Post-transcriptional regulation of RNase-L expression is mediated by the 3'-untranslated region of its mRNA

RNase-L mediates critical cellular functions including antiviral, pro-apoptotic, and tumor suppressive activities; accordingly, its expression must be tightly regulated. Little is known about the control of RNASEL expression; therefore, we examined the potential regulatory role of a conserved 3'-untranslated region (3'-UTR) in its mRNA. The 3'-UTR mediated a potent decrease in the stability of RNase-L mRNA, and of a chimeric beta-globin-3'-UTR reporter mRNA. AU-rich elements (AREs) are cis-acting regulatory regions that modulate mRNA stability. Eight AREs were identified in the RNase-L 3'-UTR, and deletion analysis identified positive and negative regulatory regions associated with distinct AREs. In particular, AREs 7 and 8 served a strong positive regulatory function. HuR is an ARE-binding protein that stabilizes ARE-containing mRNAs, and a predicted HuR binding site was identified in the region comprising AREs 7 and 8. Co-transfection of HuR and RNase-L enhanced RNase-L expression and mRNA stability in a manner that was dependent on this 3'-UTR region. Immunoprecipitation demonstrated that RNase-L mRNA associates with a HuR containing complex in intact cells. Activation of endogenous HuR by cell stress, or during myoblast differentiation, increased RNase-L expression, suggesting that RNase-L mRNA is a physiologic target for HuR. HuR-dependent regulation of RNase-L enhanced its antiviral activity demonstrating the functional significance of this regulation. These findings identify a novel mechanism of RNase-L regulation mediated by its 3'-UTR.     

The proximal region of the 3'-untranslated region of cyclooxygenase-2 is recognized by a multimeric protein complex containing HuR,TIA-1, TIAR,and the heterogeneous nuclear ribonucleoprotein U

Cyclooxygenase-2 (COX-2) is an early response gene induced in renal mesangial cells by interleukin-1beta (IL-1beta). The 3'-untranslated region (3'-UTR) of COX-2 mRNA plays an important role in IL-1beta induction by regulating message stability and translational efficiency. The first 60 nucleotides of the 3'-UTR of COX-2 are highly conserved and contain multiple copies of the regulatory sequence AUUUA. Introduction of the 60-nucleotide sequence into the 3'-UTR of a heterologous reporter gene resulted in a 70% decrease in reporter gene expression. Electrophoretic mobility shift assays (EMSAs) demonstrated that mesangial cell nuclear fractions contain a multimeric protein complex that bound this region of COX-2 mRNA in a sequence-specific manner. We identified four members of the protein-RNA complex as HuR, TIA-1, TIAR, and the heterogeneous nuclear ribonucleoprotein U (hnRNP U). Treatment of mesangial cells with IL-1beta caused an increase in cytosolic HuR, which was accompanied by an increase in COX-2 mRNA that co-immunoprecipitated with cytosolic HuR. Therefore, we propose that HuR binds to the proximal region of the 3'-UTR of COX-2 following stimulation by IL-1beta and increases the expression of COX-2 mRNA by facilitating its transport out of the nucleus.     

Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR

We purified the KH-type splicing regulatory protein (KSRP) as a protein interacting with the 3'-untranslated region (3'-UTR) of the human inducible nitric oxide (iNOS) mRNA. Immunodepletion of KSRP enhanced iNOS 3'-UTR RNA stability in in vitro-degradation assays. In DLD-1 cells overexpressing KSRP cytokine-induced iNOS expression was markedly reduced. In accordance, downregulation of KSRP expression increases iNOS expression by stabilizing iNOS mRNA. Co-immunoprecipitations showed interaction of KSRP with the exosome and tristetraprolin (TTP). To analyze the role of KSRP binding to the 3'-UTR we studied iNOS expression in DLD-1 cells overexpressing a non-binding mutant of KSRP. In these cells, iNOS expression was increased. Mapping of the binding site revealed KSRP interacting with the most 3'-located AU-rich element (ARE) of the human iNOS mRNA. This sequence is also the target for HuR, an iNOS mRNA stabilizing protein. We were able to demonstrate that KSRP and HuR compete for this binding site, and that intracellular binding to the iNOS mRNA was reduced for KSRP and enhanced for HuR after cytokine treatment. Finally, a complex interplay of KSRP with TTP and HuR seems to be essential for iNOS mRNA stabilization after cytokine stimulation.     

