Combined application of in vivo UV-crosslinking and in vitro label transfer in the examination of AU-rich sequence binding protein - RNA interactions.

A combination of in vivo UV light-induced crosslinking of nucleic acids to proteins and in vitro label transfer assay was applied to investigate specific interactions between AU-rich sequences (ARE) in the 3′ UTR of lymphokine mRNAs and cytoplasmic AU-rich sequence element binding proteins (AUBP) in normal human lymphoblasts and MLA 144 gibbon lymphoid tumor cells. We demonstrate that a pool of cytoplasmic AUBP can be effectively crosslinked to RNA in vivo , suggesting a close association of these proteins with ARE sequences in the cytoplasm. We also show that the UV-crosslinked AUBP pool is markedly reduced in malignantly transformed MLA 144 cells compared with normal lymphoblasts, indicating weaker interaction between lymphokine ARE and AUBP in these tumor cells. Similar differences in AUBP-RNA associations were found between the membrane-bound polysomal subfractions of the two cell types where most of the AUBP activity was localized. We suggest that the decreased AUBP-mRNA association in MLA 144 cells might reflect a process concerned with disturbances of mRNA metabolism in the neoplastic phenotype.     

Differentiation-dependent cytoplasmic distribution and in vivo RNA association of proteins recognized by the 3'-UTR stability element of alpha-globin mRNA in erythroleukemic cells

In this study, we analyzed subcytoplasmic distribution and in vivo RNA association of proteins with specific affinity to cytosine-rich stability determinant sequences of alpha-globin mRNA 3'-UTR in a MEL-707 erythroleukemic model. We took advantage of the possibility that these cells can be reversibly differentiated (as a continuous population, but not at the level of individual cells) which, therefore, allows analysis of various stages of erythroid differentiation within the same cell population. Label transfer experiments revealed four major complexes with molecular mass of 110-, 70-, 55- and 50-kDa in various cytoplasmic fractions. Using the combination of in vitro label transfer and in vivo UV-crosslinking techniques, we also demonstrated that subcytoplasmic distribution as well as in vivo RNA association of various complex-forming proteins is differentiation dependent in MEL-707 cells. These results indicate that changes in the cytoplasmic environment imposed by the differentiating stimulus might direct important biochemical signals as to how the stability determinant 3'UTR elements interact with their binding proteins. These data also suggest that stability complexes are dynamic macromolecular structures with high response capacity to various extra- and intracellular regulatory stimuli.     

Tissue distribution of AU-rich mRNA-binding proteins involved in regulation of mRNA decay

Short lived cytokine and proto-oncogene mRNAs are destabilized by an A+U-rich element (ARE) in the 3'-untranslated region. Several regulatory proteins bind to AREs in cytokine and proto-oncogene mRNAs, participate in inhibiting or promoting their rapid degradation of ARE mRNAs, and influence cytokine expression and cellular transformation in experimental models. The tissue distribution and cellular localization of the different AU-rich binding proteins (AUBPs), however, have not been uniformly characterized in the mouse, a model for ARE mRNA decay. We therefore carried out immunoblot and immunohistochemical analyses of the different AUBPs using the same mouse tissues. We show that HuR protein, a major AUBP that stabilizes the ARE mRNAs, is most strongly expressed in the thymus, spleen (predominantly in lymphocytic cells), intestine, and testes. AUF1 protein, a negative regulator of ARE mRNA stability, displayed strong expression in thymus and spleen cells within lymphocytic cells, moderate expression in the epithelial linings of lungs, gonadal tissues, and nuclei of most neurons in the brain, and little expression in the other tissues. Tristetraprolin, a negative regulator of ARE mRNA stability, displayed a largely non-overlapping tissue distribution with AUF1 and was predominantly expressed in the liver and testis. KH-type splicing regulatory protein, a presumptive negative regulator of ARE mRNA stability, was distributed widely in murine organs. These results indicate that HuR and AUF1, which functionally oppose each other, have generally similar distributions, suggesting that the balance between HuR and AUF1 is likely important in control of short lived mRNA degradation, lymphocyte development, and/or cytokine production, and possibly in certain aspects of neurological function.     

AUF1 Is a bcl-2 A + U-rich element-binding protein involved in bcl-2 mRNA destabilization during apoptosis

We previously identified a conserved A + U-rich element (ARE) in the 3'-untranslated region of bcl-2 mRNA. We have also recently demonstrated that the bcl-2 ARE interacts with a number of ARE-binding proteins (AUBPs) whose pattern changes during apoptosis in association with bcl-2 mRNA half-life reduction. Here we show that the AUBP AUF1 binds in vitro to bcl-2 mRNA. The results obtained in a yeast RNA three-hybrid system have demonstrated that the 1-257-amino acid portion of p37 AUF1 (conserved in all isoforms), containing the two RNA recognition motifs, also binds to the bcl-2 ARE in vivo. UVC irradiation-induced apoptosis results in an increase of AUF1. Inhibition of apoptosis by a general caspase inhibitor reduces this increase by 2-3-fold. These results indicate involvement of AUF1 in the ARE/AUBP-mediated modulation of bcl-2 mRNA decay during apoptosis.     

