Cytoplasmic stabilities of 3′UTR-polymorphic prothrombin mRNAs

The active form of prothrombin (F.II) participates in disparate biochemical pathways that either promote or inhibit hemostatic processes. Functional levels of plasma prothrombin are maintained within physiologically relevant limits by critical, yet poorly understood regulatory processes that prevent both hemorrhagic and hypercoagulable states. While the pathological effects of F.II post-translational dysregulation have been known for several decades (e.g., vitamin K deficiency), the importance of other post-transcriptional processes to regulated F.II expression have only recently been recognized. Our current understanding of these mechanisms is largely evolved from structural and functional studies of F.II mRNAs that that carry a GΠA mutation at position 20210. This polymorphism comprises a single-nucleotide transition at the nominal position where F.II pre-mRNA undergoes 3′ cleavage and polyadenylation. Heterozygotes for the F.II G20210A mutation exhibit prothrombin levels that are approximately 25% higher than normal(1), and display elevated risks for both venous(2) and possibly arterial(3) thromboses.

The positioning of the GΠA transition within the prothrombin 3′UTR suggests that the mechanism(s) responsible for hyperprothrombinemia is likely to affect the properties of nascent and/or mature F.II mRNAs. Recent studies have fulfilled this expectation, demonstrating that the G20210A mutation enhances the efficiency of 3′-end formation for pre-mRNAs carrying the F.II 3′UTR(4,5). A single study suggests that the polymorphism also increases the cytoplasmic half-life of F.II mRNA(6), a conclusion that is inconsistent with previous quantitative analyses of endogenous full-length F.II mRNAs prepared from surgical liver specimens(7).

The current manuscript attempts to resolve these discordant claims by formally testing the impact of the G20210A mutation on the stability of mRNAs carrying the F.II 3′UTR. Our studies assess the half-live values of stably expressed F.II reporter mRNAs in vivo in intact hepatophenotypical HepG2 cells, using a novel transcriptional chase approach that is specifically designed to avoid disrupting cellular homeostasis. These studies demonstrate equal stabilities for reporter mRNAs carrying G- or A-polymorphic F.II 3′UTRs, implicating the importance of mRNA processing and/or translation--and not cytoplasmic half-life--to regulated prothrombin expression.

Formal mRNA stability analyses were conducted using cultured cells engineered to express a tetracycline trans-activator (tTA) fusion protein that drives transcription of genes linked to a recombinant tetracycline response element(8) (TRE; Fig 1A). tTA activity is rapidly inhibited in the presence of doxycycline (dox), repressing transcription of TRE-linked genes. Hepatophenotypical HepG2-cell subclones were generated that stably express the tTA transactivator, from which one--exhibiting efficient dox-conditional repression--was selected for subsequent experiments (Fig 1B). The suitability of these HepG2^tTA cells for mRNA stability analyses was validated by comparing the half-life (t_1/2) value of hβ-globin (β^WT) mRNA(9) to t_1/2 values of derivative β-globin mRNAs containing a defined mRNA-destabilizing 3′UTR motif(10) (β^ARE1 and β^ARE2; Fig 1C). In these experiments, cells were co-transfected with pTRE-β^WT and either pTRE-β^ARE1 or -β^ARE2, exposed to dox, and the relative levels of the paired mRNAs established at defined intervals using a competitive RT-PCR method. In contrast to β^WT mRNA, the level of each of the unstable derivative RNAs fell rapidly (Figs 1D, E), validating the utility of the method for assessing the relative half-lives of mRNAs encoded by TRE-linked genes.

Fig 1. Generation and validation of tTA-expressing HepG2 cells.

