Abstract Genetic factors and nerve injury‐induced changes of gene expression in sensory neurons are potential contributors to tactile allodynia, a neuropathic pain state manifested as hypersensitivity to innocuous mechanical stimulation. To uncover genes relevant to neuropathic allodynia, we analyzed gene expression profiles in dorsal root ganglia (DRG) of spinal nerve‐ligated Harlan and Holtzman Sprague Dawley rats, strains with different susceptibilities to neuropathic allodynia. Using Affymetrix gene chips, we identified genes showing differential basal‐level expression in these strains without injury‐induced regulation. Of more than 8000 genes analyzed, less than 180 genes in each strain were regulated after injury, and 19–22% of that was regulated in a strain‐specific manner. Importantly, we identified functionally related genes that were co‐regulated post injury in one or both strains. In situ hybridization and real‐time PCR analyses of a subset of identified genes confirmed the patterns of the microarray data, and the former also demonstrated that injury‐induced changes occurred, not only in neurons, but also in non‐neuronal cells. Together, our studies provide a global view of injury plasticity in DRG of these rat stains and support a plasticity‐based mechanism mediating variations in allodynia susceptibility, thus providing a source for further characterization of neuropathic pain‐relevant genes and potential pathways.
Up-regulation of thrombospondin-4 (TSP4) or voltage-gated calcium channel subunit α2δ1 (Cavα2δ1) proteins in the spinal cord contributes to neuropathic pain development through an unidentified mechanism. We have previously shown that TSP4 interacts with Cavα2δ1 to promote excitatory synaptogenesis and the development of chronic pain states. However, the TSP4 determinants responsible for these changes are not known. Here, we tested the hypothesis that the Cavα2δ1-binding domains of TSP4 are synaptogenic and pronociceptive. We mapped the major Cavα2δ1-binding domains of TSP4 within the coiled-coil and epidermal growth factor (EGF)-like domains in vitro Intrathecal injection of TSP4 fragment proteins containing the EGF-like domain (EGF-LIKE) into naïve rodents was sufficient for inducing behavioral hypersensitivity similar to that produced by an equal molar dose of full-length TSP4. Gabapentin, a drug that binds to Cavα2δ1, blocked EGF-LIKE-induced behavioral hypersensitivity in a dose-dependent manner, supporting the notion that EGF-LIKE interacts with Cavα2δ1 and thereby mediates behavioral hypersensitivity. This notion was further supported by our findings that a peptide within EGF-LIKE (EGFD355-369) could block TSP4- or Cavα2δ1-induced behavioral hypersensitivity after intrathecal injections. Furthermore, only TSP4 proteins that contained EGF-LIKE could promote excitatory synaptogenesis between sensory and spinal cord neurons, which could be blocked by peptide EGFD355-369. Together, these findings indicate that EGF-LIKE is the molecular determinant that mediates aberrant excitatory synaptogenesis and chronic pain development. Blocking interactions between EGF-LIKE and Cavα2δ1 could be an alternative approach in designing target-specific pain medications.
Abstract Background Peripheral nerve injury induces up‐regulation of the calcium channel alpha‐2‐delta‐1 proteins in the dorsal root ganglia and dorsal spinal cord that correlates with neuropathic pain development. Similar behavioural hypersensitivity was also observed in injury‐free transgenic ( TG ) mice over‐expressing the alpha‐2‐delta‐1 proteins in neuronal tissues. To investigate pathways regulating alpha‐2‐delta‐1 protein‐mediated behavioural hypersensitivity, we examined whether spinal serotonergic 5‐ HT3 receptors are involved similarly in the modulation of behavioural hypersensitivity induced by either peripheral nerve injury in a nerve injury model or neuronal alpha‐2‐delta‐1 over‐expression in the TG model. Methods The effects of blocking behavioural hypersensitivity in these two models by intrathecal or systemic injections of 5‐ HT3 receptor antagonist, ondansetron, were compared. Results Our data indicated that the TG mice displayed similar behavioural hypersensitivities to non‐painful mechanical stimulation (tactile allodynia) and painful thermal stimulation (thermal hyperalgesia) as that observed in the nerve injury model. Interestingly, tactile allodynia and thermal hyperalgesia in both models can be blocked similarly by intrathecal, but not systemic, injection of ondansetron. Conclusions Our data suggest that spinal 5‐ HT3 receptors are likely to play a role in alpha‐2‐delta‐1‐mediated behavioural hypersensitivities through a descending serotonergic facilitation.