Microfilament-dependent modulation of cytoplasmic protein binding to TNFalpha mRNA AU-rich instability element in human lymphoid cells

Cytoplasmic proteins with binding capability to AU-rich instability determinant sequences (ARE) of tumour necrosis factor alpha (TNFalpha) mRNA 3' untranslated region (3'UTR) were assessed in human lymphoid cells. In vitro label transfer experiments using wild type as well as mutant sequences in which the 70 nucleotide-long AUUUA pentamer-containing portion of the 3'UTR had been deleted conferred binding specificity to five major activities of 22/25-, 38/40-, 50-, 60- and 80-kDa proteins in cytoplasmic extracts of peripheral blood mononuclear cells (PBMCs). Cytochalasin-induced disarrangement of the F-actin-based microfilament system led to a Triton X-100-insoluble to soluble redistribution of these binding activities. No such changes were observed in Jurkat tumour cells. Combination of in vivo UV-crosslinking and in vitro label transfer experiments revealed considerable differences in RNA association between proteins of the same cell type as well as between proteins of identical molecular weight (Mw) derived from either PBMCs or Jurkat cells. Our findings may explain some aspects of differential regulation of interleukin 2 (IL-2) and TNFalpha mRNA stability upon microfilament disruption in human PBMCs observed in an earlier study. These results also suggest that the physical state of cytoplasmic structural environment might contribute to important regulatory processes regarding key elements of eukaryotic mRNA metabolism, such as modulation of stability. Finally, these data highlight the possibility that the often observed disorganization of the cytoskeleton in tumour cells may partly be responsible for the maintenance of the neoplastic state, a phenomenon that potentially involves ARE-AUBP interactions.     

Aberrant regulation of mRNA 3' untranslated region in cancers and inflammation

Almost 10% of mammalian coding mRNAs contain in their 3' untranslated region a sequence rich in adenine and uridine residues known as AU-rich element (ARE). Many of them encode oncogenes (for instance c-Myc and c-Fos), cell cycle regulators (cyclin D1, A1, B1), cytokines (TNFalpha, IL2) and growth factors (GM-CSF) which are overexpressed in cancer or inflammatory diseases due to increased mRNA stability and/or translation. AREs are recognized by a group of proteins, collectively called AUBPs which display various functions. For instance, HuR/ELAV is mainly known to protect ARE-containing mRNAs from degradation, while AUF1, TTP and KSRP act to destabilize their bound target mRNAs and TIA/TIAR to inhibit their translation. Alterations in ARE sequences or AUBP abundance, cellular localization or activity due to post-translational modifications such as phosphorylation can promote or enhance malignancy or perturb immune homeostasis. Here, c-myc and TNFalpha are chosen as examples to illustrate how altered 3' UTR gene regulation impacts on pathologies.     

Facilitation of mRNA deadenylation and decay by the exosome-bound, DExH protein RHAU

The AU-rich element (ARE) in the 3' untranslated region of unstable mRNAs mediate their rapid degradation. ARE binding proteins (AUBPs) have been described that either stabilize or otherwise degrade ARE-mRNAs by recruiting the exosome, a complex of 3'-to-5' exoribonucleases. We have identified RHAU, a putative DExH RNA helicase that was isolated in association with the ARE of urokinase plasminogen activator mRNA (ARE(uPA)). RHAU physically interacts with the deadenylase PARN and the human exosome and enhances the deadenylation and decay of ARE(uPA)-mRNAs. An alternatively spliced isoform of RHAU that localized to the cytoplasm had a more pronounced effect on ARE(uPA)-mRNA destabilization than full-length RHAU. Furthermore, the ATPase activity of RHAU is essential for its mRNA-destabilizing function. ARE(uPA)-mRNA recognition by RHAU may be mediated through its RNA-dependent interaction with the AUBPs HuR and NFAR1. A model is presented to describe the action of RHAU in ARE(uPA)-directed mRNA turnover.     

Convergent actions of I kappa B kinase beta and protein kinase C delta modulate mRNA stability through phosphorylation of 14-3-3 beta complexed with tristetraprolin

Regulation of gene expression at the level of mRNA stability is a major topic of research; however, knowledge about the regulatory mechanisms affecting the binding and function of AU-rich element (ARE)-binding proteins (AUBPs) in response to extracellular signals is minimal. The beta1,4-galactosyltransferase 1 (beta4GalT1) gene enabled us to study the mechanisms involved in binding of tristetraprolin (TTP) as the stability of its mRNA is regulated solely through one ARE bound by TTP in resting human umbilical vein endothelial cells. Here, we provide evidence that the complex formation of TTP with 14-3-3beta is required to bind beta4GalT1 mRNA and promote its decay. Furthermore, upon tumor necrosis factor alpha stimulation, the activation of both Ikappabeta kinase and protein kinase Cdelta is involved in the phosphorylation of 14-3-3beta on two serine residues, paralleled by release of binding of TTP and 14-3-3beta from beta4GalT1 mRNA, nuclear sequestration of TTP, and beta4GalT1 mRNA stabilization. Thus, a key mechanism regulating mRNA binding and function of the destabilizing AUBP TTP involves the phosphorylation status of 14-3-3beta.     

