Inhibition of the Splicing of Glucose-6-phosphate Dehydrogenase Precursor mRNA by Polyunsaturated Fatty Acids
2002; Elsevier BV; Volume: 277; Issue: 34 Linguagem: Inglês
10.1074/jbc.m203196200
ISSN1083-351X
AutoresHuimin Tao, Wioletta Szeszel-Fedorowicz, Batoul Amir-Ahmady, Matthew Gibson, Laura P. Stabile, Lisa M. Salati,
Tópico(s)Hyperglycemia and glycemic control in critically ill and hospitalized patients
ResumoPolyunsaturated fatty acids inhibit the expression of hepatic glucose-6-phosphate dehydrogenase (G6PD) by changes in the amount of G6PD pre-mRNA in the nucleus in the absence of changes in the transcription rate of the gene. We have compared the nuclear accumulation of partially and fully spliced mRNA for G6PD in the livers of mice fed diets highversus low in polyunsaturated fat. Consumption of a diet high in polyunsaturated fat decreased the accumulation of partially spliced forms of the G6PD pre-mRNA. Examining the fate of multiple introns within the G6PD primary transcript indicated that in mice fed a high fat diet, G6PD pre-mRNA containing intron 11 accumulated within the nucleus, whereas G6PD mature mRNA abundance was inhibited 50% or more within the same livers. Transient transfection of RNA reporters into primary hepatocyte cultures was used to localize the cis-acting RNA element involved in this regulated splicing. Reporter RNA produced from constructs containing exon 12 were decreased in amount by arachidonic acid. The extent of this decrease paralleled that seen in the expression of the endogenous G6PD mRNA. The presence of both exon 12 and a neighboring intron within the G6PD reporter RNA was essential for regulation by polyunsaturated fatty acid. Inhibition was not dependent on the presence of the G6PD polyadenylation signal and the 3′-untranslated region, but substitution with the SV40 poly(A) signal attenuated the inhibition by arachidonic acid. Thus, exon 12 contains a putative splicing regulatory element involved in the inhibition of G6PD expression by polyunsaturated fat. Polyunsaturated fatty acids inhibit the expression of hepatic glucose-6-phosphate dehydrogenase (G6PD) by changes in the amount of G6PD pre-mRNA in the nucleus in the absence of changes in the transcription rate of the gene. We have compared the nuclear accumulation of partially and fully spliced mRNA for G6PD in the livers of mice fed diets highversus low in polyunsaturated fat. Consumption of a diet high in polyunsaturated fat decreased the accumulation of partially spliced forms of the G6PD pre-mRNA. Examining the fate of multiple introns within the G6PD primary transcript indicated that in mice fed a high fat diet, G6PD pre-mRNA containing intron 11 accumulated within the nucleus, whereas G6PD mature mRNA abundance was inhibited 50% or more within the same livers. Transient transfection of RNA reporters into primary hepatocyte cultures was used to localize the cis-acting RNA element involved in this regulated splicing. Reporter RNA produced from constructs containing exon 12 were decreased in amount by arachidonic acid. The extent of this decrease paralleled that seen in the expression of the endogenous G6PD mRNA. The presence of both exon 12 and a neighboring intron within the G6PD reporter RNA was essential for regulation by polyunsaturated fatty acid. Inhibition was not dependent on the presence of the G6PD polyadenylation signal and the 3′-untranslated region, but substitution with the SV40 poly(A) signal attenuated the inhibition by arachidonic acid. Thus, exon 12 contains a putative splicing regulatory element involved in the inhibition of G6PD expression by polyunsaturated fat. glucose-6-phosphate dehydrogenase cytomegalovirus Rous sarcoma virus chloramphenicol acetyltransferase precursor mRNA untranslated region Polyunsaturated fatty acids are potent regulators of cellular metabolism. As components of phospholipids, they are involved in signal transduction from specific plasma membrane receptors and are precursors for the synthesis of eicosanoids. When present in the diet of animals or the medium of cells in culture, polyunsaturated fats can both increase and decrease the activity or amount of specific cellular proteins. In this regard, polyunsaturated fatty acids have long been known to decrease the capacity of liver cells to synthesize fatty