Splicing of alternative exon 6 to invariant exons 2, 3, and 4 in acetylcholinesterase (AChE) pre-mRNA results in expression of the prevailing enzyme species in the nervous system and at the neuromuscular junction of skeletal muscle. The structural determinants controlling splice selection are examined in differentiating C2-C12 muscle cells by selective intron deletion from and site-directed mutagenesis in the Ache gene. Transfection of a plasmid lacking two invariant introns (introns II and III) within the open reading frame of the Ache gene, located 5′ of the alternative splice region, resulted in alternatively spliced mRNAs encoding enzyme forms not found endogenously in myotubes. Retention of either intron II or III is sufficient to control the tissue-specific pre-mRNA splicing pattern prevalent in situ. Further deletions and branch point mutations revealed that upstream splicing, but not the secondary structure of AChE pre-mRNA, is the determining factor in the splice selection. In addition, deletion of the alternative intron between the splice donor site and alternative acceptor sites resulted in aberrant upstream splicing. Thus, selective splicing of AChE pre-mRNA during myogenesis occurs in an ordered recognition sequence in which the alternative intron influences the fidelity of correct upstream splicing, which, in turn, determines the downstream splice selection of alternative exons. Splicing of alternative exon 6 to invariant exons 2, 3, and 4 in acetylcholinesterase (AChE) pre-mRNA results in expression of the prevailing enzyme species in the nervous system and at the neuromuscular junction of skeletal muscle. The structural determinants controlling splice selection are examined in differentiating C2-C12 muscle cells by selective intron deletion from and site-directed mutagenesis in the Ache gene. Transfection of a plasmid lacking two invariant introns (introns II and III) within the open reading frame of the Ache gene, located 5′ of the alternative splice region, resulted in alternatively spliced mRNAs encoding enzyme forms not found endogenously in myotubes. Retention of either intron II or III is sufficient to control the tissue-specific pre-mRNA splicing pattern prevalent in situ. Further deletions and branch point mutations revealed that upstream splicing, but not the secondary structure of AChE pre-mRNA, is the determining factor in the splice selection. In addition, deletion of the alternative intron between the splice donor site and alternative acceptor sites resulted in aberrant upstream splicing. Thus, selective splicing of AChE pre-mRNA during myogenesis occurs in an ordered recognition sequence in which the alternative intron influences the fidelity of correct upstream splicing, which, in turn, determines the downstream splice selection of alternative exons. acetylcholinesterase base pair(s) small nuclear ribonucleoprotein particle Dulbecco's modified Eagle's medium genomic DNA exon single A → C mutation four A → C mutations. Formation of a contiguous open reading frame in eukaryotic mRNA by elimination of intervening sequences (introns) through splicing is a critical step in biosynthesis of functional proteins (1Green M.R. Annu. Rev. Cell Biol. 1991; 7: 559-599Crossref PubMed Scopus (553) Google Scholar, 2Adams M.D. Rudner D.Z. Rio D.C. Curr. Opin. Cell Biol. 1996; 8: 331-339Crossref PubMed Scopus (117) Google Scholar, 3Moore M. Query C.C. Sharp P.A. Gesteland R. Atkins J.F. The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 303-358Google Scholar). Alternative splicing of pre-mRNA in various cell types generates protein isoforms with distinct activities or tissue-specific distribution patterns during development (4Andreadis A. Gallego M.E. Nadal-Ginard B. Annu. Rev. Cell Biol. 1987; 3: 207-242Crossref PubMed Scopus (112) Google Scholar, 5Smith C.W. Patton J.G. Nadal-Ginard B. Annu. Rev. Genet. 