Pre-mRNA splicing of IgM exons M1 and M2 is directed by a juxtaposed splicing enhancer and inhibitor - PubMed (original) (raw)
Pre-mRNA splicing of IgM exons M1 and M2 is directed by a juxtaposed splicing enhancer and inhibitor
J L Kan et al. Genes Dev. 1999.
Abstract
Splicing of certain pre-mRNA introns is dependent on an enhancer element, which is typically purine-rich. It is generally thought that enhancers increase the use of suboptimal splicing signals, and one specific proposal is that enhancers stabilize binding of U2AF65 to weak polypyrimidine (Py) tracts. Here, we test this model using an IgM pre-mRNA substrate, which contains a well-characterized enhancer. Although the enhancer was required for in vitro splicing, we found it had no effect on U2AF65 binding. Unexpectedly, replacement of the natural IgM Py tract, branchpoint, and 5' splice site with consensus splicing signals did not circumvent the enhancer requirement. These observations led us to identify a novel regulatory element within the IgM M2 exon that acts as a splicing inhibitor; removal of the inhibitor enabled splicing to occur in the absence of the enhancer. The IgM M2 splicing inhibitor is evolutionarily conserved, can inhibit the activity of an unrelated, constitutively spliced pre-mRNA, and acts by repressing splicing complex assembly. Interestingly, the inhibitor itself forms an ATP-dependent complex that contains U2 snRNP. We conclude that splicing of IgM exons M1 and M2 is directed by two juxtaposed regulatory elements-an enhancer and an inhibitor-and that a primary function of the enhancer is to counteract the inhibitor.
Figures
Figure 1
The IgM M2 enhancer does not function by increasing U2AF65 binding. (A) In vitro splicing of IgM substrates. (Bottom) A diagram of the RNA substrates μM and μMΔ (formerly named pμM1-2 and pμMΔ; Watakabe et al. 1993). The intron sequence deleted to generate RNA substrates lacking a Py tract, μMPy- and μMΔPy- is shown. (Top) Splicing was performed at 30°C for 90 min using standard conditions for μM (lane 1) and μMΔ (lane 2). The unspliced RNA substrates and spliced product are indicated. (B) Early splicing time course. In vitro splicing of RNA substrates was performed at 30°C for the times indicated. A portion of the reaction mixture was used to assess splicing by electrophoresis on a 13% denaturing gel. (Bottom) The remainder was UV cross-linked and immunoprecipitated as described in Materials and Methods and electrophoresed on a 10% SDS–polyacrylamide gel. (C) Specificity of U2AF65 binding. In vitro splicing reactions were performed as described above for 20 min in the presence or absence of ATP, cross-linked, and immunoprecipitated with the MC3 antibody. RNA substrates lacking (lanes 1,2) or containing (lanes 3,4) the enhancer or lacking the pyrimidine tract (lanes 1,3) are shown. (D) HeLa U2AF add-back. Cross-linking was performed as described for C. UV–RNA cross-linking/immunoprecipitation was performed in U2AF-depleted nuclear extract (NE) (lanes 1,5) and with increasing addition of U2AF purified from HeLa cells (lanes 2–4,6–8). (E) rU2AF65 add-back. (Top) UV–RNA cross-linking/immunoprecipitation in the presence of NE (lanes 1,6), with increasing amounts of rU2AF65 (lanes 2–4, 7–9), and immunoprecipition performed in the absence of the primary monoclonal antibody (lanes 5,10). (Bottom) In vitro splicing was performed in parallel and is shown. (F) Total UV–RNA cross-linking of IgM substrates. In vitro splicing and UV–RNA cross-linking was performed as described in C in the presence of NE (lanes 1,3) and with the addition of rU2AF65 (lanes 2,4). RNA substrates containing (lanes 1,2) or lacking (lanes 3,4) the enhancer are shown. (G) Splicing time course and UV–RNA cross-linking/immunoprecipitation of dsx RNA. (Top) In vitro splicing of RNA substrates, dsx–ASLV and dsxΔE (formerly named dsx–SO; Tanaka et al. 1994), was performed at 30°C for the times indicated. A portion of the reaction mixture was used to assess splicing by electrophoresis on an 8% denaturing gel. (Bottom) The remainder was UV cross-linked and immunoprecipitated as described in Materials and Methods and electrophoresed on a 10% SDS–polyacrylamide gel. Splicing substrates, intermediates, and products are indicated. Control UV–RNA cross-linking/immunoprecipitation (lanes 1,8) were performed in the absence of U2AF65 monoclonal antibody.