Overexpression of HuR, a nuclear-cytoplasmic shuttling protein, increases the in vivo stability of ARE-containing mRNAs.

The messenger RNAs of many proto-oncogenes, cytokines and lymphokines are targeted for rapid degradation through AU-rich elements (AREs) located in their 3'' untranslated regions (UTRs). HuR, a ubiquitously expressed member of the Elav family of RNA binding proteins, exhibits specific affinities for ARE-containing RNA sequences in vitro which correlate with their in vivo decay rates, thereby implicating HuR in the ARE-mediated degradation pathway. We have transiently transfected HuR into mouse L929 cells and observed that overexpression of HuR enhances the stability of beta-globin reporter mRNAs containing either class I or class II AREs. The increase in mRNA stability parallels the level of HuR overexpression, establishing an in vivo role for HuR in mRNA decay. Furthermore, overexpression of HuR deletion mutants lacking RNA recognition motif 3 (RRM 3) does not exert a stabilizing effect, indicating that RRM 3 is important for HuR function. We have also developed polyclonal anti-HuR antibodies. Immunofluorescent staining of HeLa and L929 cells using affinity-purified anti-HuR antibody shows that both endogenous and overexpressed HuR proteins are localized in the nucleus. By forming HeLa-L929 cell heterokaryons, we demonstrate that HuR shuttles between the nucleus and cytoplasm. Thus, HuR may initially bind to ARE-containing mRNAs in the nucleus and provide protection during and after their export to the cytoplasmic compartment.     

Different modes of interaction by TIAR and HuR with target RNA and DNA

TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U-rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2'-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways.     

A 32-kilodalton protein binds to AU-rich domains in the 3'' untranslated regions of rapidly degraded mRNAs.

An AU-rich sequence present within the 3' untranslated region has been shown to mark some short-lived mRNAs for rapid degradation. We demonstrate by label transfer and gel shift experiments that a 32-kDa polypeptide, present in nuclear extracts, specifically interacts with the AU-rich domains present within the 3' untranslated region of human granulocyte-macrophage colony-stimulating factor, c-fos, and c-myc mRNAs and a similar domain downstream of the poly(A) addition site of the adenovirus IVa2 mRNA. Competition experiments and partial protease analysis indicated that the same polypeptide interacts with all four RNAs. A single AUUUA sequence in a U-rich context was sufficient to signal binding of the 32-kDa polypeptide. Insertion of three copies of this minimal recognition site led to markedly reduced accumulation of beta-globin RNA, while the same insert carrying a series of U-to-G changes had little effect on RNA levels. Steady-state levels of beta-globin-specific nuclear RNA, including incompletely processed RNA, and cytoplasmic mRNA were reduced. Cytoplasmic mRNA containing the AU-rich recognition sites for the 32-kDa polypeptide exhibited a half-life shorter than that of mRNA with a mutated insert. We suggest that binding of the 32-kDa polypeptide may be involved in the regulation of mRNA half-life.     

STUDIES ON THE ORIGIN OF RIBOSOMES IN AMOEBA PROTEUS

The origin of cytoplasmic RNA and ribosomes was studied in Amoeba proteus by transplantation of a radioactive nucleus into an unlabeled cell followed by examination of the cytoplasm of the recipient for the presence of label. When a RNA-labeled nucleus was used, label appeared in the ribosomes, ribosomal RNA, and soluble RNA. Since the kinetics of appearance of labeled RNA indicates that the nucleus was not injured during the transfer, and since the transferred nuclear pool of labeled acid-soluble RNA precursors is inadequate to account for the amount of cytoplasmic RNA label, it is concluded that cytoplasmic ribosomal RNA is derived from acid-insoluble nuclear RNA and is probably transported as an intact molecule. Likewise, cytoplasmic soluble RNA probably originated in the nucleus, although labeling by terminal exchange in the cytoplasm is also possible. The results were completely different when a protein-labeled nucleus was grafted into an unlabeled host. In this case, label was found only in soluble proteins in the host cell cytoplasm, and there were no (or very few) radioactive ribosomes. This suggests that the nuclear pool of ribosomal protein and ribosomal protein precursors is relatively small and perhaps nonexistent (and, furthermore, shows that there was no cytoplasmic ribosomal contamination of the transferred nucleus).