A Novel Bipartite Intronic Splicing Enhancer Promotes the Inclusion of a Mini-exon in the AMP Deaminase 1 Gene
2001; Elsevier BV; Volume: 276; Issue: 27 Linguagem: Inglês
10.1074/jbc.m011637200
ISSN1083-351X
AutoresThomas Genetta, Hiroko Morisaki, Takayuki Morisaki, Edward W. Holmes,
Tópico(s)RNA and protein synthesis mechanisms
ResumoAlternative splicing of the 12-base exon 2 of the adenosine monophosphate deaminase (AMPD) gene is subject to regulation by both cis- and trans-regulatory signals. The extent of exon 2 inclusion is stage- and cell type-specific and is subject to the physiological state of the cell. In adult skeletal muscle, a cell type that regulates the activity of this allosteric enzyme at several levels, the exon 2-plus form of AMPD, predominates. We have performed a systematic analysis of the cis-acting regulatory sequences that reside in the intron immediately downstream of this mini-exon. A complex element comprising sequences that enhance exon 2 inclusion and sequences that counteract this effect resides in the middle of this intron. We demonstrate that the enhancing component is bipartite, with more than a kilobase of sequence separating the two functional sites. The presence of even minimal levels the mini-exon in the fully processed AMPD mRNA requires both of these sites, neither of which appears in any other published splicing enhancer. An RNA binding activity derived from a muscle cell line requires both of the enhancing sites. Mutations in either of the sites that eliminate exon 2 inclusion abrogate this binding activity. Alternative splicing of the 12-base exon 2 of the adenosine monophosphate deaminase (AMPD) gene is subject to regulation by both cis- and trans-regulatory signals. The extent of exon 2 inclusion is stage- and cell type-specific and is subject to the physiological state of the cell. In adult skeletal muscle, a cell type that regulates the activity of this allosteric enzyme at several levels, the exon 2-plus form of AMPD, predominates. We have performed a systematic analysis of the cis-acting regulatory sequences that reside in the intron immediately downstream of this mini-exon. A complex element comprising sequences that enhance exon 2 inclusion and sequences that counteract this effect resides in the middle of this intron. We demonstrate that the enhancing component is bipartite, with more than a kilobase of sequence separating the two functional sites. The presence of even minimal levels the mini-exon in the fully processed AMPD mRNA requires both of these sites, neither of which appears in any other published splicing enhancer. An RNA binding activity derived from a muscle cell line requires both of the enhancing sites. Mutations in either of the sites that eliminate exon 2 inclusion abrogate this binding activity. adenosine monophosphate deaminase kilobase(s) exon retention element reverse transcription-polymerase chain reaction base pair(s) The vast majority of metazoan genes contain short, information-encoding exons interspersed by relatively long stretches of noncoding introns. The processing of this information to yield a translatable message, including the splicing of the pre-mRNA, is subject to regulation at virtually every definable step. One such step, including or excluding a particular exon—alternative splicing—provides a way to alter the functional activity of a protein in the absence of gene duplication (1Sharp P.A. Cell. 1994; 77: 805-815Abstract Full Text PDF PubMed Scopus (461) Google Scholar, 2Adams M.D. Rudner D.Z. Rio D.C. Curr. Opin. Cell Biol. 1996; 8: 331-339Crossref PubMed Scopus (117) Google Scholar). Discreet functional domains may be added or subtracted depending on signals elaborated by the particular needs of the cell. The study of