1989; 23: 527-577Crossref PubMed Scopus (567) Google Scholar). About 15% of mammalian gene mutations associated with disease states are reported to affect RNA splicing signals (2Adams M.D. Rudner D.Z. Rio D.C. Curr. Opin. Cell Biol. 1996; 8: 331-339Crossref PubMed Scopus (117) Google Scholar). However, the molecular mechanisms underlying regulation of pre-mRNA splicing, especially in mammalian cells, are not well understood (1Green M.R. Annu. Rev. Cell Biol. 1991; 7: 559-599Crossref PubMed Scopus (553) Google Scholar, 2Adams M.D. Rudner D.Z. Rio D.C. Curr. Opin. Cell Biol. 1996; 8: 331-339Crossref PubMed Scopus (117) Google Scholar, 3Moore M. Query C.C. Sharp P.A. Gesteland R. Atkins J.F. The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 303-358Google Scholar). One such example is the selective splicing of acetylcholinesterase (AChE)1 pre-mRNA during myogenesis. The primary function of AChE is to terminate the action of the released neurotransmitter acetylcholine in the central and peripheral nervous systems. AChE exists in multiple molecular forms whose catalytic subunits are encoded by alternatively spliced mRNAs from a single gene (6Li Y. Camp S. Rachinsky T.L. Getman D. Taylor P. J. Biol. Chem. 1991; 266: 23083-23090Abstract Full Text PDF PubMed Google Scholar, 7Massoulie J. Pezzementi L. Bon S. Krejci E. Vallette F.M. Prog. Neurobiol. 1993; 41: 31-91Crossref PubMed Scopus (1058) Google Scholar, 8Karpel R. Ben Aziz-Aloya R. Sternfeld M. Ehrlich G. Ginzberg D. Tarroni P. Clementi F. Zakut H. Soreq H. Exp. Cell Res. 1994; 210: 268-277Crossref PubMed Scopus (76) Google Scholar, 9Taylor P. Radic Z. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 281-320Crossref PubMed Scopus (609) Google Scholar). The alternative gene products differ in sequence only at their very carboxyl termini, resulting in enzyme forms that differ in amphiphilic character, extent of oligomerization, and cellular disposition, but exhibit identical catalytic parameters. Within the open reading frame of the mammalian Ache gene are found three invariant exons (exons 2–4) that encode the amino-terminal 536 amino acids common to all forms of the enzyme. These invariant exons are alternatively spliced to one of three downstream sequences encoding distinct carboxyl termini and generating tissue-specific molecular forms of AChE (10Li Y. Camp S. Taylor P. J. Biol. Chem. 1993; 268: 5790-5797Abstract Full Text PDF PubMed Google Scholar). Exon 4 to 6 spliced mRNA, encoding catalytic subunits that assemble as monomers, dimers and tetramers, is the major species found in mammalian skeletal muscle and brain (10Li Y. Camp S. Taylor P. J. Biol. Chem. 1993; 268: 5790-5797Abstract Full Text PDF PubMed Google Scholar, 11Luo Z.D. Pincon-Raymond M. Taylor P. J. Neurochem. 1996; 67: 111-118Crossref PubMed Scopus (15) Google Scholar). The second splice option is extension of exon 4 to its 3′ intron, yielding a monomeric, hydrophilic species found in embryonic skeletal muscle and cells of hematopoietic origin (6Li Y. Camp S. Rachinsky T.L. Getman D. Taylor P. J. Biol. Chem. 1991; 266: 23083-23090Abstract Full Text PDF PubMed Google Scholar, 10Li Y. Camp S. Taylor P. J. Biol. Chem. 1993; 268: 5790-5797Abstract Full Text PDF PubMed Google Scholar, 12Legay C. Huchet M. Massoulie J. Changeux J.P. Eur. J. Neurosci. 1995; 7: 1803-1809Crossref PubMed Scopus (59) Google Scholar). The third splice option is the exon 4 to 5 splice that encodes an amphiphilic, glycophospholipid-linked form of AChE typically expressed in hematopoietic cells as well as pituitary and certain neuronal cells in culture (10Li Y. Camp S. Taylor P. J. Biol. Chem. 1993; 268: 5790-5797Abstract Full Text PDF PubMed Google Scholar). Splicing of pre-mRNA involves the binding of small nuclear ribonucleoprotein particles (snRNPs) to conserved intronic sequences on pre-mRNA. The binding of U1 snRNP and U2AF to the 5′ splice site and the polypyrimidine tract, respectively, promotes the binding of U2 snRNP to the branch point. The next step is the formation of spliceosome complex through the binding of the U4/U5/U6 snRNPs followed by a catalytic reaction resulting in a lariat formation between the 5′ splice site and the adenosine residue at the branch point. This, in turn, activates the cleavage of 3′ splice site and brings the