HuD, a Neuronal-specific RNA-binding Protein, Increases thein Vivo Stability of MYCN RNA

Abstract

MYCN amplification and consequent deregulated expression plays a crucial role in determining the clinical behavior of neuroblastoma. Enhanced expression of MYCN confers growth potential to neuroblastoma cells, and a direct link between MYCN expression and the development of neuroblastoma has been demonstrated in transgenic mice studies. Although the molecular pathways underlying the regulation of MYCN have not been fully elucidated, post-transcriptional mechanisms appear to be important. Previously, we reported that an embryonic lethal abnormal vision-like (ELAV) protein binds with high specificity to at least two AU-rich elements within the MYCN 3-untranslated region. In this study, we characterized the ability of cis-acting elements within the MYCN 3-untranslated region to destabilize mRNA in cells and examined the functional consequences of its interactions with the ELAV protein HuD. We show that at least 4 cis-acting elements within the MYCN 3-untranslated region are able to signal the degradation of stable heterologous mRNA. Ectopic overexpression of HuD dramatically inhibits RNA decay mediated by the full-length MYCN 3-untranslated region and cis-acting destabilizing elements that harbor HuD binding sites in vivo. HuD may contribute to the malignant phenotype of neuroblastoma cells by stabilizing MYCN mRNA, thereby enhancing steady-state levels of expression of this oncogene. Neuroblastoma (NB),1 a neoplasm that arises from embryonic neural crest tissue, is the second-most common solid pediatric tumor (1). MYCN is amplified in ϳ25% of primary NBs (2-4), and MYCN overexpression is frequently detected in NB tumors (5). Although the clinical significance of MYCN expression in NB tumors that lack MYCN amplification remains controversial (6 -10), a strong correlation between high levels of MYCN expression consequent to genomic amplification of this oncogene and aggressive disease is well established (3-5, 11). Laboratory experiments have further supported an important role for MYCN in determining NB phenotype. Ectopic overexpression of MYCN results in enhanced malignant growth (12, 13), whereas MYCN antisense studies performed by our laboratory and others have shown that down-regulation of MYCN in human NB is associated with a decrease in cellular proliferation and inhibited tumor cell growth in vitro (14 -16). Furthermore, a direct link between MYCN expression and the development of NB has been demonstrated in transgenic mice studies (17).Previously we examined MYCN regulation in N-(neuroblastic, tumorigenic) and S-type (substrate-adherent, non-tumorigenic) subclones (W-N and W-S) of the MYCN-amplified NB cell line NBL-W (18). In addition to distinct morphology and growth characteristics, the subclones have differential levels of MYCN expression despite having the same genomic MYCN copy number. We found that the disparity in steady-state levels of MYCN mRNA in the W-N and W-S cells was largely determined by differences in MYCN mRNA stability (19). Lazarova and co-workers (20) examined MYCN expression in other ...

Control of c-myc mRNA stability by IGF2BP1-associated cytoplasmic RNPs

“…Regulation via the ARE relies on at least two classes of trans-acting proteins. While AUF-1 (HNRNPD) is thought to promote destabilization of the transcript, members of the ELAV-like protein family stabilize the mRNA via the ARE (Chagnovich and Cohn 1996;DeMaria and Brewer 1996;Manohar et al 2002). In contrast, IGF2BP1 was suggested to protect the c-myc mRNA from endonucleolytic cleavage by associating with the CRD (Lemm and Ross 2002;Kobel et al 2007;Sparanese and Lee 2007).…”

Thrombospondin-1 Peptide ABT-510 Combined with Valproic Acid Is an Effective Antiangiogenesis Strategy in Neuroblastoma

“…All assays were carried out in a 96-well format. Real-time fluorescent detection of PCR products was done with an Applied Biosystems 7500 Fast Real-Time PCR System using the following thermocycling conditions: 1 cycle of 50jC for 2 min and 95jC for 20 s; 40 cycles of 95jC for 3 s and 60jC for 30 s. The sequences for the primers and probes for MYCN, TSP-1, SPARC, and b2-microglobulin were described previously (10,27,28). Data were analyzed using the comparative method (DDC t ) to calculate relative quantities of a nucleic acid sequence.…”

Cellular Retinoic Acid–Binding Protein II Is a Direct Transcriptional Target of MycN in Neuroblastoma

“…Because HuD has been shown to bind to the 3 ¶-UTR of MycN mRNA, leading to increased RNA stability (25), CRABP-II transfectants and vector control transfectants were treated with actinomycin D and MycN mRNA levels were examined by quantitative RT-PCR at different time points after transcriptional inhibition as a way of assessing MycN mRNA stability. The results showed no significant increase in MycN mRNA stability in CRABP-II transfectants compared with control transfectants (Fig.…”

“…Hel-N1 binds in vitro to the 3 ¶ untranslated regions (UTR) of several AUrich mRNAs, including c-Myc, c-Fos, G/Mcsf, and the transcriptional repressor Id leading to enhanced mRNA stability (23). Both HuD and Hel-N1 bind to the 3 ¶-UTR of the MycN mRNA and increase its stability in neuroblastoma (24,25). In other systems, HuB and HuD stabilize RNAs associated with polysomes, thereby increasing their translation (26,27).…”