Mechanisms of CDE-dependent mRNA decay

The cytokine TNFα is a potent effector of inflammation, and is causally involved in toxic shock syndrome and rheumatoid arthritis. It is vital to maintain continuous control over TNFα levels under continuous control, which can be achieved in one way by regulating the decay rate of the TNFα mRNA. One cis-element, the AU-rich element (ARE) in the TNFα 3’UTR confers to rapid degradation of this message. When the element is knocked out in mice, TNFα is overproduced resulting in the corresponding pathology. We recently described a second cis-element, the constitutive decay element (CDE), that is unique to TNFα and, compared to the ARE, is unresponsive to TNFα RNA stabilizing signals such as lipopolysaccharide (LPS) or phorbol esters. Furthermore a CDE-bearing reporter was degraded in Slow C cells that are defective in ARE dependent decay, implying that CDE mediated degradation is performed by a different mechanism. Through deletion mutation we were able to define a 40 nucleotide (nt) stretch (fragment O), located 42 nt downstream of the ARE, that is the minimal functional CDE. Any mutation to this minimal sequence was not only inactive in promoting decay, but also exhibited differences in protein binding compared to fragment O. The CDE binding proteins were assayed by UV-crosslinking assays, purified by biochemical fractionation and identified by mass spectrometry. The RNA-binding protein nucleolin was the prime candidate. We produced full length recombinant nucleolin with the baculovirus system, and could demonstrate that recombinant nucleolin binds in vitro to fragment O but not its mutated sequences by electrophoretic mobility shift assay. The nucleolin-CDE association was also observed with band shift and super shift assays in THP-1 human macrophage cytoplasmic extracts. Furthermore we could show by RNA immunoprecipitation assays in THP-1 cells, that nucleolin is specifically associated with endogenous TNFα mRNA in vivo, but not with the transcripts of IL-6 and IL-10, or GAPDH. To assess whether down regulation of nucleolin by RNA interference would affect
CDE mediated decay, we transfected nucleolin or control siRNAs into a human HT1080 GFP-CDE reporter cell line. By FACS we observed an increase in GFP protein levels with the nucleolin specific siRNAs but not with the control siRNAs, indicative of elevated CDE-reporter mRNA levels. Actinomycin D chase experiments confirmed stabilization of the CDE reporter with a 40% increase in reporter mRNA half-life from 96+2 min to 135+11 min, that was statistically significant. We conclude that nucleolin is associated with the CDE in vitro and in vivo and is be functionally involved in the CDE-mediated degradation pathway.