alternative splicing further provides an experimental tool for understanding how the participating ribonucleoprotein complexes target intron-exon boundaries (3Staley J.P. Guthrie C. Cell. 1998; 92: 315-326Abstract Full Text Full Text PDF PubMed Scopus (916) Google Scholar). Vertebrate exons are, on average, less than 300 nucleotides, whereas many introns are thousands of nucleotides in length (4Black D.L. RNA. 1995; 1: 763-771PubMed Google Scholar). Exons below an average size of 50 nucleotides have been shown to be inherently difficult to recognize by the splicing machinery (4Black D.L. RNA. 1995; 1: 763-771PubMed Google Scholar, 5Berget S.M. J. Biol. Chem. 1995; 270: 2411-2414Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar). One explanation for this “masking” of small exons, originally set forth by Berget (5Berget S.M. J. Biol. Chem. 1995; 270: 2411-2414Abstract Full Text Full Text PDF PubMed Scopus (878) Google Scholar), is that the initiation of the splicing reaction is exon-centered. There is much evidence to support this model, termedexon definition, in which ribonucleoprotein initiation complexes recognize intron-exon boundaries and bridge across the exon. As exon size decreases below 50 nucleotides, these complexes are prevented from forming a productive interaction with the 3′- and 5′-splice recognition sites on the pre-mRNA or with each other, through steric hindrance. Sequences that function either to facilitate (enhancers) or to inhibit (repressors) the recognition of alternatively spliced exons have been found in both introns (6Black D.L. Cell. 1992; 69: 795-807Abstract Full Text PDF PubMed Scopus (151) Google Scholar, 7Carlo T. Sterner D.A. Berget S.M. RNA. 1996; 2: 342-353PubMed Google Scholar, 8Huh G.S. Hynes R.O. Mol. Cell. Biol. 1993; 13: 5301-5314Crossref PubMed Scopus (59) Google Scholar, 9Hertel K.J. Maniatis T. Cell. 1998; 98: 449-455Google Scholar) and exons (9Hertel K.J. Maniatis T. Cell. 1998; 98: 449-455Google Scholar, 10Chen C.D. Kobayshi R. Helfman D.M. Genes Dev. 1999; 13: 593-606Crossref PubMed Scopus (171) Google Scholar, 11Graveley B.R. Hertel K.J. Maniatis T. EMBO. 1998; 17: 6747-6756Crossref PubMed Scopus (138) Google Scholar). Adenosine monophosphate deaminase (AMPD)1 catalyzes a key step in purine nucleotide metabolism in virtually all eukaryotes. The purine nucleotide cycle serves as the sole source (in the form of fumarate) of citric acid cycle intermediates in contracting muscle tissue. A deficiency in AMPD is the most prevalent genetic disease in humans, the number of people heterozygous approaching 10% of Caucasian and individuals of African descent (12Sabina R. Holmes E.W. Scriber C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. 2. McGraw-Hill, New York1990: 1769-1780Google Scholar, 13Sabina R. Ogasawara N. Holmes E.W. Mol. Cell. Biol. 1989; 9: 2244-2256Crossref PubMed Scopus (37) Google Scholar). A small percentage of homozygous deficient individuals, nearly 2% of the affected populations, display symptoms of chronic fatigue and lost productivity as well as a greater predisposition to stress-related ailments, including heart disease and stroke (12Sabina R. Holmes E.W. Scriber C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. 2. McGraw-Hill, New York1990: 1769-1780Google Scholar, 14Morisaki H. Morisaki T. Newby L.K. Holmes E.W. J. Clin. Invest. 1993; 91: 2275-2280Crossref PubMed Scopus (64) Google Scholar). Interestingly, a mutation in a least one AMPD allele appears to confer a protective effect on individuals at risk for one of the most prevalent diseases of industrialized nations, congestive heart failure. We have found that people harboring at least one AMPD mutant allele have a significantly prolonged probability of survival after the onset of symptoms leading to this extremely serious medical condition (15Loh E. Rebbeck T. Mahoney P. DeNofrio D. Swain J. Holmes E.W. Circulation. 