A Dimer Interface Mutation in Glyceraldehyde-3-Phosphate Dehydrogenase Regulates Its Binding to AU-rich RNA

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme best known for its role in glycolysis. However, extra-glycolytic functions of GAPDH have been described, including regulation of protein expression via RNA binding. GAPDH binds to numerous adenine-uridine rich elements (AREs) from various mRNA 3′-untranslated regions in vitro and in vivo despite its lack of a canonical RNA binding motif. How GAPDH binds to these AREs is still unknown. Here we discovered that GAPDH binds with high affinity to the core ARE from tumor necrosis factor-α mRNA via a two-step binding mechanism. We demonstrate that a mutation at the GAPDH dimer interface impairs formation of the second RNA-GAPDH complex and leads to changes in the RNA structure. We investigated the effect of this interfacial mutation on GAPDH oligomerization by crystallography, small-angle x-ray scattering, nano-electrospray ionization native mass spectrometry, and hydrogen-deuterium exchange mass spectrometry. We show that the mutation does not significantly affect GAPDH tetramerization as previously proposed. Instead, the mutation promotes short-range and long-range dynamic changes in regions located at the dimer and tetramer interface and in the NAD⁺ binding site. These dynamic changes are localized along the P axis of the GAPDH tetramer, suggesting that this region is important for RNA binding. Based on our results, we propose a model for sequential GAPDH binding to RNA via residues located at the dimer and tetramer interfaces.     

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 multifunctional protein glyceraldehyde-3-phosphate dehydrogenase is both regulated and controls colony-stimulating factor-1 messenger RNA stability in ovarian cancer

Although glyceraldehyde-3-phosphate dehydrogenase's (GAPDH) predilection for AU-rich elements has long been known, the expected connection between GAPDH and control of mRNA stability has never been made. Recently, we described GAPDH binding the AU-rich terminal 144 nt of the colony-stimulating factor-1 (CSF-1) 3' untranslated region (UTR), which we showed to be an mRNA decay element in ovarian cancer cells. CSF-1 is strongly correlated with the poor prognosis of patients with ovarian cancer. We investigated the functional significance of GAPDH's association with CSF-1 mRNA and found that GAPDH small interfering RNA reduces both CSF-1 mRNA and protein levels by destabilizing CSF-1 mRNA. CSF-1 mRNA half-lives were decreased by 50% in the presence of GAPDH small interfering RNA. RNA footprinting analysis of the 144 nt CSF-1 sequence revealed that GAPDH associates with a large AU-rich-containing region. The effects of binding of GAPDH protein or ovarian extracts to mutations of the AU-rich regions within the footprint were consistent with this finding. In a tissue array containing 256 ovarian and fallopian tube cancer specimens, we found that GAPDH was regulated in these cancers, with almost 50% of specimens having no GAPDH staining. Furthermore, we found that low GAPDH staining was associated with a low CSF-1 score (P = 0.008). In summary, GAPDH, a multifunctional protein, now adds regulation of mRNA stability to its repertoire. We are the first to evaluate the clinical role of GAPDH protein in cancer. In ovarian cancers, we show that GAPDH expression is regulated, and we now recognize that one of the many functions of GAPDH is to promote mRNA stability of CSF-1, an important cytokine in tumor progression.     

Tethering KSRP, a decay-promoting AU-rich element-binding protein, to mRNAs elicits mRNA decay

Inherently unstable mRNAs contain AU-rich elements (AREs) in their 3' untranslated regions that act as mRNA stability determinants by interacting with ARE-binding proteins (ARE-BPs). We have destabilized two mRNAs by fusing sequence-specific RNA-binding proteins to KSRP, a decay-promoting ARE-BP, in a tethering assay. These results support a model that KSRP recruits mRNA decay machinery/factors to elicit decay. The ability of tethered KSRP to elicit mRNA decay depends on functions of known mRNA decay enzymes. By targeting the Rev response element of human immunodeficiency virus type 1 by using Rev-KSRP fusion protein, we degraded viral mRNA, resulting in a dramatic reduction of viral replication. These results provide a foundation for the development of novel therapeutic strategies to inhibit specific gene expression in patients with acquired or hereditary diseases.     