acidsde novo. The pathway of de novo fatty acid biosynthesis is essential for the conversion of energy substrates, such as glucose, which are in excess of immediate needs, to fatty acids that can be stored as triacylglycerols. This pathway is most active in liver and adipose tissue and involves a family of enzymes referred to as the lipogenic enzymes (for review, see Ref. 1Hillgartner F.B. Salati L.M. Goodridge A.G. Physiol. Rev. 1995; 75: 47-76Crossref PubMed Scopus (396) Google Scholar). These enzymes include ATP-citrate lyase, acetyl-CoA carboxylase, fatty acids synthase, malic enzyme, and glucose-6-phosphate dehydrogenase (G6PD).1 Consistent with their role in energy metabolism, the activities of these enzymes are induced when animals are fed a high carbohydrate diet and decreased during starvation. Likewise, lipogenic enzyme activity is decreased by a high fat diet, particularly a diet rich in polyunsaturated fatty acids. Regulation of the activities of ATP-citrate lyase, acetyl-CoA carboxylase, fatty acid synthase, and malic enzyme involves changes in the rate of transcription of these genes (2Blake W.L. Clarke S.D. J. Nutr. 1990; 120: 1727-1729Crossref PubMed Scopus (119) Google Scholar, 3Paulauskis J.D. Sul H.S. J. Biol. Chem. 1989; 264: 574-577Abstract Full Text PDF PubMed Google Scholar, 4Stapleton S.R. Mitchell D.A. Salati L.M. Goodridge A.G. J. Biol. Chem. 1990; 265: 18442-18446Abstract Full Text PDF PubMed Google Scholar, 5Hillgartner F.B. Charron T. Chesnut K.A. Biochem. J. 1996; 319: 263-268Crossref PubMed Scopus (44) Google Scholar, 6Katsurada A. Iritani N. Fukuda H. Matsumura Y. Nishimoto N. Noguchi T. Tanaka T. Eur. J. Biochem. 1990; 190: 435-441Crossref PubMed Scopus (136) Google Scholar, 7Ma X.J. Salati L.M. Ash S.E. Mitchell D.A. Klautky S.A. Fantozzi D.A. Goodridge A.G. J. Biol. Chem. 1990; 265: 18435-18441Abstract Full Text PDF PubMed Google Scholar). In general, these changes in transcription rate fail to account for the magnitude of change in the amount of mRNA for these enzymes. Thus, posttranscriptional regulation has been proposed as an additional mechanism to account for the regulation of the amount of some of these enzymes (6Katsurada A. Iritani N. Fukuda H. Matsumura Y. Nishimoto N. Noguchi T. Tanaka T. Eur. J. Biochem. 1990; 190: 435-441Crossref PubMed Scopus (136) Google Scholar, 8Dozin B. Rall J.E. Nikodem V.M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 4705-4709Crossref PubMed Scopus (47) Google Scholar). Regulation of the expression of G6PD is in sharp contrast to other members of the lipogenic enzyme family in that starvation, refeeding, and dietary polyunsaturated fat result in large changes in the amount of G6PD mRNA but do not regulate the transcriptional activity of the G6PD gene (9Stabile L.P. Hodge D.L. Klautky S.A. Salati L.M. Arch. Biochem. Biophys. 1996; 332: 269-279Crossref PubMed Scopus (29) Google Scholar). Posttranscriptional regulation of gene expression can occur at multiple steps during RNA processing or by changes in the stability of the mature mRNA. The processing of the nascent transcript to the mature mRNA includes the addition of the 5′-m7GppG cap, splicing, and 3′-end formation. The correct processing of an mRNA is essential for its release from the site of transcription and export to the cytoplasm (10Hilleren P. McCarthy T. Rosbash M. Parker R. Jensen T.H. Nature. 2001; 413: 538-542Crossref PubMed Scopus (294) Google Scholar, 11Antoniou M. Geraghty F. Hurst J. Grosveld F. Nucleic Acids Res. 1998; 26: 721-729Crossref PubMed Scopus (65) Google Scholar, 12Custodio N. Carmo-Fonseca M. Geraghty F. Pereira H.S. Grosveld F. Antoniou M. EMBO J. 1999; 18: 2855-2866Crossref PubMed Scopus (178) Google Scholar). Thus, the efficient and complete maturation of mRNA is a potential control point of gene expression. Previous studies in our laboratory have characterized the posttranscriptional regulation of G6PD by dietary factors. The dietary paradigm of starvation then refeeding causes a 15-fold or more increase in G6PD mRNA abundance. Regulation of G6PD mRNA abundance during the refeeding of starved mice is caused by an increase in the rate of spliced RNA accumulation in the nucleus (13Amir-Ahmady B. Salati L.M. J. Biol. Chem. 2001; 276: 10514-10523Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The