Figure 1
The IgM M2 enhancer does not function by increasing U2AF65 binding. (A) In vitro splicing of IgM substrates. (Bottom) A diagram of the RNA substrates μM and μMΔ (formerly named pμM1-2 and pμMΔ; Watakabe et al. 1993). The intron sequence deleted to generate RNA substrates lacking a Py tract, μMPy- and μMΔPy- is shown. (Top) Splicing was performed at 30°C for 90 min using standard conditions for μM (lane 1) and μMΔ (lane 2). The unspliced RNA substrates and spliced product are indicated. (B) Early splicing time course. In vitro splicing of RNA substrates was performed at 30°C for the times indicated. A portion of the reaction mixture was used to assess splicing by electrophoresis on a 13% denaturing gel. (Bottom) The remainder was UV cross-linked and immunoprecipitated as described in Materials and Methods and electrophoresed on a 10% SDS–polyacrylamide gel. (C) Specificity of U2AF65 binding. In vitro splicing reactions were performed as described above for 20 min in the presence or absence of ATP, cross-linked, and immunoprecipitated with the MC3 antibody. RNA substrates lacking (lanes 1,2) or containing (lanes 3,4) the enhancer or lacking the pyrimidine tract (lanes 1,3) are shown. (D) HeLa U2AF add-back. Cross-linking was performed as described for C. UV–RNA cross-linking/immunoprecipitation was performed in U2AF-depleted nuclear extract (NE) (lanes 1,5) and with increasing addition of U2AF purified from HeLa cells (lanes 2–4,6–8). (E) rU2AF65 add-back. (Top) UV–RNA cross-linking/immunoprecipitation in the presence of NE (lanes 1,6), with increasing amounts of rU2AF65 (lanes 2–4, 7–9), and immunoprecipition performed in the absence of the primary monoclonal antibody (lanes 5,10). (Bottom) In vitro splicing was performed in parallel and is shown. (F) Total UV–RNA cross-linking of IgM substrates. In vitro splicing and UV–RNA cross-linking was performed as described in C in the presence of NE (lanes 1,3) and with the addition of rU2AF65 (lanes 2,4). RNA substrates containing (lanes 1,2) or lacking (lanes 3,4) the enhancer are shown. (G) Splicing time course and UV–RNA cross-linking/immunoprecipitation of dsx RNA. (Top) In vitro splicing of RNA substrates, dsx–ASLV and dsxΔE (formerly named dsx–SO; Tanaka et al. 1994), was performed at 30°C for the times indicated. A portion of the reaction mixture was used to assess splicing by electrophoresis on an 8% denaturing gel. (Bottom) The remainder was UV cross-linked and immunoprecipitated as described in Materials and Methods and electrophoresed on a 10% SDS–polyacrylamide gel. Splicing substrates, intermediates, and products are indicated. Control UV–RNA cross-linking/immunoprecipitation (lanes 1,8) were performed in the absence of U2AF65 monoclonal antibody.