1999; 99: 1422-1425Crossref PubMed Scopus (128) Google Scholar). In muscle cells, AMPD is fully activated only when bound to myosin heavy chain through its carboxyl terminus. This enzyme is also influenced allosterically by cellular levels of purine nucleotides, through a binding site near its amino terminus (12Sabina R. Holmes E.W. Scriber C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. 2. McGraw-Hill, New York1990: 1769-1780Google Scholar, 24Wheeler T.J. Lowenstein J.M. J. Biol. Chem. 1979; 254: 8994-8999Abstract Full Text PDF PubMed Google Scholar, 25Marquetant R. Sabina R.L. Holmes E.W. Biochemistry. 1989; 28: 8744-8749Crossref PubMed Scopus (24) Google Scholar, 26Ashby B. Frieden C. J. Biol. Chem. 1977; 252: 1869-1872Abstract Full Text PDF PubMed Google Scholar, 27Ashby B. Frieden C. Bischoff R. J. Cell Biol. 1979; 81: 361-373Crossref PubMed Scopus (38) Google Scholar, 28Koretz J.F. Frieden C. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 7186-7188Crossref PubMed Scopus (11) Google Scholar). We have shown that the four-amino acid peptide encoded by exon 2 influences both of these properties in a fiber type-dependent fashion (19Hisatome I. Morisaki T. Kamma H. Sugama T. Morisaki H. Ohtahara A. Holmes E.W. Am. J. Physiol. 1998; 275: 870-881Crossref PubMed Google Scholar). In adult, fast twitch, glycolytic myofibers, the sensitivity of AMPD to cellular ATP/GTP levels is altered significantly by the presence of the exon 2 domain (14Morisaki H. Morisaki T. Newby L.K. Holmes E.W. J. Clin. Invest. 1993; 91: 2275-2280Crossref PubMed Scopus (64) Google Scholar, 16Mineo I. Clarke P.R.H. Sabina R.L. Holmes E.W. Mol. Cell. Biol. 1990; 10: 5271-5278Crossref PubMed Scopus (41) Google Scholar, 19Hisatome I. Morisaki T. Kamma H. Sugama T. Morisaki H. Ohtahara A. Holmes E.W. Am. J. Physiol. 1998; 275: 870-881Crossref PubMed Google Scholar). In the resting state, ATP levels in the myocyte are relatively high, and a purine nucleotide binding site near the carboxyl terminus of AMPD is occupied. The exon 2-minus isoform of AMPD, which predominates in slow twitch, oxidative myofibers, can bind to myosin heavy chain and become activated in the presence of relatively high concentrations of ATP (12Sabina R. Holmes E.W. Scriber C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. 2. McGraw-Hill, New York1990: 1769-1780Google Scholar, 19Hisatome I. Morisaki T. Kamma H. Sugama T. Morisaki H. Ohtahara A. Holmes E.W. Am. J. Physiol. 1998; 275: 870-881Crossref PubMed Google Scholar). Thus, the essential role of AMPD in generating both fumarate (for use as a citric acid cycle intermediate) and IMP (from the deamination of AMP) can be retained in both fiber types, at least in part, by regulating the alternative splicing of exon 2. The AMPD gene is comprised of 16 exons and is regulated both transcriptionally (13Sabina R. Ogasawara N. Holmes E.W. Mol. Cell. Biol. 1989; 9: 2244-2256Crossref PubMed Scopus (37) Google Scholar) and post-transcriptionally (16Mineo I. Clarke P.R.H. Sabina R.L. Holmes E.W. Mol. Cell. Biol. 1990; 10: 5271-5278Crossref PubMed Scopus (41) Google Scholar, 17Mineo I. Holmes E.W. Mol. Cell. Biol. 1991; 11: 5263-5356Crossref Scopus (14) Google Scholar, 18Morisaki T. Gross M. Morisaki H. Pongrats D. Zollner N. Holmes E.