MBNL and CELF proteins regulate alternative splicing of the skeletal muscle chloride channel CLCN1

Abstract

The expression and function of the skeletal muscle chloride channel CLCN1/ClC-1 is regulated by alternative splicing. Inclusion of the CLCN1 exon 7A is aberrantly elevated in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. Increased exon 7A inclusion leads to a reduction in CLCN1 function, which can be causative of myotonia. Two RNA-binding protein families--muscleblind-like (MBNL) and CUG-BP and ETR-3-like factor (CELF) proteins--are thought to mediate the splicing misregulation in DM. Here, we have identified multiple factors that regulate the alternative splicing of a mouse Clcn1 minigene. The inclusion of exon 7A was repressed by MBNL proteins while promoted by an expanded CUG repeat or CELF4, but not by CUG-BP. Mutation analyses suggested that exon 7A and its flanking region mediate the effect of MBNL1, whereas another distinct region in intron 6 mediates that of CELF4. An exonic splicing enhancer essential for the inclusion of exon 7A was identified at the 5' end of this exon, which might be inhibited by MBNL1. Collectively, these results provide a mechanistic model for the regulation of Clcn1 splicing, and reveal novel regulatory properties of MBNL and CELF proteins.

Figure 1.

Splicing regulation of MBNL and CELF proteins. (A) Structure of chloride channel minigenes. Both human CLCN1/ClC-1 and mouse Clcn1 minigenes were subcloned between the BglII and SalI sites of pEGFP-C1. Black boxes represent exons of the minigenes. Arrows indicate the position of primers used in the splicing assays. Exon 6B is a human-specific exon and is absent in Clcn1. (B) Splicing regulation of Clcn1 by MBNL and CELF proteins. Representative results of cellular splicing assays using the Clcn1 minigene in COS-7 cells. The upper bands correspond to a splice product containing exon 7A, whereas lower bands correspond to a splice product lacking exon 7A. Bar chart shows quantified results of exon 7A inclusion (mean ± SD, n = 3). Statistical significance was analyzed by analysis of variance (ANOVA) and Dunnett's multiple comparisons. All MBNL proteins and CELF proteins except for CUG-BP and ETR-3 showed significant differences (*P < 0.0001) compared to the empty vector. (C) CUG-BP and ETR-3 increased an unspliced product of the Clcn1 minigene. Structures of PCR products are indicated.

Figure 2.

MBNL1 is antagonized by CELF4 and expanded CUG repeats. (A) Antagonistic effects of CELF4 against MBNL1 in the splicing regulation of Clcn1. Clcn1 minigene was co-transfected with MBNL1 or MBNL1 plus CELF protein into COS-7 cells. Upper panel: lane 1, the pattern of empty vector-transfected cells; lanes 2–8, a constant amount of MBNL1-encoding vector and increasing amounts of one of the CELF-encoding vectors were co-transfected at ratios of 1 : 0 (lane 2), 1 : 1 (lanes 3, 5 and 7) and 1:3 (lanes 4, 6 and 8). Lower panel: similarly, a CELF4-encoding vector and MBNL1-encoding vector were transfected at ratios of 1 : 0 (lane 9), 1 : 1 (lane 10) and 1 : 3 (lane 11). The total amount of transfected plasmids was held constant by adjusting the amounts of empty vector. (B) Effects of an expanded CUG repeat on the chloride channel splicing. Clcn1 minigene was transfected with an expression vector of DMPK harboring a normal (DM18) or expanded (DM480) CTG repeat (left panel). Either normal or expanded DMPK vector was co-transfected with MBNL1 and the Clcn1 minigene, and the splicing patterns were analyzed (right panel). (C) Results of Clcn1-splicing assays in Neuro2a cells. MBNL1-mediated repression of exon 7A inclusion in Neuro2a cells (left). ‘V’ and ‘M’ indicate empty vector and MBNL1, respectively. Note that the basal inclusion of exon 7A in Neuro2a cells was higher than that in COS-7 cells. Clcn1-splicing regulation was affected by RNAi of Mbnl1 but not Cugbp1 in Neuro2a cells (right). Bar chart shows the quantified results of exon 7A inclusion (mean ±SD, n = 4). According to ANOVA and Dunnett's test, miMbnl1-146 induced a statistically significant increase of exon 7A compared to miLuc-200 (*P < 0.0001), whereas miCugbp1-559 did not (P = 0.36).