(A) Principles of doxycycline-regulated gene expression. The tTA fusion protein comprises the Tet repressor DNA binding protein (TetR) from the tetracycline-resistance operon of E. coli, linked to the VP16 transcriptional activation domain (VP16) from the human Herpes simplex virus. The TRE element comprises tandem tetracycline operator sequences (TetO) linked to a cytomegaloviral immediate-early promoter (CMV). The strong transcriptional activating properties of the tTA fusion protein (top) are inactivated in the presence of doxycycline (dox; bottom). (B) Identification of derivative HepG2 cells exhibiting dox-responsive gene silencing. Subclones expressing the tTA fusion protein(8) were generated by transfection with pTetOff (Clontech). G418-resistant HepG2 clones were co-transfected with a TRE-linked firefly luciferase gene and a control renilla luciferase gene linked to a constitutive, dox-indifferent SV40 promoter/enhancer. Aliquots were cultured for 24 h in dox-free and dox-supplemented media, and firefly and renilla luciferase activities subsequently determined by luminometry. The ratio of firefly luciferase activities in dox-free and dox-exposed cells, normalized to renilla activities, is plotted. (C) Construction of genes encoding stable and unstable test mRNAs. The construction of pTRE-β^WT, -β^ARE1, and -β^ARE2 are described elsewhere(15). TRE-β^WT contains the full-length human β-globin gene and contiguous 3′-flanking region; TRE-β^ARE1 is identical except for the insertion of a 59-bp AU-rich mRNA instability element (ARE, indicated by ⊠), 15 nts 3′ to the translation stop codon. Both test genes abut a TRE transcriptional control element (crosshatched). Introns and 3′-flanking sequences are denoted by thin and thick lines, respectively. A third derivative β-globin test gene containing an ARE insertion at position 93 of the 3′UTR was similarly constructed (β^ARE2, not shown). (D) Stabilities of test mRNAs in HepG2^tTA cells. pTREβ^ARE1 and control pTRE-β^WT were co-transfected into HepG2^tTA cells. Cultures were amended with dox and sacrificed at the indicated intervals. [³²P]-labeled RT-PCR-amplified β-globin cDNAs, encompassing the ARE-insertion site, were resolved on a denaturing acrylamide-urea gel. mRNAs from cells transfected singly with TRE-β^WT or -β^ARE1 were analyzed in control lanes C1 and C2, respectively. (E) Decay curves for test mRNAs. Signal intensities from panel D were quantitated by PhosphorImager densitometry, and the β^ARE1: β^WT ratios plotted relative to the post-dox interval (■). Results were confirmed by parallel analysis of co-expressed β^ARE2 and β^WT mRNAs (●). (F) Construction of genes encoding βF.IIG and βF.IIA mRNAs. The construction and structural validation of reporter genes containing the F.II 5′UTR, 3′UTR, and contiguous 3′-flanking region is described elsewhere(11). pTRE-βF.IIG comprises (i) the native F.II 5′UTR, (ii) the native human β-globin coding region and included introns, and (iii) the native F.II 3′UTR and contiguous 3′ flanking region. pTRE-βF.IIA is identical except for a GΠA substitution at 3′UTR position 20210. Sequences derived from the β-globin (gray) and prothrombin genes (black) are indicated. (G) Decay analyses of stably expressed βF.II mRNAs. Total RNA was isolated from eight independent double-stable βF.IIG- and βF.IIA-expressing HepG2^tTA cell lines at baseline (t=0), and 2, 4, 9, and 24 h after exposure to dox. Levels of reporter mRNAs were determined in triplicate by qRT-PCR, using probe sets specific for β-globin or endogenous control β-actin mRNA. Samples were analyzed using an Applied Biosystems ABI 7500 system; relative quantities of βF.II mRNAs were established using the ΔΔCt method. (H) Stabilities of stably expressed βF.IIG and βF.IIA mRNAs. Half-life values for mRNAs were derived from exponential decay curves (panel G) using a least-squares method. Individual (●) and average t_1/2 values (bars, +/− 1 S.D.) are illustrated for βF.IIG and βF.IIA mRNAs. A dashed line indicates the t_1/2 value for βF.IIA mRNA required to account for the 125% F.II expression levels observed in G20210A heterozygotes.