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6457-6461Crossref PubMed Scopus (189) Google Scholar) in a development- and tissue-specific manner. The primary transcript is alternatively spliced with the exon 2-minus form predominating in all cells prenatally and in myoblasts postnatally. This 12-base mini-exon is largely, but not exclusively, retained in adult myotubes (17Mineo I. Holmes E.W. Mol. Cell. Biol. 1991; 11: 5263-5356Crossref Scopus (14) Google Scholar). Recent data suggest a role for the exon 2- encoded peptide in altering the allosteric responsiveness of AMPD to intracellular ATP levels, clearly important for the homeostasis of muscle (19Hisatome I. Morisaki T. Kamma H. Sugama T. Morisaki H. Ohtahara A. Holmes E.W. Am. J. Physiol. 1998; 275: 870-881Crossref PubMed Google Scholar). A recently discovered genetic lesion, a C-T transition that results in the introduction of a nonsense mutation at the end of exon 2, is partly responsible for human AMPD deficiency. As a consequence of alternative splicing, however, this mutated exon is excluded in 0.6–2% of the enzyme in adult muscle, resulting in a partial rescue of the deficiency. Most individuals harboring this mutation are therefore asymptomatic (14Morisaki H. Morisaki T. Newby L.K. Holmes E.W. J. Clin. Invest. 1993; 91: 2275-2280Crossref PubMed Scopus (64) Google Scholar). For all of these reasons, we are very interested in the molecular mechanisms by which the alternative splicing of AMPD is regulated. We have demonstrated recently that alternative splicing of exon 2 is driven largely by two competing factors (20Morisaki H. Morisaki T. Kariko K. Genetta T. Holmes E.W. Gene (Amst.). 2000; 246: 365-372Crossref PubMed Scopus (4) Google Scholar). First, the short distance between the suboptimal 5′-donor and 3′-acceptor splice recognition sites of this 12-base exon makes its recognition by the splicing apparatus inherently difficult. Second, sequences located roughly in the middle of the 5.2-kb downstream intron, the exon retention element (ExRE), are required for inclusion of the this exon in the final splicing product. We have shown that the strength of these two opposing influences on exon 2 inclusion is influenced greatly by cell type. In non-muscle cells, such as fibroblasts, exon 2 is included in slightly less than half of the final splice products. In myoblasts, the balance is shifted dramatically toward inclusion, about 90%, and differentiated myotubes push this even further, to a 97% inclusion rate (20Morisaki H. Morisaki T. Kariko K. Genetta T. Holmes E.W. Gene (Amst.). 2000; 246: 365-372Crossref PubMed Scopus (4) Google Scholar). We present in this report the initial characterization of sequences residing in the ExRE of intron 2 of the AMPD gene which regulate the extent of exon 2 inclusion in the final splicing product. We have narrowed the ExRE to two short, novel, discreet sequences in the middle of intron 2, separated by ∼1,150 bases. We found that this bipartite splicing enhancer functions in an orientation- and sequence-dependent manner. In addition, there is an absolute requirement for both of these enhancing elements in exon 2 inclusion. We go on to demonstrate the presence of a myocyte-specific factor that is detected only when both of the enhancing sites are present together in a binding reaction. Manipulations, deletions, and mutations of intron 2 of the human AMPD gene were made according to standard recombinant DNA protocols and procedures (21Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G Smith J.A. M.Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, New York1990Google Scholar). All constructs were sequenced by the University of Pennsylvania Medical Center DNA Sequencing Facility. The resulting intron 2 enhancer mutations were cloned into the β-actin-based expression vector (22Gunning P. Leavitt J. Muscat G. Ng S.Y. Kedes L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4831-4835Crossref PubMed Scopus (704) Google Scholar) depicted in Fig. 1A. Balb/c 3T3 fibroblasts and murine Soleus 8 myoblasts were maintained in Dulbecco's modified Eagle's medium plus 10% fetal calf serum supplemented with glutamine in an atmosphere containing 5% CO2. Cells were transfected using a standard calcium phosphate procedure (21Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G Smith J.A. M.Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, New York1990Google Scholar). After 48, transiently transfected cells were harvested, and total RNA was isolated using a guanidinium-based procedure (21Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G Smith J.A. M.Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, New York1990Google Scholar). Stable lines were established by incubating cells 48 h post-transfection in 750 μg/ml (effective concentration) Geneticin (Life Technologies, Inc.) and allowing the growth of distinct colonies (approximately 3 weeks). RNA was isolated from pooled colonies using the same guanidinium procedure. Preparation of total RNA and reverse transcription reaction conditions for the polymerase chain reaction and subsequent gel analysis of PCR products were carried out as described (20Morisaki H. Morisaki T. Kariko K. Genetta T. Holmes E.W. Gene (Amst.). 2000; 246: 365-372Crossref PubMed Scopus (4) Google Scholar). Two sets of primers, both specific for the human AMPD homolog, were used in these studies. PCR products of 69 bp (exon 2-plus) and 57 bp (exon 2-minus) were generated as described (20Morisaki H. Morisaki T. Kariko K. Genetta T. Holmes E.W. Gene (Amst.). 2000; 246: 365-372Crossref PubMed Scopus (4) Google Scholar). RT-PCR analysis yielding products of 216 bp (exon 2-plus) or 204 (exon 2-minus) was generated using the primer H6 (5′-GTCTGGATCTCATCCACATC-3′, which bound to exon 3) in the RT reaction, and H6 and TH2 (5′-GTCACCCCACAGTCTCCTC-3′, which bound to exon 1) in the PCR. The cycling conditions used with the second set of primers were: 93 °C, 3 min, 1 cycle; 93 °C, 1 min, 58 °C, 1 min, 72 °C, 30 s, 30 cycles; 72 °C, 10 min; otherwise the reagents, buffers, and conditions were identical to those described previously (20Morisaki H. Morisaki T. Kariko K. Genetta T. Holmes E.W. Gene (Amst.). 2000; 246: 365-372Crossref PubMed Scopus (4) Google Scholar). In either case, these primers were specific to the human homolog of AMPD and did not amplify endogenous murine AMPD. The plasmid p25 was constructed by cloning the 989-baseEcoRI-BamHI AC fragment from intron 2 of the human AMPD gene (14Morisaki H. Morisaki T. Newby L.K. Holmes E.W. J. Clin. Invest. 1993; 91: 2275-2280Crossref PubMed Scopus (64) Google Scholar) into the pBKS vector (Stratagene). A series of deletions, three generated from the 5′-end of the A fragment (AC1, a 150-base deletion; AC2, a 300-base deletion; AC3, a 450-base deletion) and two generated from the 3′-end of the C fragment (AC4, a 150-base deletion, and AC5, a 300-base deletion) were generated via PCR using the following primers. Top, 5′-CGCGAATTCCTTCCTGTGTTAATAATAGTAATCTCC-3′; bottom, 5′-CCAGGATCCAACAGAGAAGCCCACTATGTTGG-3′. Top, 5′-CGCGAATTCCAGTTATTATGTGGTTTGCCCAAGGC-3′; bottom, same as AC1. Top, 5′-CCAGGAATTCCACCTCCCGAGTTCAAGCAATTCTCC-3′; bottom, same as AC1. Top, 5′-CGCGAATTCCTTTGGGAGATGAAATGTGG-3′; bottom, 5′-CCAGGATCCAAATGGAACACCAAGTAAATGC-3′. Top, same as AC4; bottom, 5′- CCAGGATCCAAGCAGAAGTTGGAAGAGGCTGC-3′. All of the resulting PCR products were gel purified, digested withEcoRI and BamHI, gel purified again, and used to replace the wild-type AC region in the AMPD expression vector. The p25 plasmid (see above) was used as the starting vector from which a series of nested deletions, averaging 50 bases, was generated from either end of the AC fragment, using the exonuclease III-based Erase-a-Base kit (Promega). After digesting the single-stranded 5′-overhang with S1 nuclease, double-stranded linkers