HuD binds to three AU-rich sequences in the 3'-UTR of neuroserpin mRNA and promotes the accumulation of neuroserpin mRNA and protein

Abstract

Neuroserpin is an axonally secreted serine protease inhibitor expressed in the nervous system that protects neurons from ischemia-induced apoptosis. Mutant neuroserpin forms have been found polymerized in inclusion bodies in a familial autosomal encephalopathy causing dementia, or associated with epilepsy. Regulation of neuroserpin expression is mostly unknown. Here we demonstrate that neuroserpin mRNA and the RNA-binding protein HuD are co-expressed in the rat central nervous system, and that HuD binds neuroserpin mRNA in vitro with high affinity. Gel-shift, supershift and T1 RNase assays revealed three HuD-binding sequences in the 3'-untranslated region (3'-UTR) of neuroserpin mRNA. They are AU-rich and 20, 51 and 19 nt in length. HuD binding to neuroserpin mRNA was also demonstrated in extracts of PC12 pheochromocytoma cells. Additionally, ectopic expression of increasing amounts of HuD in these cells results in the accumulation of neuroserpin 3'-UTR mRNA. Furthermore, stably transfected PC12 cells over-expressing HuD contain increased levels of both neuroserpin mRNAs (3.0 and 1.6 kb) and protein. Our results indicate that HuD stabilizes neuroserpin mRNA by binding to specific AU-rich sequences in its 3'-UTR, which prolongs the mRNA lifetime and increases protein level.

Activity-dependent expression of RNA binding protein HuD and its association with mRNAs in neurons

“…These neuronal mRNAs involved in neuronal differentiation and synaptic plasticity contain AU-rich elements (AREs) in their 3' untranslated regions (3' UTR), [25][26][27][28][29][30][31] such as those encoding GAP-43, Neurosperin and Acetylcholinesterase mRNA. Previous studies demonstrated the importance of cytoplasmic localization of HuD RBP in differentiating PC12 cells.…”

Characterization of the Interaction between Neuronal RNA-binding Protein HuD and AU-rich RNA

“…In vitro analyses have shown that these proteins can bind to AREs (19, 22, 26 -31). Experiments in tissue culture, in vitro decay analyses, and in vivo studies have demonstrated direct effects of Hu proteins on the levels and/or stability of ARE-containing mRNAs (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46). Hu proteins have also been implicated in cancer, as they are overexpressed in neuroblastoma and small cell lung cancer, two types of cancer that show stabilization of labile proto-oncogene mRNAs such as those of the MYC, MYCN, and FOS genes (35,46,47).…”

Anti-Mullerian-hormone-dependent regulation of the brain serine-protease inhibitor neuroserpin

“…The nuclei are counterstained with DAPI (blue; A-F). following binding of a cytoplasmic protein, the RNA-binding protein HuD, to the neuroserpin 3Ј-UTR, leading to an increased accumulation of neuroserpin mRNA (Cuadrado et al, 2002;Navarro-Yubero et al, 2004). In the present study, we provide evidence that a 5Ј regulatory element (-317 to -310) of the rat neuroserpin promoter, characterized as a Smad-binding element (SBE), positively controls the transcription of neuroserpin.…”

“…Using luciferase reporter constructs that contained segments of the promoter region of the neuroserpin gene, Berger and collaborators identified a 200-bp segment, near the transcription initiation site, that was responsible for both the neuron-specific expression and the enhanced transcription following depolarization. Similarly, neuroserpin mRNA and protein expression are induced by the thyroid hormone in rat PC12 cells (Cuadrado et al, 2002;NavarroYubero et al, 2004). Although there is no direct effect on the transcription rate, the neuroserpin mRNA half-life is increased …”

“…In mouse hippocampal neurons, elevated extracellular concentrations of KCl induce neuroserpin gene transcription, whereas zif/268 (also known as Krox-24 and EGR1) has a repressive effect (Berger et al, 1999). Neuroserpin gene expression is also regulated at the level of RNA stability by the binding of HuD (also known as ELAVL4; an RNAbinding protein) to AU-rich sequences in its 3Ј-UTR or by a thyroid hormone (triiodothyronine, T 3 ) in PC12 cells and in the rat brain (Cuadrado et al, 2002;Navarro-Yubero et al, 2004). More recently,…”