spliced exons together (3Moore M. Query C.C. Sharp P.A. Gesteland R. Atkins J.F. The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 303-358Google Scholar, 13Black D.L. RNA. 1995; 1: 763-771PubMed Google Scholar, 14Fu X.D. RNA. 1995; 1: 663-680PubMed Google Scholar). Thus, intronic sequences play an important role in molecular interactions during constitutive splicing. One mechanism for the control of alternative splicing involves interaction of tissue or cell-specific factors with specificcis-elements in pre-mRNA (14Fu X.D. RNA. 1995; 1: 663-680PubMed Google Scholar). This interaction may influence the relative strength and, consequently, recognition of a given splice site as demonstrated in regulated splicing in vitro (13Black D.L. RNA. 1995; 1: 763-771PubMed Google Scholar, 14Fu X.D. RNA. 1995; 1: 663-680PubMed Google Scholar). A large body of evidence indicates that flanking intronic sequences are important in such an interaction (1Green M.R. Annu. Rev. Cell Biol. 1991; 7: 559-599Crossref PubMed Scopus (553) Google Scholar, 15Helfman D.M. Roscigno R.F. Mulligan G.J. Finn L.A. Weber K.S. Genes Dev. 1990; 4: 98-110Crossref PubMed Scopus (66) Google Scholar). One such example is the splicing of rat β-tropomyosin in which downstream intron splicing events are critical in upstream alternative splicing regulation (16Helfman D.M. Ricci W.M. Finn L.A. Genes Dev. 1988; 2: 1627-1638Crossref PubMed Scopus (69) Google Scholar). Since a single gene encodes all tissue-specific forms of AChE, tissue-specific factors interacting with specificcis-elements can be expected to regulate alternative splicing. Within the Ache gene, three invariant introns are found upstream of the alternative splice region where three splice options occur from a donor site. Two of the upstream invariant introns are located within the open reading frame of the gene (Fig. 1). As the first step toward understanding the alternative splicing of AChE pre-mRNA, we examined the role of these introns in the regulation of AChE pre-mRNA splicing during myogenesis. Fortuitously, the mammalian Ache gene is relatively compact with only 6 kilobase pairs separating the CAP site for transcription and the first polyadenylation signal. This enabled us to transfect the entireAche genomic DNA with its accompanying promoter as well as specified deletion constructs directly into differentiating myocytes. Cell culture supplies were from Fisher, and components of culture medium were from Life Technologies, Inc. [32P]UTP (specific activity: 800 Ci/mmol) was purchased from NEN Research Products (Wilmington, DE). Sucrose (ultrapure) was from ICN Biomedicals (Aurora, OH). Other chemicals were from Sigma. Mouse myoblast C2-C12 cells and human embryonic kidney cells (HEK-293) were from American Type Culture Collection. C2-C12 cells were cultured at 37 °C, with 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing 20% fetal bovine serum, 0.5% chick embryo extract and 1% penicillin, streptomycin, and amphotericin B stock solution (Antibiotic-Antimycotic, Life Technologies, Inc.). Cells were passed either two or three times before plating onto 100-mm culture dishes. Differentiation from myoblasts to myotubes was induced at about 70% confluence by replacing the high serum medium with DMEM containing 2% horse serum. HEK cells were maintained under the same conditions as the C2-C12 cells, except that the culture medium contained 10% fetal bovine serum. Constructions of Achegenomic expression plasmids with the endogenous promoter and their branch point mutants are described in the legends to Figs. 1 and 3. cDNA expression constructs under CMV promoters were described previously (10Li Y. Camp S. Taylor P. J. Biol. Chem. 1993; 268: 5790-5797Abstract Full Text PDF PubMed Google Scholar). Proliferating C2-C12 myoblasts transfect poorly, so to optimize transfection efficiencies, cells were transfected 1 day after induction of differentiation. Transfection of 10 μg/100-mm plate of the designated expression plasmids was performed by standard calcium phosphate procedures followed by glycerol shock 4–5 h later (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Codl Spring Harbor, NY1989: 16.30-16.81Google Scholar). In some experiments, LipofectAMINE (Life Technologies, Inc.)