increase in spliced RNA accumulation cannot be accounted for by the rate of accumulation of polyadenylated RNA nor does it involve changes in the length of the poly(A) tail. Thus, the G6PD gene is transcribed at a continuous and unchanging rate, and the amount of mature G6PD mRNA produced is regulated during the splicing of the primary transcript to the mature mRNA. The absence of transcriptional regulation of G6PD makes it an ideal model to study this unique posttranscriptional mechanism. We asked if inhibition of G6PD expression by polyunsaturated fatty acids occurs at the same step as positive regulatory stimuli. In liver, G6PD activity and mRNA amount decrease 80% in mice consuming a diet high in polyunsaturated fat versus those fed a low fat diet (9Stabile L.P. Hodge D.L. Klautky S.A. Salati L.M. Arch. Biochem. Biophys. 1996; 332: 269-279Crossref PubMed Scopus (29) Google Scholar). The decrease in cytoplasmic RNA is preceded by a similar decrease in the amount of mRNA in the nucleus (14Hodge D.L. Salati L.M. Arch. Biochem. Biophys. 1997; 348: 303-312Crossref PubMed Scopus (21) Google Scholar). This effect of dietary fat is recapitulated in hepatocytes in primary culture. In this regard, incubation of primary rat hepatocytes with arachidonic acid results in a parallel decrease in the amount of G6PD enzyme activity and mRNA abundance in the absence of a change in the transcriptional activity of the gene (15Stabile L.P. Klautky S.A. Minor S.M. Salati L.M. J. Lipid Res. 1998; 39: 1951-1963Abstract Full Text Full Text PDF PubMed Google Scholar). Similar to the inhibition by dietary fat, the decrease in G6PD mRNA by arachidonic acid is preceded by a decrease in the amount of mRNA in the nucleus (15Stabile L.P. Klautky S.A. Minor S.M. Salati L.M. J. Lipid Res. 1998; 39: 1951-1963Abstract Full Text Full Text PDF PubMed Google Scholar). Thus, inhibition of G6PD expression by polyunsaturated fatty acids occurs in the nucleus and is the result of the intracellular actions of fatty acids. Determining the molecular details of this regulation will provide new information on the breadth of mechanisms by which nutrients control gene expression. In this paper, we present data that define a new mechanism by which polyunsaturated fats inhibit gene expression. We demonstrate that inhibition of G6PD expression involves a decrease in the rate of splicing of the G6PD RNA transcript. Using intact mice, we have identified a slowly spliced intron, intron 11, in mice consuming a high fat diet. We have extended these results using primary rat hepatocytes and have demonstrated that transcripts from pre-mRNA reporters containing exon 12 and a neighboring intron are inhibited by polyunsaturated fatty acids in a manner quantitatively similar to the endogenous gene. Male C57BL/6 mice 4 weeks of age were obtained from Charles River Corp. Mice were adapted to reverse cycle room (lights on 7:00 p.m., lights off 7:00 a.m.) for 7 days while maintained on standard chow diet. Mice were switched to a purified basal diet containing 58.43% by weight glucose, 16.45% celufil, 4% salt mix, and 0.02% vitamin mix (Amersham Biosciences) supplemented with 1% (low fat) or 6% (high fat) by weight polyunsaturated fat in the form of safflower oil (Sigma) for 7 days. Mice were sacrificed, and nuclear RNA was isolated at the times indicated in the figures. Male Sprague-Dawley rats (∼200 g from Harlan Laboratories, Indianapolis, IN) fed a standard chow diet (Harlan Teklad) were used for all experiments. Rats were starved for 16 h, refed the high glucose, fat free diet for 8 h, and then starved for 16 h before use as hepatocyte donors. Hepatocytes were isolated by a modification of the technique of Seglen (16Seglen P.O. Exp. Cell Res. 1973; 82: 391-398Crossref PubMed Scopus (1027) Google Scholar) as previously described (15Stabile L.P. Klautky S.A. Minor S.M. Salati L.M. J. Lipid Res. 1998; 39: 1951-1963Abstract Full Text Full Text PDF PubMed Google Scholar). Hepatocytes (3.3 × 106) were placed in 60-mm dishes coated with rat tail collagen in Hi/Wo/Ba medium (Waymouth's MB752/1 plus 20 mm HEPES, pH 7.4, 0.5 mm serine, 0.5 mm alanine, 0.2% bovine serum albumin) plus 5% newborn calf serum (37 °C, 5% CO2). Cell viability in all experiments was 90% or greater as estimated by trypan blue (0.04%) exclusion. After 3–4 h, the medium was replaced with serum-free medium. After an additional 16 h of incubation, the medium was replaced with medium containing the treatments indicated in the figure legends and a Matrigel overlay (0.3 mg/ml; BD PharMingen) (17Shih H.M. Towle H.C. Biotechniques. 