Figure 1
The IgM M2 enhancer does not function by increasing U2AF65 binding. (A) In vitro splicing of IgM substrates. (Bottom) A diagram of the RNA substrates μM and μMΔ (formerly named pμM1-2 and pμMΔ; Watakabe et al. 1993). The intron sequence deleted to generate RNA substrates lacking a Py tract, μMPy- and μMΔPy- is shown. (Top) Splicing was performed at 30°C for 90 min using standard conditions for μM (lane 1) and μMΔ (lane 2). The unspliced RNA substrates and spliced product are indicated. (B) Early splicing time course. In vitro splicing of RNA substrates was performed at 30°C for the times indicated. A portion of the reaction mixture was used to assess splicing by electrophoresis on a 13% denaturing gel. (Bottom) The remainder was UV cross-linked and immunoprecipitated as described in Materials and Methods and electrophoresed on a 10% SDS–polyacrylamide gel. (C) Specificity of U2AF65 binding. In vitro splicing reactions were performed as described above for 20 min in the presence or absence of ATP, cross-linked, and immunoprecipitated with the MC3 antibody. RNA substrates lacking (lanes 1,2) or containing (lanes 3,4) the enhancer or lacking the pyrimidine tract (lanes 1,3) are shown. (D) HeLa U2AF add-back. Cross-linking was performed as described for C. UV–RNA cross-linking/immunoprecipitation was performed in U2AF-depleted nuclear extract (NE) (lanes 1,5) and with increasing addition of U2AF purified from HeLa cells (lanes 2–4,6–8). (E) rU2AF65 add-back. (Top) UV–RNA cross-linking/immunoprecipitation in the presence of NE (lanes 1,6), with increasing amounts of rU2AF65 (lanes 2–4, 7–9), and immunoprecipition performed in the absence of the primary monoclonal antibody (lanes 5,10). (Bottom) In vitro splicing was performed in parallel and is shown. (F) Total UV–RNA cross-linking of IgM substrates. In vitro splicing and UV–RNA cross-linking was performed as described in C in the presence of NE (lanes 1,3) and with the addition of rU2AF65 (lanes 2,4). RNA substrates containing (lanes 1,2) or lacking (lanes 3,4) the enhancer are shown. (G) Splicing time course and UV–RNA cross-linking/immunoprecipitation of dsx RNA. (Top) In vitro splicing of RNA substrates, dsx–ASLV and dsxΔE (formerly named dsx–SO; Tanaka et al. 1994), was performed at 30°C for the times indicated. A portion of the reaction mixture was used to assess splicing by electrophoresis on an 8% denaturing gel. (Bottom) The remainder was UV cross-linked and immunoprecipitated as described in Materials and Methods and electrophoresed on a 10% SDS–polyacrylamide gel. Splicing substrates, intermediates, and products are indicated. Control UV–RNA cross-linking/immunoprecipitation (lanes 1,8) were performed in the absence of U2AF65 monoclonal antibody.
Figure 1
The IgM M2 enhancer does not function by increasing U2AF65 binding. (A) In vitro splicing of IgM substrates. (Bottom) A diagram of the RNA substrates μM and μMΔ (formerly named pμM1-2 and pμMΔ; Watakabe et al. 1993). The intron sequence deleted to generate RNA substrates lacking a Py tract, μMPy- and μMΔPy- is shown. (Top) Splicing was performed at 30°C for 90 min using standard conditions for μM (lane 1) and μMΔ (lane 2). The unspliced RNA substrates and spliced product are indicated. (B) Early splicing time course. In vitro splicing of RNA substrates was performed at 30°C for the times indicated. A portion of the reaction mixture was used to assess splicing by electrophoresis on a 13% denaturing gel. (Bottom) The remainder was UV cross-linked and immunoprecipitated as described in Materials and Methods and electrophoresed on a 10% SDS–polyacrylamide gel. (C) Specificity of U2AF65 binding. In vitro splicing reactions were performed as described above for 20 min in the presence or absence of ATP, cross-linked, and immunoprecipitated with the MC3 antibody. RNA substrates lacking (lanes 1,2) or containing (lanes 3,4) the enhancer or lacking the pyrimidine tract (lanes 1,3) are shown. (D) HeLa U2AF add-back. Cross-linking was performed as described for C. UV–RNA cross-linking/immunoprecipitation was performed in U2AF-depleted nuclear extract (NE) (lanes 1,5) and with increasing addition of U2AF purified from HeLa cells (lanes 2–4,6–8). (E) rU2AF65 add-back. (Top) UV–RNA cross-linking/immunoprecipitation in the presence of NE (lanes 1,6), with increasing amounts of rU2AF65 (lanes 2–4, 7–9), and immunoprecipition performed in the absence of the primary monoclonal antibody (lanes 5,10). (Bottom) In vitro splicing was performed in parallel and is shown. (F) Total UV–RNA cross-linking of IgM substrates. In vitro splicing and UV–RNA cross-linking was performed as described in C in the presence of NE (lanes 1,3) and with the addition of rU2AF65 (lanes 2,4). RNA substrates containing (lanes 1,2) or lacking (lanes 3,4) the enhancer are shown. (G) Splicing time course and UV–RNA cross-linking/immunoprecipitation of dsx RNA. (Top) In vitro splicing of RNA substrates, dsx–ASLV and dsxΔE (formerly named dsx–SO; Tanaka et al. 1994), was performed at 30°C for the times indicated. A portion of the reaction mixture was used to assess splicing by electrophoresis on an 8% denaturing gel. (Bottom) The remainder was UV cross-linked and immunoprecipitated as described in Materials and Methods and electrophoresed on a 10% SDS–polyacrylamide gel. Splicing substrates, intermediates, and products are indicated. Control UV–RNA cross-linking/immunoprecipitation (lanes 1,8) were performed in the absence of U2AF65 monoclonal antibody.
