The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA

The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3'UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT-qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.

Combinatorial effects of splice variants modulate function of Aiolos

The transcription factor Aiolos (also known as IKZF3), a member of the Ikaros family of zinc-finger proteins, plays an important role in the control of B lymphocyte differentiation and proliferation. Previously, multiple isoforms of Ikaros family members arising from differential splicing have been described and we now report a number of novel isoforms of Aiolos. It has been demonstrated that full-length Ikaros family isoforms localize to heterochromatin and that they can associate with complexes containing histone deacetylase (HDAC). In this study, for the first time we directly investigate the cellular localization of various Aiolos isoforms, their ability to heterodimerize with Ikaros and associate with HDAC-containing complexes, and the effects on histone modification and binding to putative targets. Our work demonstrates that the cellular activities of Aiolos isoforms are dependent on combinations of various functional domains arising from the differential splicing of mRNA transcripts. These data support the general principle that the function of an individual protein is modulated through alternative splicing, and highlight a number of potential implications for Aiolos in normal and aberrant lymphocyte function.

Extracellular tumor-related mRNA in plasma of lymphoma patients and survival implications.

BACKGROUND We studied anomalous extracellular mRNAs in plasma from patients with diffuse large B-cell lymphoma (DLBCL) and their survival implications. mRNAs studied have been reported in the literature as markers of poor (BCL2, CCND2, MYC) and favorable outcome (LMO2, BCL6, FN1) in tumors. These markers were also analyzed in lymphoma tissues to test possible associations with their presence in plasma. METHODOLOGY/PRINCIPAL FINDINGS mRNA from 42 plasma samples and 12 tumors from patients with DLBCL was analyzed by real-time PCR. Samples post-treatment were studied. The immunohistochemistry of BCL2 and BCL6 was defined. Presence of circulating tumor cells was determined by analyzing the clonality of the immunoglobulin heavy-chain genes by PCR. In DLBCL, MYC mRNA was associated with short overall survival. mRNA targets with unfavorable outcome in tumors were associated with characteristics indicative of poor prognosis, with partial treatment response and with short progression-free survival in patients with complete response. In patients with low IPI score, unfavorable mRNA targets were related to shorter overall survival, partial response, high LDH levels and death. mRNA disappeared in post-treatment samples of patients with complete response, and persisted in those with partial response or death. No associations were found between circulating tumor cells and plasma mRNA. Absence of BCL6 protein in tumors was associated with presence of unfavorable plasma mRNA. CONCLUSIONS/SIGNIFICANCE Through a non-invasive procedure, tumor-derived mRNAs can be obtained in plasma. mRNA detected in plasma did not proceed from circulating tumor cells. In our study, unfavorable targets in plasma were associated with poor prognosis in B-cell lymphomas, mainly MYC mRNA. Moreover, the unfavorable targets in plasma could help us to classify patients with poor outcome within the good prognosis group according to IPI.

Localization of peroxisome proliferator-activated receptor alpha (PPARα) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.

The N-acylethanolamines (NAEs), oleoylethanolamide (OEA) and palmithylethanolamide (PEA) are known to be endogenous ligands of PPARα receptors, and their presence requires the activation of a specific phospholipase D (NAPE-PLD) associated with intracellular Ca(2+) fluxes. Thus, the identification of a specific population of NAPE-PLD/PPARα-containing neurons that express selective Ca(2+)-binding proteins (CaBPs) may provide a neuroanatomical basis to better understand the PPARα system in the brain. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the co-existence of NAPE-PLD/PPARα and the CaBPs calbindin D28k, calretinin and parvalbumin in the rat hippocampus. PPARα expression was specifically localized in the cell nucleus and, occasionally, in the cytoplasm of the principal cells (dentate granular and CA pyramidal cells) and some non-principal cells of the hippocampus. PPARα was expressed in the calbindin-containing cells of the granular cell layer of the dentate gyrus (DG) and the SP of CA1. These principal PPARα(+)/calbindin(+) cells were closely surrounded by NAPE-PLD(+) fiber varicosities. No pyramidal PPARα(+)/calbindin(+) cells were detected in CA3. Most cells containing parvalbumin expressed both NAPE-PLD and PPARα in the principal layers of the DG and CA1/3. A small number of cells containing PPARα and calretinin was found along the hippocampus. Scattered NAPE-PLD(+)/calretinin(+) cells were specifically detected in CA3. NAPE-PLD(+) puncta surrounded the calretinin(+) cells localized in the principal cells of the DG and CA1. The identification of the hippocampal subpopulations of NAPE-PLD/PPARα-containing neurons that express selective CaBPs should be considered when analyzing the role of NAEs/PPARα-signaling system in the regulation of hippocampal functions.

Role of Sam68 in post-transcriptional gene regulation.

The STAR family of proteins links signaling pathways to various aspects of post-transcriptional regulation and processing of RNAs. Sam68 belongs to this class of heteronuclear ribonucleoprotein particle K (hnRNP K) homology (KH) single domain-containing family of RNA-binding proteins that also contains some domains predicted to bind critical components in signal transduction pathways. In response to phosphorylation and other post-transcriptional modifications, Sam68 has been shown to have the ability to link signal transduction pathways to downstream effects regulating RNA metabolism, including transcription, alternative splicing or RNA transport. In addition to its function as a docking protein in some signaling pathways, this prototypic STAR protein has been identified to have a nuclear localization and to take part in the formation of both nuclear and cytosolic multi-molecular complexes such as Sam68 nuclear bodies and stress granules. Coupling with other proteins and RNA targets, Sam68 may play a role in the regulation of differential expression and mRNA processing and translation according to internal and external signals, thus mediating important physiological functions, such as cell death, proliferation or cell differentiation.

Localization of the cannabinoid CB1 receptor and the 2-AG synthesizing (DAGLα) and degrading (MAGL, FAAH) enzymes in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.

The retrograde suppression of the synaptic transmission by the endocannabinoid sn-2-arachidonoylglycerol (2-AG) is mediated by the cannabinoid CB1 receptors and requires the elevation of intracellular Ca(2+) and the activation of specific 2-AG synthesizing (i.e., DAGLα) enzymes. However, the anatomical organization of the neuronal substrates that express 2-AG/CB1 signaling system-related molecules associated with selective Ca(2+)-binding proteins (CaBPs) is still unknown. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the expression of the 2-AG/CB1 signaling system (CB1 receptor, DAGLα, MAGL, and FAAH) and the CaBPs calbindin D28k, calretinin, and parvalbumin in the rat hippocampus. CB1, DAGLα, and MAGL labeling was mainly localized in fibers and neuropil, which were differentially organized depending on the hippocampal CaBPs-expressing cells. CB(+) 1 fiber terminals localized in all hippocampal principal cell layers were tightly attached to calbindin(+) cells (granular and pyramidal neurons), and calretinin(+) and parvalbumin(+) interneurons. DAGLα neuropil labeling was selectively found surrounding calbindin(+) principal cells in the dentate gyrus and CA1, and in the calretinin(+) and parvalbumin(+) interneurons in the pyramidal cell layers of the CA1/3 fields. MAGL(+) terminals were only observed around CA1 calbindin(+) pyramidal cells, CA1/3 calretinin(+) interneurons and CA3 parvalbumin(+) interneurons localized in the pyramidal cell layers. Interestingly, calbindin(+) pyramidal cells expressed FAAH specifically in the CA1 field. The identification of anatomically related-neuronal substrates that expressed 2-AG/CB1 signaling system and selective CaBPs should be considered when analyzing the cannabinoid signaling associated with hippocampal functions.