incorporating an 8-base ASC restriction site (5′-GCTGACGCCCGGCG-3′) were ligated onto the resulting blunt ends. After digesting the deletions with ASC I (New England Biolabs) and SacI (which cuts only in the ampicillin resistance gene of the vector) they were sized on agarose gels, and the appropriate deletion pairs were ligated. Using the EcoRI and BamHI sites, each of the resulting 22 ASC mutations replaced the wild-type AC region in the AMPD mini-gene expression vector. The mutations, designed to alter contiguous bases in groups of four, were introduced in and around the mutation 7 and 8 ASC sites and were generated using the QuikChange site-directed mutagenesis kit (Stratagene). The top strand of each of the 10 pairs of double-stranded oligonucleotides used in this procedure, incorporating the 4 mutated bases (underlined) flanked by 15 wild-type nucleotides, were as follows (the numbers correspond to the number of the mutation in Fig. 6A). 1) 5′-GAGTCTTGCTCTGTCATTAAGGCTGGAGTCCAGT 3′. 2) 5′-TCTGTCGCCCAGGCTATTATGCAGTAGCATAATC-3′. 3) 5′-CCCAGGCTGGAGTGCTAATGCATAATCTCGGCTC-3′. 4) 5′-GAGTGCAGTAGCATATAAACGGCTCATTGCAAGC-3′. 5) 5′-AGCATAATCTCGGCTATAAGCAAGCTCCACCTCC-3′. 6) 5′-CGAGGGTGTGGAGGCTAATTCAGGCCATCGAATG-3′. 7) 5′-GGAGGCATTATCAGGTATACGAATGCATTTACTT-3′. 8) 5′-ATCAGGCCATCGAATTATATTACTTGGTGTTCCA- 3′. 9) 5′-CCATCGAATGCATTTTAAAGGTGTTCCATTTGTT-3′. 10) 5′-GCATTTACTTGGTGTATATTTTGTTCCTTCATGG-3′. The bottom strand for each oligonucleotide is simply its complement. All mutations were generated in the p25 construct and verified by sequencing. As in the previous manipulations of this region, each of the 10 mutations was subcloned using the 5′-EcoRI and 3′-BamHI sites flanking the AC region into the AMPD mini-gene expression vector. Confluent cultures of murine Soleus 8 myoblasts were allowed to fuse into myotubes by reducing the fetal calf serum levels in the culture medium from 20% to 2% (23Monterras D. Pinset C. Chelly J. Kahn A. Gros F. EMBO J. 1989; 8: 2203-2207Crossref PubMed Scopus (66) Google Scholar). 72 h after the medium switch, the culture dishes were cooled on ice, and the cells were lysed in ice-cold Triton extraction buffer (T buffer) (20 mmHEPES (pH 7.9), 10 mm NaCl, 3 mmMgCl2, 0.2 mm EDTA, 0.1% Triton X-100, 20% glycerol, 1 mm dithiothreitol, 1 mmphenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml pepstatin, 10 μg/ml aprotinin) with 20 strokes of a Dounce homogenizer (type B pestle). Nuclei were pelleted by centrifuging the lysed cells at 3,000 × g for 15 min at 4 °C. Nuclei were washed once in T buffer, re-pelleted, and then extracted by resuspending in 1 ml of TS buffer (T buffer with high salt, 500 mm NaCl and 0.4 m KCl)/3 × 107 cells, in a 15-ml screw-capped tube. After gently rocking the nuclear extraction at 4 °C for 1 h, it was spun at 13,000 × g for 10 min at 4 °C, and the supernatant was aliquoted, snap frozen in liquid nitrogen, and stored at −80 °C. Aliquots of this extract were used only once in subsequent experiments. The following oligonucleotides were designed with a T7 promoter at the 5′-end (italicized) followed by 30 bases from intron 2 of the AMPD gene. Centered in these 30 bases were the wild-type sequences defined by either ASC mutation 7 or 18 (underlined; see Fig.4A). Top, 5′-GTAATACGACTCACTATAGGGCCCAGGCTGGAGTGCAGTAGCATAATCTC-3′; bottom, 5′-GAGATTATGCTACTGCACTCCAGCCTGGGCCCTATAGTGAGTCGTATTAC-3′. Top, 5′-GTAATACGACTCACTATAGGCCATCGAATGCATTTACTTGGTGTTCCATT-3′; bottom, 5′-AATGGAACACCAAGTAAATGCATTCGATGGCCTATAGTGAGTCGTATTAC-3′. Two additional pairs of oligonucleotides were synthesized, identical to the sequences above, except that the 8 bases defining mutation 7 and 18 sites were altered to match their cognate linker scan mutations. The mutated bases are indicated below in lowercase letters. Top, 5′-GTAATACGACTCACTATAGGGCCCAGGCTGGgGcGCgGcAGCATAATCTC-3′; bottom, 5′-GAGATTATGCTgCcGCgCcCCAGCCTGGGCCCTATAGTGAGTCGTATTAC-3′. Top, 5′-GTAATACGACTCACTATAGGCCATCGAATGCggcgcgccGGTGTTCCATT-3′; bottom, 5′-AATGGAACACCggcgcgccGCATTCGATGGCCTATAGTGAGTCGTATTAC-3′. Equimolar amounts of the appropriate oligonucleotide pairs were annealed together by being placed in a boiling water bath for 1 min, and the bath was removed to the bench top and allowed to come to room temperature. 