Figure 1
The IgM M2 enhancer does not function by increasing U2AF65 binding. (A) In vitro splicing of IgM substrates. (Bottom) A diagram of the RNA substrates μM and μMΔ (formerly named pμM1-2 and pμMΔ; Watakabe et al. 1993). The intron sequence deleted to generate RNA substrates lacking a Py tract, μMPy- and μMΔPy- is shown. (Top) Splicing was performed at 30°C for 90 min using standard conditions for μM (lane 1) and μMΔ (lane 2). The unspliced RNA substrates and spliced product are indicated. (B) Early splicing time course. In vitro splicing of RNA substrates was performed at 30°C for the times indicated. A portion of the reaction mixture was used to assess splicing by electrophoresis on a 13% denaturing gel. (Bottom) The remainder was UV cross-linked and immunoprecipitated as described in Materials and Methods and electrophoresed on a 10% SDS–polyacrylamide gel. (C) Specificity of U2AF65 binding. In vitro splicing reactions were performed as described above for 20 min in the presence or absence of ATP, cross-linked, and immunoprecipitated with the MC3 antibody. RNA substrates lacking (lanes 1,2) or containing (lanes 3,4) the enhancer or lacking the pyrimidine tract (lanes 1,3) are shown. (D) HeLa U2AF add-back. Cross-linking was performed as described for C. UV–RNA cross-linking/immunoprecipitation was performed in U2AF-depleted nuclear extract (NE) (lanes 1,5) and with increasing addition of U2AF purified from HeLa cells (lanes 2–4,6–8). (E) rU2AF65 add-back. (Top) UV–RNA cross-linking/immunoprecipitation in the presence of NE (lanes 1,6), with increasing amounts of rU2AF65 (lanes 2–4, 7–9), and immunoprecipition performed in the absence of the primary monoclonal antibody (lanes 5,10). (Bottom) In vitro splicing was performed in parallel and is shown. (F) Total UV–RNA cross-linking of IgM substrates. In vitro splicing and UV–RNA cross-linking was performed as described in C in the presence of NE (lanes 1,3) and with the addition of rU2AF65 (lanes 2,4). RNA substrates containing (lanes 1,2) or lacking (lanes 3,4) the enhancer are shown. (G) Splicing time course and UV–RNA cross-linking/immunoprecipitation of dsx RNA. (Top) In vitro splicing of RNA substrates, dsx–ASLV and dsxΔE (formerly named dsx–SO; Tanaka et al. 1994), was performed at 30°C for the times indicated. A portion of the reaction mixture was used to assess splicing by electrophoresis on an 8% denaturing gel. (Bottom) The remainder was UV cross-linked and immunoprecipitated as described in Materials and Methods and electrophoresed on a 10% SDS–polyacrylamide gel. Splicing substrates, intermediates, and products are indicated. Control UV–RNA cross-linking/immunoprecipitation (lanes 1,8) were performed in the absence of U2AF65 monoclonal antibody.
