoding, presumably regulatory, regions. There is every possibility that many of these variations will alter the interactions between regulatory RNAs and their targets, a prospect that should be borne in mind in future functional analyses

Structural and Functional Characterization of Noncoding Repetitive RNAs Transcribed in Stressed Human Cells

Thermal and chemical stresses induce the formation in human cells of novel and transient nuclear structures called nuclear stress bodies (nSBs). These contain heat shock factor 1 (HSF-1) and a specific subset of pre-mRNA processing factors. Nuclear stress bodies are assembled on specific pericentromeric heterochromatic domains containing satellite III (SatIII) DNA. In response to stress, these domains change their epigenetic status from heterochromatin to euchromatin and are transcribed in poly-adenylated RNAs that remain associated with nSBs. In this article, we describe the cloning, sequencing, and functional characterization of these transcripts. They are composed of SatIII repeats and originate from the transcription of multiple sites within the SatIII arrays. Interestingly, the level of SatIII RNAs can be down-regulated both by antisense oligonucleotides and small interfering RNAs (siRNA). Knockdown of SatIII RNA by siRNAs requires the activity of Argonaute 2, a component of the RNA-induced silencing complex. Down-regulation of satellite III RNAs significantly affects the recruitment of RNA processing factors to nSBs without altering the association of HSF-1 with these structures nor the presence of acetylated histones within nSBs. Thus, satellite III RNAs have a major role in the formation of nSBs