1995; 18: 813-816PubMed Google Scholar). Subsequently, the medium was changed every 12–24 h to one of the same composition but without Matrigel. Arachidonic acid (Nu-Check Prep, West Elysian, MN) was bound to bovine serum albumin (18Mooney R.A. Lane M.D. J. Biol. Chem. 1981; 256: 11724-11733Abstract Full Text PDF PubMed Google Scholar). The fatty acid (4 mm), albumin (1 mm) stocks contained butylated hydroxytoluene (0.01%), and the medium contained α-tocopherol phosphate, disodium (10 μg/liter), to minimize oxidation of fatty acids. Transfection was done using Lipofectin following the manufacturer's protocol (Invitrogen) and using 2–4 μg of test DNA and 1 μg of RSV CAT to control for transfection efficiency. The ratio of DNA to liposome reagent was 1:6.7 in all experiments. Transfection was begun after 4 h of culture, and the transfection medium remained on the hepatocytes for 16 h. In transfection experiments the Matrigel overlay was added 4 h after the transfection began (17Shih H.M. Towle H.C. Biotechniques. 1995; 18: 813-816PubMed Google Scholar). Plasmids used in the analysis of cis-acting elements in the G6PD primary transcript were made using pGL3-Basic (Promega Corp., Madison, WI) as the vector backbone. The luciferase gene was removed from this vector, and portions of G6PD genomic DNA or the G6PD cDNA were inserted in its place. Either the CMV promoter/enhancer sequences (1–640 bp from the pCMVβ vector, CLONTECH) or the G6PD promoter sequences (−780 to +3) were inserted into the multiple cloning site to drive expression of the test sequences. The plasmids contained either the SV40 polyadenylation signal provided in the plasmid or the G6PD polyadenylation signal. The RSV CAT plasmid used to control for transfection efficiency was constructed using the pCAT3 Basic vector (Promega) and inserting the RSV long terminal repeat from pRSV CAT (19Gorman C.M. Merlino G.T. Willingham M.C. Pastan I. Howard B.H. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6777-6781Crossref PubMed Scopus (880) Google Scholar). Total RNA from 2–3 plates per treatment was isolated by the method of Chomczynski and Sacchi (20Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63187) Google Scholar). Quantitation of RNA using Northern analysis was done as previously described (9Stabile L.P. Hodge D.L. Klautky S.A. Salati L.M. Arch. Biochem. Biophys. 1996; 332: 269-279Crossref PubMed Scopus (29) Google Scholar). Nuclei from liver were isolated by a modification (13Amir-Ahmady B. Salati L.M. J. Biol. Chem. 2001; 276: 10514-10523Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) of the method of Leppard and Shenk (21Leppard K.N. Shenk T. EMBO J. 1989; 8: 2329-2336Crossref PubMed Scopus (126) Google Scholar). This protocol results in the isolation of nuclear RNA that is in the processing pathway and is essentially devoid of cytoplasmic contamination (13Amir-Ahmady B. Salati L.M. J. Biol. Chem. 2001; 276: 10514-10523Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In later experiments using probes with two introns (Figs. 3 and 4), the DNase I digestion and high salt extraction steps were eliminated because these probes permit the detection of pre-mRNA at two different steps in splicing. The absence of cytoplasmic contamination in this fraction was assessed as described (13Amir-Ahmady B. Salati L.M. J. Biol. Chem. 2001; 276: 10514-10523Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Cytoplasmic RNA in the nuclear RNA preparations was less than 1%.FIG. 4Pre-mRNA containing intron 11 selectively accumulates in mice fed the high fat diet. RNA isolation and the dietary protocol were as described in Fig. 3. The isolated RNA (30 μg) was analyzed by RNase protection assay using the pJW1 probe, depicted in Fig. 1. A, a representative assay is shown. The size and structure of the protected fragments are shown on theright of the figure. Single lines represent introns, and rectangles represent exons. Eachlane represents RNA from a single mouse from the 2-h time point shown in