Figure 1
The IgM M2 enhancer does not function by increasing U2AF65 binding. (A) In vitro splicing of IgM substrates. (Bottom) A diagram of the RNA substrates μM and μMΔ (formerly named pμM1-2 and pμMΔ; Watakabe et al. 1993). The intron sequence deleted to generate RNA substrates lacking a Py tract, μMPy- and μMΔPy- is shown. (Top) Splicing was performed at 30°C for 90 min using standard conditions for μM (lane 1) and μMΔ (lane 2). The unspliced RNA substrates and spliced product are indicated. (B) Early splicing time course. In vitro splicing of RNA substrates was performed at 30°C for the times indicated. A portion of the reaction mixture was used to assess splicing by electrophoresis on a 13% denaturing gel. (Bottom) The remainder was UV cross-linked and immunoprecipitated as described in Materials and Methods and electrophoresed on a 10% SDS–polyacrylamide gel. (C) Specificity of U2AF65 binding. In vitro splicing reactions were performed as described above for 20 min in the presence or absence of ATP, cross-linked, and immunoprecipitated with the MC3 antibody. RNA substrates lacking (lanes 1,2) or containing (lanes 3,4) the enhancer or lacking the pyrimidine tract (lanes 1,3) are shown. (D) HeLa U2AF add-back. Cross-linking was performed as described for C. UV–RNA cross-linking/immunoprecipitation was performed in U2AF-depleted nuclear extract (NE) (lanes 1,5) and with increasing addition of U2AF purified from HeLa cells (lanes 2–4,6–8). (E) rU2AF65 add-back. (Top) UV–RNA cross-linking/immunoprecipitation in the presence of NE (lanes 1,6), with increasing amounts of rU2AF65 (lanes 2–4, 7–9), and immunoprecipition performed in the absence of the primary monoclonal antibody (lanes 5,10). (Bottom) In vitro splicing was performed in parallel and is shown. (F) Total UV–RNA cross-linking of IgM substrates. In vitro splicing and UV–RNA cross-linking was performed as described in C in the presence of NE (lanes 1,3) and with the addition of rU2AF65 (lanes 2,4). RNA substrates containing (lanes 1,2) or lacking (lanes 3,4) the enhancer are shown. (G) Splicing time course and UV–RNA cross-linking/immunoprecipitation of dsx RNA. (Top) In vitro splicing of RNA substrates, dsx–ASLV and dsxΔE (formerly named dsx–SO; Tanaka et al. 1994), was performed at 30°C for the times indicated. A portion of the reaction mixture was used to assess splicing by electrophoresis on an 8% denaturing gel. (Bottom) The remainder was UV cross-linked and immunoprecipitated as described in Materials and Methods and electrophoresed on a 10% SDS–polyacrylamide gel. Splicing substrates, intermediates, and products are indicated. Control UV–RNA cross-linking/immunoprecipitation (lanes 1,8) were performed in the absence of U2AF65 monoclonal antibody.
Figure 1
The IgM M2 enhancer does not function by increasing U2AF65 binding. (A) In vitro splicing of IgM substrates. (Bottom) A diagram of the RNA substrates μM and μMΔ (formerly named pμM1-2 and pμMΔ; Watakabe et al. 1993). The intron sequence deleted to generate RNA substrates lacking a Py tract, μMPy- and μMΔPy- is shown. (Top) Splicing was performed at 30°C for 90 min using standard conditions for μM (lane 1) and μMΔ (lane 2). The unspliced RNA substrates and spliced product are indicated. (B) Early splicing time course. In vitro splicing of RNA substrates was performed at 30°C for the times indicated. A portion of the reaction mixture was used to assess splicing by electrophoresis on a 13% denaturing gel. (Bottom) The remainder was UV cross-linked and immunoprecipitated as described in Materials and Methods and electrophoresed on a 10% SDS–polyacrylamide gel. (C) Specificity of U2AF65 binding. In vitro splicing reactions were performed as described above for 20 min in the presence or absence of ATP, cross-linked, and immunoprecipitated with the MC3 antibody. RNA substrates lacking (lanes 1,2) or containing (lanes 3,4) the enhancer or lacking the pyrimidine tract (lanes 1,3) are shown. (D) HeLa U2AF add-back. Cross-linking was performed as described for C. UV–RNA cross-linking/immunoprecipitation was performed in U2AF-depleted nuclear extract (NE) (lanes 1,5) and with increasing addition of U2AF purified from HeLa cells (lanes 2–4,6–8). (E) rU2AF65 add-back. (Top) UV–RNA cross-linking/immunoprecipitation in the presence of NE (lanes 1,6), with increasing amounts of rU2AF65 (lanes 2–4, 7–9), and immunoprecipition performed in the absence of the primary monoclonal antibody (lanes 5,10). (Bottom) In vitro splicing was performed in parallel and is shown. (F) Total UV–RNA cross-linking of IgM substrates. In vitro splicing and UV–RNA cross-linking was performed as described in C in the presence of NE (lanes 1,3) and with the addition of rU2AF65 (lanes 2,4). RNA substrates containing (lanes 1,2) or lacking (lanes 3,4) the enhancer are shown. (G) Splicing time course and UV–RNA cross-linking/immunoprecipitation of dsx RNA. (Top) In vitro splicing of RNA substrates, dsx–ASLV and dsxΔE (formerly named dsx–SO; Tanaka et al. 1994), was performed at 30°C for the times indicated. A portion of the reaction mixture was used to assess splicing by electrophoresis on an 8% denaturing gel. (Bottom) The remainder was UV cross-linked and immunoprecipitated as described in Materials and Methods and electrophoresed on a 10% SDS–polyacrylamide gel. Splicing substrates, intermediates, and products are indicated. Control UV–RNA cross-linking/immunoprecipitation (lanes 1,8) were performed in the absence of U2AF65 monoclonal antibody.