-mediated transfection with 5 μg of DNA/plate was utilized. Control cells were mock-transfected with the plasmid vectors without the Acheinserts. Cells were allowed to differentiate for 2–3 additional days in normal differentiation medium. To measure AChE secreted into the medium, cells were differentiated in serum-free DMEM after transfection. To correct differences in transfection efficiency, 2–5 μg of a pcDNA3 expression vector containing the Escherichia coli lacZ gene (18Kalnins A. Otto K. Ruther U. Muller-Hill B. EMBO J. 1983; 2: 593-597Crossref PubMed Scopus (327) Google Scholar) were co-transfected, and β-galactosidase activity was assayed spectrophotometrically. HEK cells were transfected by standard calcium phosphate method without glycerol shock. Total RNA was extracted from previously transfected myotubes as described previously (19Luo Z. Fuentes M.E. Taylor P. J. Biol. Chem. 1994; 269: 27216-27223Abstract Full Text PDF PubMed Google Scholar) or with TRIZol reagent (Life Technologies, Inc.), treated with 10 units of RQ1 DNase (Promega) for 30 min at 37 °C, and stored at −20 °C. AChE mRNA species were quantified by RNase protection assays as described (11Luo Z.D. Pincon-Raymond M. Taylor P. J. Neurochem. 1996; 67: 111-118Crossref PubMed Scopus (15) Google Scholar). For detecting exon 4 to 6 spliced mRNA, a mouse Ache cDNA containing the sequence of exons 4 and 6 was subcloned in a Bluescript SK II plasmid and linearized withXhoI. After in vitro transcription with [32P]UTP, a 458-bp labeled antisense cRNA probe was used for RNase protection. Similarly, for detecting exon 4 and its retained 3′ intron, a XhoI to ApaI fragment of Ache genomic DNA was subcloned into Bluescript SK II and linearized with XhoI. Upon transcription it gives rise to a 464-bp antisense probe. For detecting exon 4 to 5 spliced species, an antisense probe was transcribed from a MscI linearized mouse AChE cDNA containing the exon 4 to 5 spliced sequence in an expression vector (pRc/CMV, Invitrogen, San Diego, CA). To normalize for transfection efficiencies, an antisense probe of β-galactosidase was transcribed from a BamHI to SstI fragment of the E. coli lacZ gene, subcloned into Bluescript SK II, and linearized with Eco47III. A tRNA lane was included in each RNase protection assay to ensure the complete digestion of the free probes. Molecular masses of the protected probes were estimated by electrophoresis on polyacrylamide gels, and protected bands were exposed to BioMax films (Kodak) and quantified by densitometry (UltroScan XL, Amersham Pharmacia Biotech). AChE was extracted from rinsed C2-C12 myotubes in 0.01 m sodium phosphate buffer (pH 7.0) containing 1 m NaCl, 0.01 m EGTA, 1% Triton X-100, and a spectrum of protease inhibitors (20Silman I. Lyles J.M. Barnard E.A. FEBS Lett. 1978; 94: 166-170Crossref PubMed Scopus (70) Google Scholar). Culture media were centrifuged at 1000 × g for 30 min to remove cell debris. AChE in the media was concentrated over 10-fold in Centriprep 30 concentrators (Amicon, Inc., Beverly, MA). Enzyme activity was determined at room temperature in 0.1 m sodium phosphate buffer (pH 7.0) using 0.05–0.1 ml of cell extract (21Ellman G. Courtney K.D. Andres Jr., V. Featherstone R.M. Biochem. Pharmacol. 1961; 7: 88-95Crossref PubMed Scopus (21719) Google Scholar). Exogenous AChE activity was estimated by subtracting the activity in mock-transfected cells from the total activity in transfected cells. Differences in transfection efficiency were corrected from the ratios of AChE to β-galactosidase activities. AChE species were distinguished on the basis of their sedimentation coefficients in 5–30% sucrose gradients as described previously (22Duval N. Massoulie J. Bon S. J. Cell Biol. 1992; 118: 641-653Crossref PubMed Scopus (69) Google Scholar, 23Coleman B.A. Taylor P. J. Biol. Chem. 1996; 271: 4410-4416Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Briefly, cell extracts or concentrated media were layered onto sucrose gradients containing 0.01m sodium phosphate buffer (pH 7.0), 1 m NaCl, 0.01 m EGTA, and 1% Triton X-100 or Brij-96 (v/v). Carbonic anhydrase (3.3 S), alkaline phosphatase (6.1 S), and β-galactosidase (16 S) were added to the extracts as sedimentation markers. A 0.2-ml cushion of 50% sucrose was layered at the bottom of the gradients. The gradients were centrifuged