0.5 μg of these double-stranded oligonucleotides was added to a prewarmed (42 °C) transcription mixture (40 mm Tris-Cl (pH 8.0), 8 mm MgCl2, 2 mm spermadine, 50 mm NaCl; 1 mmeach ATP, UTP, and GTP; 60 units of RNasin (Promega); 10 mmdithiothreitol; and 50 μCi of [α-32P]CTP (400–800 Ci/mmol, Amersham Pharmacia Biotech)). 10 units of T7 RNA polymerase (Promega) was added, and the reaction was incubated at 42 °C for 30 min. After digestion of the template DNA with DNase I, the RNA was gel purified on a 6% denaturing gel. RNA probes eluted from the gel were resuspended in RNase-free water and used directly in the mobility shift assay. Binding reactions included the following components (added in this order): 3 μl of 5 × binding buffer (25 mmHEPES (pH 7.9), 125 mm KCl, 10 mmMgCl2, 15% glycerol, 2.5 mmphenylmethylsulfonyl fluoride); 1 μl of nuclear extract, water to 15 μl, and ∼2.5 × 105 cpm of probe was incubated on ice for 20 min and then loaded directly onto a pre-run 5% nondenaturing gel (80 mm Tris-borate, 2 mmEDTA). Using a mini-gene construct comprising part of exon 1, exon 2, intron 2, and part of exon 3 (Fig.1A), we had demonstrated previously, in both fibroblasts and myocytes, an absolute requirement for the 5.2-kb second intron for inclusion of exon 2 in the three-exon splicing product (20Morisaki H. Morisaki T. Kariko K. Genetta T. Holmes E.W. Gene (Amst.). 2000; 246: 365-372Crossref PubMed Scopus (4) Google Scholar). Interestingly, as the phenotype of a cell became more muscle-like, the requirement for intron 1 for the inclusion of exon 2 in the final splicing product dropped dramatically; that is, when exon 1 was deleted from the mini-gene construct, 3T3 fibroblasts excluded exon 2 totally, whereas skeletal myoblasts included exon 2 slightly greater than half of the time. When these same myoblasts (Soleus 8 cells) were allowed to differentiate and fuse into myotubes, greater than 90% of final splicing products included the mini-exon (20Morisaki H. Morisaki T. Kariko K. Genetta T. Holmes E.W. Gene (Amst.). 2000; 246: 365-372Crossref PubMed Scopus (4) Google Scholar). As a first approach at identifying the sequences responsible for this effect, we constructed several mini-gene constructs carrying gross deletions of intron 2 and performed qualitative RT-PCR analysis to test the effects of each on exon 2 inclusion. The primers employed in these assays were specific for the human AMPD gene and did cross-hybridize with the murine homolog (see “Experimental Procedures” and Ref.20Morisaki H. Morisaki T. Kariko K. Genetta T. Holmes E.W. Gene (Amst.). 2000; 246: 365-372Crossref PubMed Scopus (4) Google Scholar). Nearly half of the sequences of intron 2, those closest to the flanking exons, could be eliminated without an effect on the wild-type ratio of exon 2-included to exon 2-excluded splicing products. Deletions that remove virtually all of the sequences flanking this central 2.7 kb (Del 1/3, Fig. 1B) retain the wild-type splicing pattern in both myoblast and fibroblast cells (Fig.1C, Del 1/3 lanes). Located in the middle of intron 2, this 2.7-kb fragment must, in addition, be oriented in the wild-type 5′ → 3′ direction because flipping it 180° caused a total elimination of exon 2 in the final splicing product (Fig. 1,panels