Detection and Characterization of a 3′ Untranslated Region Ribonucleoprotein Complex Associated with Human α-Globin mRNA Stability

128

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/...

contrasting

confidence: 56%

“…A similar procedure was used previously to purify the protein factor binding to the cytidine-rich elements in ␣ 2 -globin mRNA (9,10). As expected, we purified the 40-kDa poly(C)-binding protein that corresponds to PCBP (12), also known as ␣CP (9, 10) or hnRNPE (17,18), as identified by the specific antibodies.…”

mentioning

confidence: 99%

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

Paulding¹,

Czyzyk-Krzeska

1999

128

“…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).…”

Section: Discussionmentioning

confidence: 99%

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

Lindquist

Parsons

Stefanovic

et al. 2004

Liver fibrosis is characterized by an increased deposition of extracellular matrix proteins, including collagen type I, by activated hepatic stellate cells (HSCs).Previous studies have shown that this increase is mediated primarily by a post-transcriptional mechanism. In particular, the RNA-binding protein ␣CP binds to the ␣1(I) collagen 3 -untranslated region (UTR) and stabilizes this RNA in activated, but not quiescent, HSCs. This study examines the role of ␣CP in the decay of transcripts containing the collagen 3 -UTR in extracts obtained from NIH fibroblasts and quiescent and activated HSCs. Using an in vitro decay system, ␣CP binding activity was competed out with the addition of wild type oligonucleotides, but not with mutant oligonucleotides. Competition of ␣CP binding activity increased the rate of decay of wild type transcripts containing the ␣CP 3 -UTR binding site, but not of transcripts containing a mutated binding site. Quiescent HSC extracts contain no ␣CP binding activity and have no difference in the rate of decay of transcripts with wild type and mutant binding sites for ␣CP. The addition of recombinant ␣CP was sufficient to increase the half-life of the wild type transcript, whereas that of the mutant transcript was minimally changed. In vitro decay assays performed with activated HSC extracts that contain ␣CP binding activity demonstrate a markedly reduced decay rate of wild type compared with mutant transcripts. In vivo small interfering RNA experiments targeting ␣CP showed a reduction of the binding activity of ␣CP and a concomitant reduction in intracellular levels of ␣1(I) collagen messenger RNA. In conclusion, this study demonstrates the direct role of ␣CP in the stabilization of ␣1(I) collagen messenger RNA by blocking RNA degradation in activated HSCs.Hepatic fibrosis is a result of excess deposition of extracellular matrix proteins, including type I collagen, by activated hepatic stellate cells (HSCs) 1 (1-3). As a general response to most chronic stimuli, HSCs are transformed from a quiescent to that of an activated phenotype (4, 5). Isolation of HSCs from normal livers and plating them on plastic results in a spontaneous activation of the HSCs that closely mimics the activation observed in the liver in vivo (6). This culture-induced activation includes an increase in ECM production, including ␣1(I) collagen, loss of vitamin A stores, increased proliferation rate, and increased expression of smooth muscle ␣-actin (7, 8). The increase in ␣1(I) collagen expression is primarily because of an increase in the half-life of the ␣1(I) collagen mRNA molecule, from 1.5 h in quiescent HSCs to greater than 24 h in activated HSCs (9, 10). The increase in ␣1(I) collagen mRNA stability coincides with an increase in the binding activity of ␣CP (hnRNP E2, PCBP2) to a C-rich region in the ␣1(I) collagen 3Ј-untranslated region (UTR) (10). Mutant mRNAs lacking this binding region do not demonstrate increased stability in activated HSCs. ␣CP binds to the 3Ј-UTRs of several other mRNAs, including ␣-globin,...

“…Both the 3Ј-CR and 3Ј-UTR sequence in the 68-nt fragment are necessary for assembly of a unique C-binding RNP complex at this site (1). Interestingly, the stability of the ␣-globin mRNA is also regulated by a determinant on the proximal edge of the 3Ј-UTR that is dependent upon sequence in the adjacent 3Ј-CR (25,26).…”

Section: Discussionmentioning

confidence: 99%

Stability Determinants Are Localized to the 3′-Untranslated Region and 3′-Coding Region of the Neurofilament Light Subunit mRNA Using a Tetracycline-inducible Promoter

Cañete‐Soler

Schwartz

Yang

et al. 1998

The tetracycline-responsive expression system of Bujard was used to compare rates of decay of wild-type and mutant neurofilament (NF) light subunit (NF-L) mRNAs. Optimal conditions for activation and inactivation of the target transgene were determined using a luciferase reporter gene. Analyses of mRNA stability were thereupon conducted on cells that were doubly transfected with transactivator and inducible target genes and derived from pooled clones of transfected cells. Rates of mRNA decay were compared upon inactivation of the transgenes after high levels of mRNA had been induced. Altering mRNA stability is a universal post-transcriptional mechanism for modulating gene expression during growth, development and differentiation. Post-transcriptional regulation may be particularly important in cells, such as neurons, that undergo extensive differentiation and change after cessation of cell division. Alterations in mRNA stability are known to affect the levels of several key proteins in post-mitotic neurons (2-5). Increases in mRNA stability accompany the postnatal up-regulation in neurofilament (NF) 1 expression and are, therefore, instrumental in determining the size and shape of neuronal processes (6, 7). Moreover, levels of NF expression have been implicated in motoneuron disease by virtue of the selective motoneuron degeneration that occurs when a light (NF-L) or heavy (NF-H) subunit transgene is overexpressed in transgenic mice (8, 9). A transient up-regulation of endogenous NF mRNA also precedes the spontaneous motoneuron degeneration in the Wobbler mice (10).Functional assays for mapping stability determinants use a variety of methods to compare stabilities of mutant versus wild-type mRNAs (11). Most of these methods are limited by their disruptive or pleiotrophic effects on the host cells and are, therefore, especially problematic for assessing stabilities of long-lived mRNAs. An alternative method is provided by the development of the tetracycline-responsive promoter system whereby target gene expression is stringently controlled by a non-toxic antibiotic ligand (12-19). Here, we have adapted this system to assess mRNA stability in neuronal and non-neuronal cell lines and to map stability determinants in the NF-L transcript. The method has enabled the identification of stability determinants in the 3Ј-CR and 3Ј-UTR of the NF-L mRNA and have localized a key determinant to a 68-nt sequence at the junction of the 3Ј-CR and 3Ј-UTR. EXPERIMENTAL PROCEDURESPlasmids-pUHD15.1, pUHD10.3, and pUHD13.3 were a generous gift of Dr. H. Bujard. pUHD15.1M is a modified expression vector that was rendered autoinducible (see Ref. 16) by placing an EcoRI site and consensus translation start sequence immediately upstream of the tTA open reading frame and subcloning the EcoRI/BamHI PCR fragment from pUHD15.1 into the polylinker of pUHD10.3. Fig. 1) were constructed by splicing together three PCR fragments. Fragment 1 extended from ϩ1 to a BglII site at ϩ829 in exon 1, fragment 2 extended from ϩ829 to a KpnI site at ϩ1208, a...