at 41,000 rpm for 24 h at 4 °C in a SW41 Ti rotor (Beckman Instruments, Palo Alto, CA) and upward fractionated. Aliquots of each fraction were assayed for AChE and marker activities using a 96-well microtiter plate reader. To examine the role of invariant introns of theAche gene in pre-mRNA splice selection, we constructed from genomic DNA five mouse AChE expression plasmids containing the endogenous promoter (Fig. 1). The genomic DNA construct (gDNA), upon transfection into C2-C12 cells, should express a pattern of AChE mRNA species similar to the endogenous AChE transcripts. A second construct lacks both invariant introns II and III within the open reading frame of the Ache gene (Δi2–3,3–4), but contains the entire alternative splicing region 3′ of exon 4. The other three expression constructs contain the gDNA with one of the three 5′ introns in the gene deleted. As shown in Fig. 2 A, transient transfection of gDNA resulted in enhanced expression of exon 4 to 6 spliced mRNA (E4–6) as expected in myotubes. This indicates that the splicing machinery in the transfected cells has the capacity to process the additional AChE transcripts correctly. However, when the same plasmid DNA was transfected into human embryonic kidney cells, only small fraction of the AChE transcripts was spliced from exon 4 to 6, indicating that selective exon 4 to 6 splicing may be intrinsic to certain differentiated cells, such as C2-C12 muscle cells. When the transfected cells were maintained in an early stage of differentiation by returning to high serum conditions immediately after transfection, AChE mRNA transcribed from the transfected gDNA was only 37% (average of two independent transfections) of the level found in fully differentiated myotubes (Fig. 2 A). This suggests that, similar to the endogenous AChE mRNA (19Luo Z. Fuentes M.E. Taylor P. J. Biol. Chem. 1994; 269: 27216-27223Abstract Full Text PDF PubMed Google Scholar, 24Fuentes M.E. Taylor P. Neuron. 1993; 10: 679-687Abstract Full Text PDF PubMed Scopus (62) Google Scholar), mRNA from the transfected gene underwent stabilization during myogenesis. By contrast, transient transfection of Δi2–3,3–4 resulted in additional protected bands corresponding to the individual sizes of exon 4 (E4) and 6 (E6) in the probe. This severing of the exon 4 to 6 linkage indicates that, in addition to exon 4 to 6 splice, exon 4 with retained 3′ intron or/and exon 4 to 5 spliced species are expressed. These alternatively spliced species are confirmed by two probes shown in Fig. 2 (B and C). The full-length protected probes indicate the presence of mRNA with exon 4 either linked to its 3′ intron (E4-RI in Fig. 2 B) or spliced to exon 5 (E4–5 in Fig. 2 C), respectively. These alternatively spliced species were also confirmed by the protected exon 5 sequences (E5) in these probes reflecting exon 4 to 5 splice (Fig. 2 B) or exon 4 linked to its 3′ intron (Fig. 2 C). Protected exon 4 sequences (E4) in these probes appearing in the absence of protected E5 demonstrates the presence of only the exon 4 to 6 splice (Fig. 2, B and C). These data show that removal of introns II and III results in expression of alternatively spliced AChE mRNA species, which are normally not seen in myotubes. To examine the influence of individual intron II or III on pre-mRNA splicing, AChE expression constructs devoid of either intron II (Δi2–3) or III (Δi3–4) were transfected into differentiating C2-C12 cells. Total AChE mRNA levels accumulating in differentiated myotubes were lower than that in myotubes transfected with either gDNA or Δi2–3,3–4 (Table I). However, the majority of the transcripts were spliced between exon 4 and 6, as seen for the endogenous mRNA (Fig. 2, B and C, and Table I). Thus, the presence of either intron II or III is sufficient to direct exon 4 to 6 splicing during myogenesis and yield a pattern approaching that of endogenous pre-mRNA splicing. Cumulative data on the pattern of spliced AChE mRNA species in transfected C2-C12 myotubes are summarized in Table I.Table IEnhanced mRNA expression in C2-C12 myotubes after transfection of the respective plasmids into myoblastsPlasmid transfectedgDNAΔi2–3,3–4Δi2–3Δi3–4mRNA total expression10096 ± 950 ± 566 ± 11(% of transfected gDNA)Relative mRNA species(% total AChE mRNA) E4–687 ± 531 ± 782 ± 373 ± 11 E4-RI1-aRetained intron (intron IV).3.0 ± 0.742 ± 49 ± 116 ± 4 E4–51.3 ± 0.526 ± 19 ± 213 ± 3Genomic DNA encoding AChE and its respective deletion constructs are described in Fig. 1. Protected antisense mRNA probes were analyzed by densitometric analysis. After subtracting the density of the endogenous AChE bands quantified from mock transfected myotubes, band densities are normalized to the densities of protected β-galactosidase probes. Uracil content in each protected probe of designated length was used to normalize for the differences in sizes of protected bands. Total mRNA levels were determined by averaging the sum of AChE mRNA levels detected by three probes. The levels of different mRNA species were determined by protected bands from the respective probes and shown as percentage of the total AChE mRNA detected by the same probe. Values reported are the means ± S.E. averaged from four to six independent transfections.1-a Retained intron (intron IV). Open table in a new tab Genomic DNA encoding AChE and its respective deletion constructs are described in Fig. 1. Protected antisense mRNA probes were analyzed by densitometric analysis. After subtracting the density of the endogenous AChE bands quantified from mock transfected myotubes, band densities are normalized to the densities of protected β-galactosidase probes. Uracil content in each protected probe of designated length was used to normalize for the differences in sizes of protected bands. Total mRNA levels were determined by averaging the sum of AChE mRNA levels detected by three probes. The levels of different mRNA species were determined by protected bands from the respective probes and shown as percentage of the total AChE mRNA detected by the same probe. Values reported are the means ± S.E. averaged from four to six independent transfections. To examine the influence of the first intron in the Ache gene on its expression and splicing, differentiating C2-C12 cells were transiently transfected with an Ache expression construct devoid of this intron (Δi1–2). However, differentiated myotubes did not express the transfected plasmid at levels above the endogenous AChE mRNA (Fig. 2 A, representative data from three independent transfection experiments), indicating that the first intron residing 5′ of the ATG start site is necessary for Ache gene transcription or RNA stabilization. The following experiments were directed to resolving the sequence of splicing events occurring around the alternative splice region of AChE pre-mRNA. Helfman et al. (16Helfman D.M. Ricci W.M. Finn L.A. Genes Dev. 1988; 2: 1627-1638Crossref PubMed Scopus (69) Google Scholar) reported that downstream splicing in rat β-tropomyosin pre-mRNA is critical to upstream splice site selection. To examine the order of intron splicing around the alternative splice region and the influence of downstream intron on upstream splicing, we used the probe shown in Fig. 4 to detect unspliced and spliced transcripts, and the presence of splicing intermediates in cells transfected with gDNA or expression constructs in which the upstream invariant exons were spliced to either exon 5 (Δi4–5) or exon 6 (Δi4–6) (Fig. 3). Transfection of gDNA resulted in expression of a 540-bp species representing unspliced pre-mRNA, a 480-bp splice intermediate devoid of intron III, and a 171-bp species representing exon 4 to 6 splice (Fig. 4,A and B; note that retention of intron IV and exon 4 to 5 splicing should yield a protected exon 5 species). We did not see a protected species that would indicate production of splice intermediates with only intron III attached after gDNA transfection, suggesting that, at steady state, splicing of intron III precedes splicing of intron IV. However, transient transfection of Δi4–5 and Δi4–6 resulted in expression of a 231-bp species representing an unspliced transcript or an aberrant mRNA species spliced from a cryptic splice site 5′ of exon 4 (Fig. 4 B). These data indicate that intron IV is critical in controlling the efficiency of correct upstream splicing and further suggest that splicing of the invariant intron occurs prior to splicing of intron IV, the site of alternative splicing. The influence of upstream introns on downstream splice selection could result from a secondary structure in