B. This assay is representative of two separate experiments and nuclear RNA preparations. B, RNase protection products representing unspliced and partially spliced RNA were quantified using ImageQuant software. Each barrepresents the mean ± S.E. of n = 4 mice with the exception of the 4-h low fat bars, which are n = 3 mice. The phosphorimaging units were normalized for the C content of the fragment (PI Un/# C). The identity of thebars is listed on the right of the figure. HF, high fat; LF, low fat; FL, full-length protected fragment.View Large Image Figure ViewerDownload (PPT) The following probes were designed for use in the ribonuclease protection assay (Fig. 1). The exon 2-intron 2 (E2-I2) and exon 8-intron 8-exon 9-intron 9 (pBG2) probes have been previously described (13Amir-Ahmady B. Salati L.M. J. Biol. Chem. 2001; 276: 10514-10523Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Probes to intron 6-exon 7-intron 7-exon 8 (pBG1) and exon 10-intron 11 (p200) were constructed by subcloning G6PD genomic DNA into pBluescript KS+ (Stratagene, La Jolla, CA). An additional probe, pJW1, was designed that hybridized to exon 10-intron 10-exon 11-intron 11 and exon 12. The template for this probe was synthesized from a genomic subclone by PCR amplification. The 5′ primer was 5′-CGGAATTCAACTTATGGCAACAG-3′; the underlined sequence is an EcoRI site for subcloning followed by G6PD exon 10 sequence. The 3′ primer was 5′-AAGGATTCCTCTCGATCAATCTTG-3′; the underlined sequence is a BamHI site for subcloning followed by G6PD exon 12 sequence. After amplification, the DNA was subcloned into pBluescript KS+, and the authenticity of these sequences was verified by sequencing. Two templates were designed for use in ribonuclease protection assays with RNA from transfected rat hepatocytes. Rat and mouse G6PD exon 13 templates were synthesized by PCR amplification of genomic DNA. These probes were targeted to a region of exon 13 that contains substantial mismatch between the rat and mouse sequences. In this way the rat probe detects mRNA produced from the endogenous G6PD gene but not the transfected gene, whereas the mouse probe detects mRNA produced from the transfected DNA and not the endogenous gene. The primer pairs for the rat template were 5′-CGGAATTCTGGAGGGACAATGACCAAACC-3′ and 5′-CGCGGATTC¯¯CGGTTGGATGAGGCTGACTATGGAC-3′, where the single and double underline represent EcoRI and BamHI restriction sites, respectively. The fragment was subcloned into pBluescript KS+ and sequenced. The mouse exon 13 template was as previously described (14Hodge D.L. Salati L.M. Arch. Biochem. Biophys. 1997; 348: 303-312Crossref PubMed Scopus (21) Google Scholar). Templates for probes to rat β-actin, CAT, and 18 S were purchased from Ambion, Inc. (Austin, TX). All templates were linearized before use in thein vitro transcription reaction. Antisense RNA probes were synthesized in an in vitrotranscription reaction. The probe and RNA were hybridized at 45 °C overnight, and RNase digestion was as previously described (14Hodge D.L. Salati L.M. Arch. Biochem. Biophys. 1997; 348: 303-312Crossref PubMed Scopus (21) Google Scholar). The resulting hybridization products protected from RNase digestion were separated in a 5% denaturing polyacrylamide gel. The gel was dried and placed in a storage phosphor cassette for 1–3 days. Images were quantified using ImageQuant software by Molecular Dynamics (AmershamBiosciences). In FIG. 2, FIG. 3, FIG. 4, the units obtained from the image analysis were expressed per number of cytosines in the protected fragment to permit comparison of the amount of RNA between fragments of different size. RNA from hepatocytes that had undergone transient transfection frequently contained significant amounts of residual plasmid DNA. Because this DNA would also bind to probes and result in protected fragments of the same size as the target RNA, the contaminating DNA was eliminated by digestion of the RNA with RNase-free DNase 1 (10 units/10-μg aliquot of hepatocyte RNA in 10 mm Tris, pH 7.5, 25 mm MgCl2, 5 mmCaCl2) for 30 min at 37 °C before hybridization with the radioactive probes. To investigate if dietary polyunsaturated fatty acids inhibit the efficiency of splicing of the G6PD transcript, we compared the amount of unspliced and spliced G6PD mRNA in the livers of mice fed the low fat versus the high fat diet. The amount of hepatic G6PD mRNA varies in amount in response to the normal feeding behavior of the mouse. Mice eat during the dark cycle and are relatively inactive during the light cycle. At the start of the dark cycle, the level of G6PD mRNA is very low. As the mice consume the low fat diet, the amount of G6PD mRNA increases 7-fold or more (14Hodge D.L. Salati L.M. Arch. Biochem. Biophys. 