Figure 3.

Intronic deletion analysis of Clcn1. (A) Structure of Clcn1 deletion mutants. Deletion series of the Clcn1 minigene were generated by PCR-mediated mutagenesis. FL corresponds to the full-length Clcn1 minigene covering exons 6–7 and is the same construct as that used in Figures 1 and 2. The positions of nucleotides at the termini of exons and the junction of deleted regions are indicated. (B) Splicing analysis of Clcn1 deletion mutants in COS-7 cells. Cellular-splicing assays were performed using deletion mutants Δ1–Δ9 and MBNL1. Lanes ‘V’ and ‘M’ indicate the co-expression of each deletion minigene with empty vector and MBNL1, respectively. Splicing patterns were detected by RT–PCR, as in Figure 1B.

Figure 4.

Involvement of exon 7A sequence in splicing regulation by MBNL1. (A) Response of truncated Clcn1 minigenes to MBNL1 is dependent on exon 7A. Structures of 6-7A, 7A-7 and 6/7 deletion mutants are indicated in the upper left. Splicing analysis of these mutants shows both spliced and unspliced products as indicated. The bar chart shows the quantified ratio of spliced products. (B) Deletion in the 3′ region of exon 7A did not abolish the response to MBNL1. The structure of the 6-7AΔ mutant is shown (left). Splicing products of 6-7AΔ and quantification are shown as in (A). (C) Splicing regulation of the 7A/(−52)7A minigene. 7A/(−52)7A was made from 7A-7 by replacing exon 7 and its upstream 52 nt with the corresponding region of exon 7A (left). Splicing of 7A/(−52)7A was responsive to MBNL1 (right). Lanes ‘V’ and ‘M’ indicate co-transfection with the empty vector and MBNL1, respectively. Bars represent the ratio of spliced bands. Statistical analysis was performed by two-tailed t-test in comparison with the empty vector. *P < 0.05 from three independent experiments.

Figure 5.

Exon 7A splicing regulation in heterologous minigenes. (A) Structure of the Tpm2-based heterologous minigene. Fragments of Tpm2 covering exons 1 to 2 were inserted downstream of EGFP. Test exons together with their flanking regions were inserted into intron 1 of Tpm2. Intronic fragments derived from Clcn1 are indicated by thick lines, whereas those derived from Tpm2 (regions flanking exon 9) are indicated by thin lines. Exonic sequences of Clcn1 exon 7A and Tpm2 exon 9 are indicated by grey and white boxes, respectively. (B) Splicing assay results using Tpm2-based heterologous minigenes in COS-7 cells. Upper bands correspond to the spliced products containing an exon inserted between Tpm2 exon 1 and 2. ‘V’ and ‘M’ indicate empty vector and MBNL1, respectively. Compared with Tpm2 ex9, Clcn1-derived 414–720 and 451–720 minigenes exhibited evident responses to MBNL1. Bar chart shows quantified results of the splicing assay (n = 3). (C) Splicing regulation of heterologous minigenes containing a portion of Clcn1 intron 6. Results of the splicing assay are shown as in B. The structures of minigenes are shown in A. (D) Determination of the Clcn1 region responsible for CELF4-mediated exon 7A inclusion. Tpm2-based heterologous minigenes covering a Clcn1 region indicated by the numbers were tested for their responsiveness to CELF4 (C4). The splicing assay was performed as in B, except that CELF4 was used in place of MBNL1. In the case of 181–720, CELF4 expression induced a significant increase compared to control (P < 0.05, n = 3, two-tailed t-test). (E) Splicing analysis of the Δ4 mutant minigene and CELF4. The structure of Δ4 is described in Figure 3A. Splicing analysis results are shown as in D. CELF4 did not significantly alter the splicing of Δ4 (P = 0.97, n = 3, two-tailed t-test).

Figure 6.