the pre-mRNA or be linked to upstream splicing events. To distinguish these possibilities, we deleted a large part of intron III from the construct Δi2–3, but kept the splicing machinery including the splice junctions and the branch point intact (P3–4Δi2–3 in Fig. 3). Expression of this deletion construct should result in more dramatic changes in pre-mRNA structure, but not upstream splicing, compared with the expression of Δi2–3. If the secondary structure of pre-mRNA plays a major role on downstream splicing selection, expression should result in a splicing pattern similar to that of Δi2–3,3–4. By contrast, if splicing events play a predominate role in downstream splice selection, expression should result in a splicing pattern similar to that of Δi2–3, which is close to the splice pattern of the genomic construct. As indicated in Fig. 4 A, transfection of P3–4Δi2–3 resulted in a 513-bp unspliced species, shorter than the 540-bp unspliced species seen in gDNA-transfected cells as expected from deletion of the intronic sequences. The absence of a protected exon 5 species in P3–4Δi2–3-transfected cells, similar to the gDNA, indicates that neither the intron IV retained species nor the exon 4 to 5 spliced mRNA is expressed. Therefore, in gDNA and P3–4Δi2–3-transfected cells, the 480- and 171-bp protected species represent a splicing intermediate devoid of intron III and exon 4 to 6 spliced mRNA, respectively. Accordingly, partial deletion of intron III in the Δi2–3 plasmid resulted in a splicing pattern similar to that of gDNA, rather than the splicing pattern seen for Δi2–3,3–4. To confirm the linkage of upstream splicing to downstream splice selection, the branch point adenosine in P3–4Δi2–3 was mutated to cytosine (single A → C mutation, SA/C). Since such a change in mammalian branch sites may activate abnormal branch formation through cryptic branch points (25Ruskin B. Greene J.M. Green M.R. Cell. 1985; 41: 833-844Abstract Full Text PDF PubMed Scopus (146) Google Scholar, 26Rautmann G. Breathnach R. Nature. 1985; 315: 430-432Crossref PubMed Scopus (34) Google Scholar, 27Padgett R.A. Konarska M.M. Aebi M. Hornig H. Weissmann C. Sharp P.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8349-8353Crossref PubMed Scopus (67) Google Scholar), four adenosines proximal to the branch point in the intronic sequence were also mutated to cytosines (four adenosine mutations, QUA/C) (Fig. 3). If upstream splicing is linked to downstream splice selection, elimination of splicing in these constructs should render a splicing pattern similar to that of Δi2–3,3–4. As shown in Fig. 4, transfection of SA/C resulted in a splice pattern similar to that of the parental construct P3–4Δi2–3 with a slight reduction in the density of the 480-bp band and appearance of an aberrant mRNA species. The presence of the 480-bp species indicates that intron III can still be spliced out despite mutation of the branch point. The presence of unspliced 513-bp species and the absence of a 501-bp species expected from the single point mutation suggest that the hybridized probe RNA with the single point mismatch is not sensitive to RNase digestion. By contrast, transfection of QUA/C resulted in diminished upstream splicing as indicated by the disappearance of the 480-bp splice intermediate, and the appearance of the 499-bp unspliced species containing sequences of intron III downstream of the point mutations. This interruption of upstream splicing resulted in aberrant splicing at the alternative splice region, as indicated by the lack of protected exon 4 sequences over the endogenous level and the appearance of aberrant mRNAs. The similar densities of the 171- and 191-bp species (over the endogenous level) suggest a low level of exon 4 to 5 splicing, but we cannot rule out the possibility that the 191-bp species results from aberrant splicing. Using the exon 4 to 6 spliced probe shown in Fig. 2 A, we confirmed that normal exon 4 to 6 splicing is diminished in QUA/C-transfected cells (data not shown). The co-existence of the 513- and 499-bp species suggests that only partial digestion occurred in the region of single point mutations. Thus, the 499-bp species, based on the assumption that the probe was excised at the first mism
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