1997; 348: 303-312Crossref PubMed Scopus (21) Google Scholar); this increase occurs after a lag of 2–4 h. As the light cycle begins, the amount of G6PD mRNA decreases, returning to a very low level. Consumption of a diet high in polyunsaturated fat results in a 50% decrease in the amount of hepatic G6PD mRNA at the beginning of the dark cycle, and an attenuation of the feeding induced increase to less than 2-fold (14Hodge D.L. Salati L.M. Arch. Biochem. Biophys. 1997; 348: 303-312Crossref PubMed Scopus (21) Google Scholar). We reasoned that the continued increase in G6PD mRNA in the livers of mice fed a high fat diet reflected the stimulation in gene expression due to the carbohydrate in the diet. Thus, to measure only gene expression effects due to dietary fat, we measured G6PD mRNA abundance during the first 4 h of the feeding cycle, a time before the major increase in mRNA accumulation. RNA was isolated from both the cytoplasm and the nuclear insoluble fractions of mouse liver after the animals were adapted to either a low fat or a high fat diet for 7 days. The nuclear insoluble fraction is enriched in nascent RNA being transcribed and undergoing RNA processing (22Ciejek E.M. Nordstrom J.L. Tsai M.J. O'Malley B.W. Biochemistry. 1982; 21: 4945-4953Crossref PubMed Scopus (114) Google Scholar, 23Zeitlin S. Parent A. Silverstein S. Efstratiadis A. Mol. Cell. Biol. 1987; 7: 111-120Crossref PubMed Scopus (146) Google Scholar, 24Fey E.G. Krochmalnic G. Penman S. J. Cell Biol. 1986; 102: 1654-1665Crossref PubMed Scopus (330) Google Scholar). Two probes separated by 12 kilobases were used to detect G6PD RNA (Fig. 1, E2-I2 and p200 probes). Each probe hybridized across an exon/intron junction and, thus, measured G6PD RNA that contained that intron and RNA from which that intron had been spliced. The protected fragments are referred to as unspliced and spliced RNA, respectively, even though both protected fragments represent a mix of RNA containing one or more of the G6PD 12 introns. Feeding mice a high fat diet resulted in a 60–70% decrease in the amount of spliced G6PD RNA in both the nuclear and cytoplasmic fractions of mouse liver (Fig. 2, 0 h time point). Between 0 and 2 h of feeding, the amount of unspliced RNA (E2-I2 protected fragment) in the nucleus remained very low and was not different between mice fed the high fat and the low fat diet. This lack of difference in the amount of pre-mRNA is consistent with our previous data demonstrating that transcription of the G6PD gene is not regulated by polyunsaturated fat (9Stabile L.P. Hodge D.L. Klautky S.A. Salati L.M. Arch. Biochem. Biophys. 1996; 332: 269-279Crossref PubMed Scopus (29) Google Scholar). By 4 h, the amount of G6PD unspliced mRNA had increased in mice fed the low fat diet, whereas in mice fed the high fat diet, the amount of unspliced mRNA remained at a low basal level. At all time points, the amount of spliced RNA (E2 protected fragment) in the nucleus was 5–10-fold greater than the amount of unspliced RNA; however, this increase in the amount of spliced RNA was attenuated at all time points by the high fat diet. The changes in the amount of spliced RNA in the nucleus were similar to the changes in the amount of mature mRNA in the cytoplasm. Furthermore, the specific activity (phosphorimaging units/number of cytosines) of the spliced RNA was the same in both the cytoplasmic and nuclear pools, consistent with our previous results demonstrating that G6PD regulation by polyunsaturated fatty acids occurs in the nucleus (14Hodge D.L. Salati L.M. Arch. Biochem. Biophys. 