Rapid deadenylation was impaired, indicating a crucial role for KSRP in this step of mRNA degradation. The two IL-8 ARE domains both contribute to interaction with KSRP, corresponding to the importance of both domains for rapid degradation. Exposure to the inflammatory cytokine IL-1 has been shown to stabilize IL-8 mRNA through p38 mitogen-activated protein (MAP) kinase and MK2. IL-1 treatment impaired the interaction of KSRP with the IL-8 ARE in a manner dependent on p38 MAP kinase but apparently independent of MK2. Instead, evidence that TTP, a target of MK2, can also destabilize the IL-8 ARE reporter mRNA is presented. In a comprehensive approach to identify mRNAs controlled by KSRP, two criteria were evaluated by microarray analysis of (i) association of mRNAs with KSRP in pulldown assays and (ii) increased amounts in KSRP knockdown cells. According to both criteria, a group of 100 mRNAs is controlled by KSRP, many of which are unstable and encode proteins involved in inflammation. These results indicate that KSRP functions as a limiting factor in inflammatory gene expression.Rapid degradation controls the levels of many mRNAs that are translated into transiently expressed proteins. These include cytokines, growth factors, proto-oncogene products, and other proteins participating in acute reactions. The short half-lives of their mRNAs depend on regulatory RNA sequences, the most widely distributed being AU-rich elements (AREs) located in their 3Ј untranslated regions (UTRs) (8,39). AREs have been divided into three classes, differing in the sequences and modes of degradation imposed by them (8). Class I AREs contain one to three scattered AUUUA motifs, and class II AREs contain multiple overlapping AUUUA motifs. Class III AREs are less well defined and lack an AUUUA motif. With the search pattern WWWU(AUUUA)UUUW, 4,000 human mRNAs have been reported to contain AREs and grouped into the ARED database (1), where the class II AREs are further subdivided into different groups, depending on the number of AUUUA motifs present in an ARE.Interleukin-8 (IL-8) is a member of the CXC chemokine family, released from different types of cells in response to direct cell stress, pathogens, or the proinflammatory cytokines tumor necrosis factor (TNF) and IL-1 (reference 25 and references therein). It attracts and activates leukocytes and also plays a role in angiogenesis. Studying its induction in response to IL-1, we previously observed that in addition to transcriptional activation of the IL-8 gene, its mRNA is stabilized (26,46). The latter response involves the activation of p38 mitogenactivated protein (MAP) kinase and its substrate kinase MK2. Stabilization of IL-8 mRNA can contribute to enhanced IL-8 expression, e.g., in viral infection (22). Our recent studies showed that the IL-8 mRNA contains an ARE which consists of two functionally distinct domains. They cooperate for maximal destabilization and interaction with cytoplasmic proteins in vitro (44).Control of mRNA degradation by AREs involves the function of protei...