CNOT7/hCAF1 is involved in ICAM-1 and IL-8 regulation by tristetraprolin

Tristetraprolin (TTP) is an RNA-binding protein which can bind to the AU-rich elements (AREs) at the 3′-untranslated region (3′-UTR) of target mRNA and promote mRNA deadenylation and degradation. We have shown in a previous study that TTP regulates tumor necrosis factor-α (TNF-α)-induced expression of intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8), both of whose mRNAs have AREs in the 3′-UTR, in human pulmonary microvascular endothelial cells (HPMEC) through destabilizing target mRNAs, nevertheless, the mechanism by which TTP promotes mRNA decay remains unclear. Observations have indicated that TTP can interact with CAF1 (CNOT7/hCAF1 in human), a subunit of the CCR4-NOT complex with deadenylase activity. Another study illustrated that TTP can directly bind to CNOT1, the scaffold subunit of the CCR4-NOT complex. The present study showed that TTP bound to the AREs of ICAM-1 and IL-8 mRNAs and was coimmunoprecipitated with intracellular ICAM-1 and IL-8 mRNAs. TTP, CNOT7 and CNOT1 were coimmunoprecipitated in HPMEC. CNOT7 silencing stabilized ICAM-1 and IL-8 mRNAs and increased ICAM-1 and IL-8 production following TNF-α stimulation. These results, together with our previous study, suggest that CNOT7/hCAF1 is involved in ICAM-1 and IL-8 regulation by TTP in HPMEC.     

Coupled mRNA stabilization and translational silencing of cyclooxygenase-2 by a novel RNA binding protein,CUGBP2

Cyclooxygenase-2 (COX-2) expression is translationally silenced in epithelial cells undergoing radiation-induced apoptosis. CUGBP2, a predominantly nuclear protein, is also rapidly induced in response to radiation and translocates to the cytoplasm. Antisense-mediated suppression of CUGBP2 renders radioprotection through a COX-2-dependent prostaglandin pathway, providing an in vivo demonstration of translation inhibition activity for CUGBP2. CUGBP2 binds to two sets of AU-rich sequences (AREs) located within the first sixty nucleotides of the COX-2 3' untranslated region (3'UTR). Upon binding, CUGBP2 stabilizes a chimeric luciferase-COX-2 3'UTR mRNA but inhibits its translation. These findings identify a novel paradigm for RNA binding proteins in facilitating opposing functions of mRNA stability and translation inhibition and reveal a mechanism for inhibiting COX-2 expression in cancer cells.     

The association of Alu repeats with the generation of potential AU-rich elements (ARE) at 3' untranslated regions.

Background  

A significant portion (about 8% in the human genome) of mammalian mRNA sequences contains AU (Adenine and Uracil) rich elements or AREs at their 3' untranslated regions (UTR). These mRNA sequences are usually stable. However, an increasing number of observations have been made of unstable species, possibly depending on certain elements such as Alu repeats. ARE motifs are repeats of the tetramer AUUU and a monomer A at the end of the repeats ((AUUU)_nA). The importance of AREs in biology is that they make certain mRNA unstable. Proto-oncogene, such as c-fos, c-myc, and c-jun in humans, are associated with AREs. Although it has been known that the increased number of ARE motifs caused the decrease of the half-life of mRNA containing ARE repeats, the exact mechanism is as of yet unknown. We analyzed the occurrences of AREs and Alu and propose a possible mechanism for how human mRNA could acquire and keep AREs at its 3' UTR originating from Alu repeats.     

Endothelin-I induces gene expression through stimulation of endothelin type a receptors in normal rat kidney cells

RNA blots of total cellular RNA isolated from quiescent and endothelin (ET-1)-stimulated normal rat kidney (NRK) cells demonstrated that ET-1 induced the expression of c-jun, jun B, and c-fos mRNA in a time-dependent manner with maximal expression of mRNA by 1 hr after the addition of ET-1. Five hundred picomolal ET-1 was sufficient to induce maximal mRNA expression. These data agreed with saturation experiments which demonstrated that maximal binding of [¹²⁵I]ET-1 was achieved at concentrations greater than 100 pM. The K_d and B_max values for [¹²⁵I]ET-1 binding to NRK membranes were 20.5 pM and 22.2 fmol/mg protein, respectively. Competition experiments for the binding of [¹²⁵I]ET-1 to NRK membranes demonstrated that ET-1 was a more potent inhibitor (K_i = 0.047 nM) than ET-3 (K_i = 10.8 nM). No specific binding of [¹²⁵I]ET-3 (40 or 500 pM) to NRK membranes could be observed. The expression of c-jun, jun B, and c-fos mRNA was inhibited by the endothelin type A receptor (ET)-selective antagonist, BQ-123. Thus, these data demonstrate that ET-1 mediates the expression of immediate response gene mRNA in NRK cells via the ET_A receptor. ET-1 stimulation of NRK cells also upregulated EGF receptors, providing a possible mechanism for ET-1 complementation of epidermal growth factor (EGF) mitogenicity in NRK cells. © 1995 Wiley-Liss, Inc.