Analyses of 20210G- or 20210A-variant prothrombin 3′UTRs utilized reporter genes derived from pTRE-β^WT. The parental gene was modified to contain 5′UTR, 3′UTR, and contiguous 3′-flanking region sequences from both the wild-type and G20210A-variant F.II genes, in place of the corresponding β-globin regions(11) (pTRE-βF.IIG and -βF.IIA, respectively; Fig 1F). Double-stable HepG2^tTA cells were generated that constitutively express either βF.IIG or βF.IIA mRNAs from the cognate reporter genes(11). Of 35 subclones expressing the TRE-linked genes, eight--representing a range of steady-state expression levels--were selected for metabolic t_1/2 analyses (not shown). Analyses were conducted by exposing cells to dox, and determining reporter mRNA levels at defined intervals by qRT-PCR. Under these formal conditions, the average stabilities of βF.IIG and βF.IIA mRNAs did not differ significantly (p>0.1, Fig 1G). Importantly, all four independently measured t_1/2 values for βF.IIA mRNA were lower than the average t_1/2 value of 11.3 h that would be required to account for the pathological 25% elevation in plasma prothrombin levels observed in G20210A heterozygotes (Fig 1H).

The prothrombin G20210A polymorphism is believed to interfere with post-transcriptional processes that specify the efficiency of F.II mRNA 3′-end formation(4,5). One study suggests that the mutation additionally prolongs F.II mRNA stability(6), although this effect was not observed in a similar transcriptional chase analysis that used the RNA-polymerase inhibitor DRB (5,6-dichloro-1-β-D-ribobenzimidazole)(12). Hyperstable F.IIA mRNAs might also be anticipated to accumulate to greater levels than normostable F.IIG mRNAs, an expectation that conflicts with previous observations that the polymorphic mRNAs are present in equal concentrations in intact liver obtained from a prothrombin G20210A heterozygote(7). Consequently, there remains significant uncertainty regarding the biochemical effect of the G20210A mutation on F.II mRNA stability. The formal analyses reported here indicate that that the G20210A polymorphism does not materially alter the half-life of F.II mRNA, endorsing the notion that previously reported defects in the post-transcriptional processing of F.IIA mRNA are solely responsible for its attendant hyperprothrombinemia.

The discordance between our observations and those reported earlier may result from features of our method that reduce the likelihood of experimental artifact. Like others(6,12), we conducted stable-cell analyses to permit half-life values to be formally calculated, rather than simply inferred from non-steady-state levels of mRNAs transcribed from episomal DNAs in transiently transfected cells. Like others(6,12), we also employed hepatophenotypical HepG2 cells to respect strong evidence that post-transcriptional events affecting eukaryotic mRNAs can be highly cell-type specific(13). Our methods diverge from earlier analyses, however, in both the gene specificity of the transcriptional chase method, as well as in the verisimilitude of the F.II reporter mRNAs that we characterize. First, our dox-conditional experimental design permits pTRE gene-specific transcriptional control, in contrast to methods utilizing actinomycin D(6) or DRB(12). Both of these agents are non-specific in effect, disrupting normal cellular homeostasis by globally arresting the transcription of all mRNAs. Second, our reporter genes contain F.II 3′UTRs contiguous with native F.II 3′-flanking sequence, so that the transcribed mRNAs undergo 3′-cleavage/polyadenylation at their normal position(11). Reporter genes used elsewhere are ligated to heterologous flanking region, transcribing mRNAs that are polyadenylated at an artificial downstream site, and that bury the 20210 polymorphism within unrelated primary sequence(6). Finally, our genes are engineered to contain the F.II 5′UTR (rather than a heterologous 5′UTR) to account for potential regulatory interactions between trans-acting factors that bind to the F.II 5′ and 3′UTRs(14). While technical differences may explain the disparate results obtained in independent studies of F.II mRNA stability, the methodologies we employ provide a high level of confidence in both the reliability of our data, as well as their biological implications.

Our previously published data suggest a mechanism for the gain-of-function characteristics of G20210A F.II mRNA, demonstrating that variations in the secondary structure of the F.II 3′UTR can alter productive binding of trans-acting regulatory factors(11). The current data indicates that structural features of F.IIG and F.IIA 3′UTRs that exert an effect on the efficiency of 3′-end formation do not alter overall stability of the prothrombin mRNA. Further studies are required to assess whether the G20210A mutation alters either the binding or the function of trans-acting factors that mediate this aspect of post-transcriptional mRNA regulation.