B and C, Rev). A myoblast/myocyte-specific enhancement of exon 2 inclusion was observed consistently throughout these studies. Using convenient restriction sites, the 2.7-kb region was divided into four fragments and analyzed further through their systematic deletion (Fig. 2A). Only when the 600-bp fragment A and 400-bp fragment C were both present in the mini-gene construct did exon 2 appear in the final splicing product in either myoblasts or fibroblasts (Fig. 2). The 800-bp fragment separating A and C, fragment B (the lane labeledΔA ΔC ΔD) cannot, by itself, promote exon 2 inclusion to any extent measured by this assay. Interestingly, in the absence of the B, the presence of A and C together promoted the inclusion of exon 2 to greater than 95% in fibroblasts (over double the rate in the presence of fragment B), suggesting that B may function to attenuate that effect in these cells (see “Discussion”). Taken as a whole, these data indicate that the regulation of exon 2 inclusion in the final splicing product is complex, involving both positive and negative influences. We chose to focus our efforts on narrowing down those sequences in A and C responsible for the enhancement of exon 2 inclusion in the final splicing product. To simplify the identification of the sequences responsible for this effect, we began with the mini-gene splicing substrate in which the 2.7-kb fragment in intron 2 is replaced with the positive acting A and C elements (the ΔB ΔDconstruct in Fig. 2A). In this context, the combined 1,000 bases (approximately) of sequence promotes virtually total inclusion of exon 2, independent of cell type (see Fig. 2B). Using PCR-generated fragments, we created a series of progressive deletions in A and C in ∼150-base increments (the end points of which are demarcated by upward arrows in Fig. 4A). Thus, the 600-base A fragment was subjected to four incremental deletions, starting from its 5′-end, whereas the 400-base C fragment had three, proceeding in the opposite direction (Fig.3A). These deletions were then used, in turn, to replace the wild-type A and C elements for subsequent splicing analysis. The results in Fig. 3B clearly show that the sequences responsible for the enhancing activity of A reside within the second and third deletion fragments (labeled 2 and3 in the schematic in Fig. 3A), whereas the positive acting sequences in fragment C lie within 3′-most 150 bases of that fragment (labeled 5 in Fig. 3A). In addition to revealing that the first 150 bases of the A fragment were dispensable for the promotion of exon 2 inclusion, the gross deletion analysis indicated approximately where two critical regulatory sequences in A and C resided. To narrow these further, we performed a saturation mutagenesis on the entire A–C region (minus the 5′-most 150 bases) using a standard linker scan procedure. In the process, we introduced an ASC restriction site (5′-GGCGCGCC-3′) every 38 bases, on average, for a total of 22 different mutations (Fig.4A; the end points of the 150 base gross deletions are indicated by upward arrows). Each of these was then tested, as before, in the context of the AMPD mini-gene splicing substrate. In only two cases, 7 and 18, was exon 2 excluded to any extent from the final splicing product (Fig.4B). Both of these mutations had a more subtle effect on exon 2 inclusion than the150-bp deletions depicted in Fig. 3. The overall results are consistent between the two approaches. One apparent distinction is that the large deletion of 300 bases at the 5′-end of fragment A (Fig. 3A, construct 2) reduced exon 2 inclusion by about half, whereas the individual linker scan mutations in this same region (Fig. 4, mutations 1–6) produced no e
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