Poly(ADP-ribose) polymerase 1 protein (PARP1) mediates chromatin loosening and activates the transcription of inducible genes, but the mechanism of PARP1 regulation in chromatin is poorly understood. We have found that PARP1 interaction with chromatin is dynamic and that PARP1 is exchanged continuously between chromatin and nucleoplasm, as well as between chromatin domains. Specifically, the PARP1 protein preferentially interacts with nucleosomal particles, and although the nucleosomal linker DNA is not necessary for this interaction, we have shown that the core histones, H3 and H4, are critical for PARP1 binding. We have also demonstrated that the histones H3 and H4 interact preferentially with the C-terminal portion of PARP1 protein and that the N-terminal domain of PARP1 negatively regulates these interactions. Finally, we have found that interaction with the N-terminal tail of the H4 histone triggers PARP1 enzymatic activity. Therefore, our data collectively suggests a model in which both the regulation of PARP1 protein binding to chromatin and the enzymatic activation of PARP1 protein depend on the dynamics of nucleosomal core histone mediation.Eukaryotic chromatin organization involves the fundamental nucleosomal unit, which consists of four core histones plus a linker histone (1). Recently, it has been shown that the activity of transcription complexes at nucleosomes is regulated by the PARP1 3 protein (2, 3). Notwithstanding these findings, major gaps in our present understanding exist that involve the mechanism by which the PARP1 protein binds to specific chromatin domains and the mechanism by which the local PARP1 protein is activated in response to developmental and environmental stimuli.After histones, PARP1 is the most abundant nuclear protein (4). The distribution of PARP1 in chromatin is broad and occurs in regions characterized by distinct cell types (2, 3, 5). Nevertheless, exactly how the PARP1 enzyme interacts with chromatin in vivo has not been thoroughly investigated, and the molecular basis for PARP1 binding to chromatin remains poorly understood. Although zinc fingers within the PARP1 protein contribute to DNA binding in vitro, they specifically recognize damaged DNA (6) and therefore do not contribute to the association of PARP1 with intact chromatin. Moreover, a PARP1 paralog, PARP2, that has no zinc fingers and no direct DNA binding capability, nevertheless exhibits a pattern of chromatin association similar to PARP1 and is able to partially complement PARP1 functions in a PARP1 null mutant (7-9). This suggests that PARP1 and PARP2 both bind chromatin indirectly, through an interaction with one or more DNA-binding proteins.A key aim of this study is to determine the specific mechanisms by which PARP1 protein associates with chromatin in vivo. Considerable evidence now suggests that PARP1 interacts with chromatin by binding to histones (10). For example, histones H1, H2A, and H2B are efficient targets for PARP1 binding in vitro (11) and are enzymatically modified by . This idea is, howev...

The multisubunit eukaryotic translation initiation factor eIF3 is thought to assist in the recruitment of ribosomes to mRNA. The expression of eIF3 subunits is frequently disrupted in human cancers, but the specific roles of individual subunits in mRNA translation and cancer remain elusive. Using global transcriptomic, proteomic, and metabolomic profiling, we found a striking failure of Schizosaccharomyces pombe cells lacking eIF3e and eIF3d to synthesize components of the mitochondrial electron transport chain, leading to a defect in respiration, endogenous oxidative stress, and premature aging. Energy balance was maintained, however, by a switch to glycolysis with increased glucose uptake, upregulation of glycolytic enzymes, and strict dependence on a fermentable carbon source. This metabolic regulatory function appears conserved in human cells where eIF3e binds metabolic mRNAs and promotes their translation. Thus, via its eIF3d-eIF3e module, eIF3 orchestrates an mRNA-specific translational mechanism controlling energy metabolism that may be disrupted in cancer.

The transcription factor ATF2 has been shown to attenuate melanoma susceptibility to apoptosis and to promote its ability to form tumors in xenograft models. To directly assess ATF2's role in melanoma development, we crossed a mouse melanoma model (NrasQ61K::Ink4a−/−) with mice expressing a transcriptionally inactive form of ATF2 in melanocytes. In contrast to 7/21 of the NrasQ61K::Ink4a−/− mice, only 1/21 mice expressing mutant ATF2 in melanocytes developed melanoma. Gene expression profiling identified higher MITF expression in primary melanocytes expressing transcriptionally inactive ATF2. MITF downregulation by ATF2 was confirmed in the skin of Atf2−/− mice, in primary human melanocytes, and in 50% of human melanoma cell lines. Inhibition of MITF transcription by MITF was shown to be mediated by ATF2-JunB–dependent suppression of SOX10 transcription. Remarkably, oncogenic BRAF (V600E)–dependent focus formation of melanocytes on soft agar was inhibited by ATF2 knockdown and partially rescued upon shMITF co-expression. On melanoma tissue microarrays, a high nuclear ATF2 to MITF ratio in primary specimens was associated with metastatic disease and poor prognosis. Our findings establish the importance of transcriptionally active ATF2 in melanoma development through fine-tuning of MITF expression.