MBNL1 associates with Clcn1 RNA. (A) The intracellular association between EGFP-MBNL1₄₀ and the transcript of endogenous Clcn1 was analyzed by RIP. The cell lysate of a cell line stably expressing EGFP-MBNL1 was used for immunoprecipitation. The RNA fraction co-precipitated with anti-GFP antibody or IgG was reverse-transcribed. The amount of Clcn1 (pre-)mRNA was quantified by real-time quantitative PCR using a primer set for the indicated regions of the Clcn1 gene (n = 5). Bars represent the amount of Clcn1 RNA co-precipitated with anti-GFP antibody or control IgG normalized by the amount of the Clcn1 RNA in the input fraction (mean ± SD). Left: EGFP-MBNL1 was co-immunoprecipitated with RNA fragments containing Clcn1 intron 6 by GFP antibody but not control IgG (*P < 0.01, two-tailed t-test). Right: RIP analysis of multiple Clcn1 regions. Three regions of Clcn1 (intron 6, intron 12 and 3′-UTR) were amplified from immunoprecipitates of the GFP antibody. Intron 6 was significantly more enriched than the other regions (*P < 0.01, ANOVA and Tukey's test). (B) A hairpin structure predicted in the putative MBNL1-responsive region of Clcn1. Nucleotides 473–518 of Clcn1 are indicated in boldface (upper). Predicted secondary structure of the fragment Clcn1(473–518) (lower). (C) Binding between GST-MBNL1 and Clcn1(473–518) or its mutants was examined by gel shift analysis (left). ³²P-labeled probes were incubated with or without GST-MBNL1 (0.225, 0.45, 0.9, 1.8 μM) or GST (1.8 μM). The reaction mixture was separated by native PAGE and visualized by autoradiography. Structure of probes used in gel shift analysis (right). In the Clcn1(GAA) mutant, the first 12 nt of exon 7A are substituted by (GAA)₄ repeats. The Clcn1(a504c) mutant contains a point mutation of the first nucleotide of exon 7A but is otherwise the same as Clcn1(473–518).

Figure 7.

Analysis of the 5′ region of exon 7A in the Clcn1 minigene. (A) Sequence of nt 451–533 of the Clcn1 minigene that corresponds to a 3′ portion of intron 6 and a 5′ portion of exon 7A. Substituted nucleotides in Mut 1 are shown in boldface. The first 15 nt of exon 7A were deleted in Mut 2. (B) Splicing assays results for Mut 1 and Mut 2 minigenes in COS-7 cells. Mut 2 exhibited a complete loss of exon 7A inclusion. (C) Effects of deletions in the 5′ region of Clcn1 exon 7A on basal-splicing efficiency. Deleted regions in Mut 2, Mut 3 and Mut 4 are indicated (upper). Splicing assay results of Mut 2/3/4/minigenes transfected into COS-7 cells are shown (lower). WT: wild type. (D) Point mutations at the 5′ end of exon 7A disrupted the basal inclusion of exon 7A. Alignment of part of Mut3 and Mut4 sequences (upper). Different nucleotides between Mut 3 and Mut 4 are indicated by boldface and underlined text. Point mutations that partly mimic the difference between Mut 3 and Mut 4 were introduced in the Mut 5a, Mut 5b, a504c and t512g mutants, as indicated by boldface and underlined text (middle). Splicing assay results of the mutant minigenes in COS-7 cells (lower).

Figure 8.

Model of mouse Clcn1 splicing regulation by multiple factors. Cis- and trans-acting factors involved in the splicing regulation of Clcn1 exon 7A are depicted. MBNL1 represses exon 7A inclusion through a region containing the 5′ region of exon 7A as well as its flanking intronic sequence. An exonic splicing enhancer (ESE) is located at the 5′ end of exon 7A. An antisense oligonucleotide (AON) previously reported (38), as well as MBNL1, might act in part through inhibiting this ESE. The facilitation of exon 7A inclusion by CELF4 is mediated by a region located in intron 6. Expanded CUG/CCUG repeat RNA may deplete MBNL proteins, resulting in the facilitation of exon 7A inclusion.