Figure 2
The IgM M2 enhancer can function in the absence of U2AF35. (A) Quantitative immunoblotting of U2AF-depleted HeLa nuclear extract (NE). Immunoblot analysis of HeLa NE immunodepleted with the MC3 monoclonal antibody to U2AF65. Depleted NE (ΔNE, lane 1) and a concentration curve of varying amounts of HeLa NE (lanes 2–7) were probed with the U2AF65 and U2AF35 antibodies. The numbers above each lane represent the percentage of HeLa NE in the sample. (B) Far Western analysis of the rU2AF65Δ95–138–U2AF35 interaction. Far Western analysis with 35S-labeled in vitro-translated U2AF35 against rU2AF65 (lane 1) and rU2AF65Δ95–138 (lane 2). (C) In vitro splicing of IgM M2 splicing enhancer substrate in ΔNE. Splicing of IgM substrate in the absence of ATP (lane 1, control), in NE (lane 2), in ΔNE (lane 3), with increasing rU2AF65 (lanes 4–6), with rU2AF65Δ95–138 (lanes 7–9). The amount of recombinant protein used in these splicing assays is the same as those used for UV–RNA cross-linking in Fig. 1E. Equivalent levels of rU2AF65 and rU2AF65Δ95–138 were used in this experiment, as indicated by silver staining of protein added (bottom).
Figure 3
Substitution of IgM splicing signals with consensus elements does not relieve the enhancer requirement. (Bottom) Schematic representation of the replacements made to the IgM RNA substrate Py tract, branchpoint, and 5′ splice site. The sequence removed from the RNA substrate is underlined and the replacement sequence is in boldface type and underlined. (Top) In vitro splicing with different compensatory replacements of splice signals is shown. The presence or absence of the enhancer and the compensatory change made is indicated above the lanes.
Figure 4
Identification of a region within IgM exon M2 that inhibits splicing. (A)(Left) Schematic diagram of IgM substrates. (ENH) The purine-rich element; (inhibitor) the region encompassing the inhibitory element. (Right) RNA substrates for in vitro splicing were generated by use of the restriction endonuclease shown above each lane: (X) _Xba_I; (B) _Bst_NI; and (S) _Spe_I for μM (lanes 1–3) and μMΔE (lanes 4–6). Spliced products are indicated by arrows. The splicing substrate and intermediates are indicated. (B) The splicing inhibitor function is orientation-dependent. In vitro splicing of RNA substrates are μM (lane 1), μMΔE (lane 2), and with the inhibitor in reverse orientation, μMΔE+I(r) (lane 3). For in vitro transcription, μM and μMΔE were linearized with _Xba_I, and μMΔE+I(r) was linearized with _Hin_cII. (C) U2AF65 binding. IgM substrate in the absence of the inhibitor with (lane 1) and without the enhancer (lane 2).
Figure 4
Identification of a region within IgM exon M2 that inhibits splicing. (A)(Left) Schematic diagram of IgM substrates. (ENH) The purine-rich element; (inhibitor) the region encompassing the inhibitory element. (Right) RNA substrates for in vitro splicing were generated by use of the restriction endonuclease shown above each lane: (X) _Xba_I; (B) _Bst_NI; and (S) _Spe_I for μM (lanes 1–3) and μMΔE (lanes 4–6). Spliced products are indicated by arrows. The splicing substrate and intermediates are indicated. (B) The splicing inhibitor function is orientation-dependent. In vitro splicing of RNA substrates are μM (lane 1), μMΔE (lane 2), and with the inhibitor in reverse orientation, μMΔE+I(r) (lane 3). For in vitro transcription, μM and μMΔE were linearized with _Xba_I, and μMΔE+I(r) was linearized with _Hin_cII. (C) U2AF65 binding. IgM substrate in the absence of the inhibitor with (lane 1) and without the enhancer (lane 2).