1997; 348: 303-312Crossref PubMed Scopus (21) Google Scholar). Similar results were obtained with a probe (p200) that hybridizes to the exon 10-intron 10 splice junction of the G6PD RNA (data not shown). The decrease in the amount of spliced RNA in the nucleus could reflect a decrease in the rate of splicing of the primary transcript or a decrease in the stability of the fully spliced transcript. To discriminate between these possibilities, the amount of RNA early in the splicing process was measured using RNase protection assays and a probe that hybridized across two introns (exon 8, intron 8, exon 9, and intron 9, pBG2). Four protected fragments were detected representing G6PD pre-mRNA that contained both introns (unspliced), pre-mRNA that had only intron eight spliced (partially spliced), and two fragments representing G6PD RNA that had both introns removed (fully spliced). Protected fragments corresponding to spliced introns were not detected. At 0 h, the amount of unspliced RNA was similar in mice fed both the low fat and high fat diets despite a 50% decrease in the amount of fully spliced RNA (Fig.3A, overlapping circle and square, and data not shown). The amount of partially spliced RNA was greater at all time points than the amount of unspliced RNA. As the mice consumed the low fat diet, the amount of partially spliced RNA increased 7–8-fold, whereas only a 2–3-fold increase in partially spliced RNA was observed in mice fed the high fat diet. The attenuation of partially spliced mRNA accumulation with the high fat diet resulted in a decrease in the ratio of partially spliced to unspliced RNA between the two dietary groups (Fig.3B). Thus, dietary polyunsaturated fat caused a decrease in the accumulation of partially spliced RNA. These data are consistent with regulation at an early step in RNA processing rather than a change in stability of the mature mRNA in the nucleus. A decrease in the ratio of the amount of partially spliced to unspliced RNA in mice fed the high fat diet compared with the low fat diet could indicate that the splicing reaction itself is inhibited by dietary polyunsaturated fatty acids. In such a case the improperly spliced RNA would be targeted for degradation in the nucleus (11Antoniou M. Geraghty F. Hurst J. Grosveld F. Nucleic Acids Res. 1998; 26: 721-729Crossref PubMed Scopus (65) Google Scholar, 12Custodio N. Carmo-Fonseca M. Geraghty F. Pereira H.S. Grosveld F. Antoniou M. EMBO J. 1999; 18: 2855-2866Crossref PubMed Scopus (178) Google Scholar). Thus, we examined other intron-exon boundaries to determine whether any intron was selectively retained in the G6PD transcript when mice were consuming a high fat diet. A probe to intron 6, exon 7, intron 7, and exon 8 (pBG1) detected RNA with both introns present, RNA from which intron only 7 has been spliced and RNA from which both introns are removed. Partially spliced RNA representing retention of intron 6 increased 3-fold in mice fed the low fat diet (Fig. 3C). Consumption of the high fat diet attenuated the accumulation of this partially spliced RNA, consistent with the previous results. A distinctly different result was obtained when a probe was used to the exon 10, intron 10, exon 11, intron 11, and exon 12 (pJW1) region of the pre-mRNA. Use of this probe with nuclear RNA resulted in protected fragments representing RNA containing both intron 10 and 11, RNA from which intron 10 had been spliced, RNA from which intron 11 had been spliced, and RNA from which both introns had been removed. Thus two partially spliced RNA intermediates were detected in contrast to the results with other probes where the partially spliced RNA consistently contained only one of the two introns represented in the probe (Fig. 4A). Even more striking was the increase in abundance of partially spliced RNA containing intron 11 in mice fed the high fat diet (Fig.4B, NoI/O HF bars). The increase in abundance of this partially spliced RNA occurred despite a decrease of 61% or more in fully spliced RNA detected in the nucleus (Fig. 4A, exon 11 and 12 bands, and data not shown for the other time points). Likewise, the amount of unspliced RNA increased with time in mice fed the low fat diet, and this increase was inhibited in mice fed the high fat diet. This result was reproducible both between mice within a single experiment and between different experiments with different groups of mice. In the mice fed the high fat diet, the pre-mRNA retaining intron 11 must ultimately be degraded since the amount of fully spliced RNA (protected fragments repre
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