Poly(A)-binding protein-interacting protein 2, a strong regulator of vascular endothelial growth factor mRNA

Expression of vascular endothelial growth factor (VEGF) is tightly regulated, particularly at the level of its mRNA stability, which is essentially mediated through the 3'-untranslated region (3'-UTR) of VEGF mRNA. To identify new protein partners regulating VEGF mRNA stability, we screened a cDNA expression library with an RNA probe corresponding to the entire VEGF mRNA 3'-UTR. We identified the "poly(A)-binding protein-interacting protein 2" (PAIP2) as a new VEGF mRNA 3'-UTR interacting protein. By RNA electromobility shift assays, we showed that PAIP2 binds to two distinct regions of a domain encompassing base 1 to 1280 of the VEGF 3'-UTR. Such in vitro interaction was confirmed using cell extracts in which PAIP2 expression is induced by tetracycline (Tet-on cells). Moreover, we demonstrated by RNA affinity purification as well as by ribonucleoprotein complexes immunoprecipitation, that PAIP2 interacts with VEGF mRNA in vivo. Using an in vitro RNA degradation assay, the half-life of VEGF 3'-UTR was found to be increased by overexpressing PAIP2. PAIP2 stabilizes endogenous VEGF mRNA in Tet-on cells, leading to increased VEGF secretion. Moreover, RNAi-mediated knock-down of PAIP2 significantly reduces the steady-state levels of endogenous VEGF mRNA. We also showed, by in vitro protein-protein interactions and co-immunoprecipitation experiments, that PAIP2 interacts with HuR, an already known VEGF mRNA-binding protein, suggesting cooperation of both proteins for VEGF mRNA stabilization. Hence, PAIP2 appears to be a crucial regulator of VEGF mRNA and as a consequence, any variation in its expression could modulate angiogenesis.     

Purification and characterization of beta-adrenergic receptor mRNA-binding proteins

Beta-adrenergic receptors (beta-ARs), like other G-protein-coupled receptors, can undergo post-transciptional regulation at the level of mRNA stability. In particular, the human beta(1)- and beta(2)-ARs and the hamster beta(2)-AR mRNA undergo beta-agonist-mediated destabilization. By UV cross-linking, we have previously described an approximately M(r) 36,000 mRNA-binding protein, betaARB, that binds to A/C+U-rich nucleotide regions within 3'-untranslated regions. Further, we have demonstrated previously that betaARB is immunologically distinct from AUF1/heterogeneous nuclear ribonucleoprotein (hnRNP) D, another mRNA-binding protein associated with destabilization of A+U-rich mRNAs (Pende, A., Tremmel, K. D., DeMaria, C. T., Blaxall, B. C., Minobe, W., Sherman, J. A., Bisognano, J., Bristow, M. R., Brewer, G., and Port, J. D. (1996) J. Biol. Chem. 271, 8493-8501). In this report, we describe the peptide composition of betaARB. Mass spectrometric analysis of an approximately M(r) 36,000 band isolated from ribosomal salt wash proteins revealed the presence of two mRNA-binding proteins, hnRNP A1, and the elav-like protein, HuR, both of which are known to bind to A+U-rich nucleotide regions. By immunoprecipitation, HuR appears to be the biologically dominant RNA binding component of betaARB. Although hnRNP A1 and HuR can both be immunoprecipitated from ribosomal salt wash proteins, the composition of betaARB (HuR alone versus HuR and hnRNP A1) appears to be dependent on the mRNA probe used. The exact role of HuR and hnRNP A1 in the regulation of beta-AR mRNA stability remains to be determined.     

