Regulation of Tyrosine Hydroxylase mRNA Stability by Protein-binding, Pyrimidine-rich Sequence in the 3′-Untranslated Region

Abstract

The stability of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is regulated by oxygen tension in the pheochromocytomaderived PC12 cell line. We previously identified a pyrimidine-rich 27-base-long protein-binding sequence in the 3-untranslated region of TH mRNA that is associated with hypoxia-inducible formation of a ribonucleoprotein complex (hypoxia-inducible protein-binding site (HIPBS)). In this study, we show that HIPBS is an mRNA stabilizing element necessary for both constitutive and hypoxia-regulated stability of TH mRNA. The mutations within this sequence that abolish protein binding markedly decrease constitutive TH mRNA stability and ablate its hypoxic regulation. A short fragment of TH mRNA that contains the wild-type HIPBS confers the increased mRNA stability to the reporter chloramphenicol acetyltransferase mRNA. However, it is not sufficient to confer hypoxic regulation. The HIPBS element binds two isoforms of a 40-kDa poly(C)-binding protein (PCBP). Hypoxia induces expression of the isoform 1, PCBP 1 , but not the isoform 2, PCBP 2 , in PC12 cells.Tyrosine hydroxylase (TH), 1 the rate-limiting enzyme in the biosynthesis of catecholamines, is expressed in specific populations of neurons in the central and peripheral nervous systems, in the neuroendocrine cells of the adrenal medulla and carotid body, and in cultured cell lines such as the pheochromocytomaderived PC12 cell line. Regulation of TH gene expression at the level of gene transcription is well documented. Recently, there has been growing evidence that TH mRNA is also regulated at the level of mRNA stability. TH mRNA is a stable message with a half-life that varies from 9 to 16 h in various subclones of PC12 cells (1-3). It is enhanced during differentiation of neuroblastoma cells (1) and during stimulation of the protein kinase C pathway in PC12 cells (2). In contrast, the stability of TH mRNA does not change in PC12 cells during stimulation of TH mRNA expression by dexamethasone or forskolin (3). A recent study demonstrated substantial differences in basal TH mRNA turnover rates between different neuronal populations from as short a time as 6 -7 h, in the dopaminergic neurons of the arcuate nucleus, to as long as 11-23 h, in the dopaminergic midhypothalamic neurons (4). In addition, TH mRNA is destabilized in the dopaminergic cells of the arcuate nucleus in a manner that corresponds to the rhythmic output displayed by these neurons (4).Our laboratory demonstrated that hypoxia augments the stability of TH mRNA in PC12 cells (5). We identified a 27-base-long pyrimidine-rich sequence within the TH mRNA 3Ј-untranslated region (UTR) (1552-1578 bases of TH mRNA) that binds protein factors in a hypoxia-inducible manner (hypoxia-inducible protein-binding sequence (HIPBS)) in PC12 cells (6, 7), catecholaminergic cells of the superior cervical ganglia, and the dopaminergic cells of the carotid body (8). Mutational analysis revealed that the optimal protein-binding site is represented by the motif (U/C)(C/...

Control of Insulin mRNA Stability in Rat Pancreatic Islets

“…1A). Protein binding to 3Ј-UTR pyrimidine-rich segments of for example TH and erythropoetin mRNA has been implicated in both constitutive and regulated stability control of the messenger (25,29). In line with this notion, we have presently observed specific binding of a 55-60-kDa protein to the ins-PRS RNA oligonucleotide.…”

“…Thus, there may be a requirement of other cis-acting mRNA elements to obtain full control of glucoseinduced changes in mRNA stability. The induced stability of TH mRNA in response to hypoxia is for instance dependent of protein interactions with both the 3Ј-UTR and the coding region of the mRNA (29).…”

Regulation of α1(I) Collagen Messenger RNA Decay by Interactions with αCP at the 3′-Untranslated Region

“…There are many similarities between the regulation of ␣1(I) collagen mRNA in activated HSCs and the regulation of ␣-globin and lipoxygenase mRNAs in erythroid cells and the tyrosine hydroxylase mRNA in response to hypoxia (17,(47)(48)(49). These mRNAs form a class of molecules post-transcriptionally regulated via 3Ј-UTR interactions with protein(s).…”

Differential utilization of poly(rC) binding protein 2 in translation directed by picornavirus IRES elements

“…In fact, much of the evidence compiled to date suggests that the internal initiation of translation on both type I and type II picornavirus IRES elements requires certain trans-acting cellular proteins in addition to canonical translation initiation factors (for reviews, see Jackson & Kaminski, 1995;Stewart & Semler, 1997)+ In this study, we investigated the process of translation as directed by the IRES elements of poliovirus (PV), coxsackievirus strain B (CVB), human rhinovirus (HRV), encephalomyocarditis virus (EMCV), and foot-andmouth disease virus (FMDV)+ Specifically, we examined whether these picornaviruses, representing four of the six picornavirus genera, require the cellular protein, PCBP2, for viral translation+ PCBP2 is a cellular RNA-binding protein that interacts with the 59 NCR of poliovirus RNA (Dildine & Semler, 1992;Blyn et al+, 1995Blyn et al+, , 1996+ The protein contains three hnRNP K-homologous (KH) RNA-binding domains and has a binding preference for poly(rC)+ PCBP2 mRNA is widely expressed and has been localized both in the cytoplasm and the nucleus (Leffers et al+, 1995)+ To date, the only known functions attributed to PCBP2 are its functional associations with the 39 NCRs of the a-globin and the tyrosine hydroxylase mRNAs in complexes that impart stability (Kiledjian et al+, 1995;Paulding & Czyzyk-Krzeska, 1999)+ Similar complexes have also been found to form on the 39 NCRs of lipoxygenase, a(I)-collagen, and erythropoietin mRNAs (Holcik & Liebhaber, 1997;Czyzyk-Krzeska & Bendixen, 1999)+ In addition to the role of PCBP2 in cellular mRNA stability, a number of additional functions relating to viral infection have been ascribed to the protein+ We have previously shown that the interaction of PCBP2 with the poliovirus 59 NCR performs dual functions in the life cycle of the virus by facilitating the initiation of both viral protein synthesis and viral RNA synthesis (Blyn et al+, 1997;Parsley et al+, 1997)+ Likewise, Andino and colleagues confirmed this functional duality and proposed that PCBP could also function in a mechanism involving accessory proteins of viral origin to regulate the processes of translation and replication (Gamarnik & Andino, 1997)+ In addition to its documented functions in the poliovirus life cycle, PCBP2 also plays a role in the translation of HAV genomic RNA (Graff et al+, 1998)+ Furthermore, human papillomavirus type 16 (HPV16), a member of the Papovaviridae, has evolved a mechanism to utilize PCBP2+ For HPV16, PCBP2 acts as a regulator of late gene expression (Collier et al+, 1998)+ This work investigates the possibility that RNA binding by PCBP2 is a general requirement for the internal initiation of translation on picornavirus IRES elements+ Competition RNA mobility-shift assays conducted with 32 P-labeled PV stem-loop IV RNA, recombinant PCBP2 (rPCBP2), and five different picornavirus 59 NCRs confirmed a conserved physical interacti...…”