Figure 4
Identification of a region within IgM exon M2 that inhibits splicing. (A)(Left) Schematic diagram of IgM substrates. (ENH) The purine-rich element; (inhibitor) the region encompassing the inhibitory element. (Right) RNA substrates for in vitro splicing were generated by use of the restriction endonuclease shown above each lane: (X) _Xba_I; (B) _Bst_NI; and (S) _Spe_I for μM (lanes 1–3) and μMΔE (lanes 4–6). Spliced products are indicated by arrows. The splicing substrate and intermediates are indicated. (B) The splicing inhibitor function is orientation-dependent. In vitro splicing of RNA substrates are μM (lane 1), μMΔE (lane 2), and with the inhibitor in reverse orientation, μMΔE+I(r) (lane 3). For in vitro transcription, μM and μMΔE were linearized with _Xba_I, and μMΔE+I(r) was linearized with _Hin_cII. (C) U2AF65 binding. IgM substrate in the absence of the inhibitor with (lane 1) and without the enhancer (lane 2).
Figure 5
Function of the IgM M2 splicing inhibitor in a heterologous substrate. (Left) Schematic diagram of human β-globin constructs. (Right) In vitro splicing of human β-globin (lane 1) or human β-globin containing the IgM M2 splicing inhibitor in various positions (lanes 2–4). Spliced products and intermediates are indicated.
Figure 6
Enhancer-dependent splicing of a human β-globin pre-mRNA derivative. (Top) In vitro splicing of human β-globin pre-mRNA (lane 1), human β-globin pre-mRNA containing the IgM M2 splicing inhibitor (lane 2), and human β-globin pre-mRNA containing both splicing inhibitor and enhancer (lane 3). Spliced products and intermediates are indicated. (Bottom) For in vitro transcription, RNA substrates were linerized with _Bst_YI.
Figure 7
The IgM M2 splicing inhibitor represses splicing complex assembly. (A) Complex assembly of IgM substrates. Time course of splicing complex assembly for IgM pre-mRNA substrates with enhancer and inhibitor, μM/_Xba_I (lanes 1–4), without enhancer and with inhibitor, μMΔE/_Xba_I (lanes 5–8), with enhancer and without inhibitor, μM/_Bst_NI (lanes 9–12), or without enhancer and inhibitor, μMΔE/_Bst_NI (lanes 13–16). The identities of the complexes are indicated. (B) Complex assembly of human β-globin substrates. Time course of complex formation for human β-globin (lanes 1–4), for human β-globin with one copy of the inhibitor (lanes 5–8), and for human β-globin with two tandem copies of the inhibitor (lanes 9–12).
Figure 8
The IgM M2 splicing inhibitor forms an ATP-dependent complex that contains U2 snRNA. (A) Complex assembly of IgM M2 splicing inhibitor. μM RNA substrate with enhancer (lanes 1,2) and the inhibitor only (INH; lanes 3,4) incubated in the absence (lanes 1,3) or presence (lanes 2,4) of ATP. (B) Northern blot analysis of inhibitor complex. Biotinylated RNA substrates were incubated in standard in vitro splicing reaction mixtures and purified on streptavidin beads. Samples were treated with proteinase K and electrophoresed on a 10% urea–polyacrylamide gel. (Top) RNAs purified from nuclear extract (NE; lane 1), nonspecific RNA (lane 2), splicing inhibitor (lane 3), splicing enhancer and inhibitor (lane 4), splicing enhancer (lane 5), or exon sequence from AdML (lane 6) were probed with a 32P-end-labeled α–U2 snRNA oligonucleotide. (Bottom) RNAs purified from NE (lane 1), nonspecific RNA (lane 2), splicing inhibitor (lane 3), or exon sequence from AdML (lane 4) were probed with a 32P-end-labeled α-U1 snRNA oligonucleotide.
Figure 9
Sequence comparison of mouse and human IgM exon M2. Conserved elements, Py tract , enhancer, inhibitor, and polyadenylation signal are shaded and conserved sequence immediately upstream of the inhibitor is boxed.
Figure 10
Model of splicing enhancer function in IgM pre-mRNA. In the absence of the enhancer (right), a U2 snRNP-inhibitor complex pairs with the 5′ splice site complex resulting in a dead-end complex. In the presence of the enhancer (left), a functional U2 snRNP–branchpoint complex productively pairs with the 5′ splice site complex, which is subsequently assembled into an active spliceosome.
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