Apoptotic cell-derived sphingosine-1-phosphate promotes HuR-dependent cyclooxygenase-2 mRNA stabilization and protein expression

Removal of apoptotic cells by phagocytes is considered a pivotal immune regulatory process. Although considerable knowledge has been obtained on the postphagocytic macrophage phenotype, there is little information on molecular mechanisms, which provoke macrophage polarization. In this study, we show that human apoptotic Jurkat cells (AC) or AC-conditioned medium (CM) rapidly induces cyclooxygenase-2 (COX-2) expression in mouse RAW264.7 macrophages via sphingosine-1-phosphate (S1P). Pharmacological inhibition of S1P release from AC or using CM from cells with a knockdown of sphingosine kinase 2 in human MCF-7 cells abrogates this effect. Expression of COX-2 resulted from an increase in mRNA stability via its 3'-untranslated region (UTR), shown by COX-2-3'-UTR and AU-rich element-driven reporter assays. Western analysis corroborated increased nucleocytoplasmic shuttling of the RNA-binding protein HuR after CM treatment. RNA EMSA analysis revealed an S1P- and CM-mediated increase in HuR-RNA binding to a COX-2-specific UTR, whereas HuR knockdown pointed to its importance for S1P in CM-induced COX-2 expression. Immunofluorescence microscopy of phospholipase A2 (PLA2) and ELISA analysis of PGE2 revealed activation of PLA2 and production of PGE2 in response to CM but not S1P. S1P, released from AC, uses HuR to stabilize COX-2 mRNA and thus to increase COX-2 protein expression. However, only CM also activates PLA2 to provide the substrate for COX-2. Our data underscore the importance of S1P in AC-mediated immune regulation, by stabilizing COX-2 mRNA in macrophages, a prerequisite for PGE2 formation.     

The ELAV RNA-stability factor HuR binds the 5'-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation

The type I insulin-like growth factor receptor (IGF-IR) is an integral component in the control of cell proliferation, differentiation and apoptosis. The IGF-IR mRNA contains an extraordinarily long (1038 nt) 5'-untranslated region (5'-UTR), and we have characterized a diverse series of proteins interacting with this RNA sequence which may provide for intricate regulation of IGF-IR gene expression at the translational level. Here, we report the purification and identification of one of these IGF-IR 5'-UTR-binding proteins as HuR, using a novel RNA crosslinking/RNase elution strategy. Because HuR has been predominantly characterized as a 3'-UTR-binding protein, enhancing mRNA stability and generally increasing gene expression, we sought to determine whether HuR might serve a different function in the context of its binding the IGF-IR 5'-UTR. We found that HuR consistently repressed translation initiation through the IGF-IR 5'-UTR. The inhibition of translation by HuR was concentration dependent, and could be reversed in trans by addition of a fragment of the IGF-IR 5'-UTR containing the HuR binding sites as a specific competitor, or abrogated by deletion of the third RNA recognition motif of HuR. We determined that HuR repressed translation initiation through the IGF-IR 5'-UTR in cells as well, and that siRNA knockdown of HuR markedly increased IGF-IR protein levels. Interestingly, we also found that HuR potently inhibited IGF-IR translation mediated through internal ribosome entry. Kinetic assays were performed to investigate the mechanism of translation repression by HuR and the dynamic interplay between HuR and the translation apparatus. We found that HuR, occupying a cap-distal position, significantly delayed translation initiation mediated by cap-dependent scanning, but was eventually displaced from its binding site, directly or indirectly, as a consequence of ribosomal scanning. However, HuR perpetually blocked the activity of the IGF-IR IRES, apparently arresting the IRES-associated translation pre-initiation complex in an inactive state. This function of HuR as a 5'-UTR-binding protein and dual-purpose translation repressor may be critical for the precise regulation of IGF-IR expression essential to normal cellular homeostasis.