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• W2092831145 abstract "Article15 July 2004free access Muscleblind proteins regulate alternative splicing Thai H Ho Thai H Ho Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Nicolas Charlet-B Nicolas Charlet-B Department of Pathology, Baylor College of Medicine, Houston, TX, USAPresent address: IGBMC, 1 Rue Laurent Fries, 67400 Illkirch, France Search for more papers by this author Michael G Poulos Michael G Poulos Department of Molecular Genetics and Microbiology, College of Medicine, Powell Gene Therapy Center, University of Florida, Gainesville, FL, USA Search for more papers by this author Gopal Singh Gopal Singh Department of Pathology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Maurice S Swanson Maurice S Swanson Department of Molecular Genetics and Microbiology, College of Medicine, Powell Gene Therapy Center, University of Florida, Gainesville, FL, USA Search for more papers by this author Thomas A Cooper Corresponding Author Thomas A Cooper Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA Department of Pathology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Thai H Ho Thai H Ho Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Nicolas Charlet-B Nicolas Charlet-B Department of Pathology, Baylor College of Medicine, Houston, TX, USAPresent address: IGBMC, 1 Rue Laurent Fries, 67400 Illkirch, France Search for more papers by this author Michael G Poulos Michael G Poulos Department of Molecular Genetics and Microbiology, College of Medicine, Powell Gene Therapy Center, University of Florida, Gainesville, FL, USA Search for more papers by this author Gopal Singh Gopal Singh Department of Pathology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Maurice S Swanson Maurice S Swanson Department of Molecular Genetics and Microbiology, College of Medicine, Powell Gene Therapy Center, University of Florida, Gainesville, FL, USA Search for more papers by this author Thomas A Cooper Corresponding Author Thomas A Cooper Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA Department of Pathology, Baylor College of Medicine, Houston, TX, USA Search for more papers by this author Author Information Thai H Ho1, Nicolas Charlet-B2, Michael G Poulos3, Gopal Singh2, Maurice S Swanson3 and Thomas A Cooper 1,2 1Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA 2Department of Pathology, Baylor College of Medicine, Houston, TX, USA 3Department of Molecular Genetics and Microbiology, College of Medicine, Powell Gene Therapy Center, University of Florida, Gainesville, FL, USA *Corresponding author. Department of Pathology, Room 268B, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Tel.: +1 713 798 3141; Fax: +1 713 798 5838; E-mail: [email protected] The EMBO Journal (2004)23:3103-3112https://doi.org/10.1038/sj.emboj.7600300 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Although the muscleblind (MBNL) protein family has been implicated in myotonic dystrophy (DM), a specific function for these proteins has not been reported. A key feature of the RNA-mediated pathogenesis model for DM is the disrupted splicing of specific pre-mRNA targets. Here we demonstrate that MBNL proteins regulate alternative splicing of two pre-mRNAs that are misregulated in DM, cardiac troponin T (cTNT) and insulin receptor (IR). Alternative cTNT and IR exons are also regulated by CELF proteins, which were previously implicated in DM pathogenesis. MBNL proteins promote opposite splicing patterns for cTNT and IR alternative exons, both of which are antagonized by CELF proteins. CELF- and MBNL-binding sites are distinct and regulation by MBNL does not require the CELF-binding site. The results are consistent with a mechanism for DM pathogenesis in which expanded repeats cause a loss of MBNL and/or gain of CELF activities, leading to misregulation of alternative splicing of specific pre-mRNA targets. Introduction Alternative splicing is thought to occur in 41–60% of genes in the human genome (Black, 2003; Herbert, 2004). Pre-mRNAs can give rise to multiple protein isoforms with different functions and these variations contribute to protein diversity (Black, 2003). Alternative splicing also provides an additional regulatory mechanism by which vertebrates can control the expression of tissue-specific or developmental stage-specific protein isoforms. RNA-binding proteins that regulate alternative splicing bind to sequence-specific elements in the pre-mRNA to enhance or repress inclusion of alternative exons. Aberrant regulation of alternative splicing can cause the expression of inappropriate splicing patterns leading to human disease (Faustino and Cooper, 2003). An example of a disease that alters the function of RNA-binding proteins to cause misregulated alternative splicing is myotonic dystrophy (DM). Interestingly, the effects on splicing in DM1 are thought to be limited to specific pre-mRNA targets rather than a general disruption of splicing. DM is a multisystemic disorder caused by two different microsatellite expansions. Type I myotonic dystrophy (DM1) is caused by a CTG trinucleotide expansion in the 3′ untranslated region (UTR) of the DMPK gene on chromosome 19 (Brook et al, 1992; Fu et al, 1992; Mahadevan et al, 1992), while type 2 (DM2) is caused by a CCTG expansion in intron 1 of the ZNF9 gene on chromosome 3 (Liquori et al, 2001). Although the expansions are located on different chromosomes, there appears to be a common pathogenic mechanism involving the accumulation of transcripts into discrete nuclear RNA foci containing long tracts of CUG or CCUG repeats expressed from the expanded allele (Taneja et al, 1995; Davis et al, 1997; Liquori et al, 2001). The RNA gain-of-function hypothesis proposes that mutant DM transcripts alter the function and localization of alternative splicing regulators, which are critical for normal RNA processing. Consistent with this proposal, misregulated alternative splicing in DM1 has been demonstrated for six pre-mRNAs: cardiac troponin T (cTNT), insulin receptor (IR), muscle-specific chloride channel (ClC-1), tau, myotubularin-related protein 1 (MTMR1) and fast skeletal troponin T (TNNT3) (Philips et al, 1998; Savkur et al, 2001; Sergeant et al, 2001; Buj-Bello et al, 2002; Charlet-B et al, 2002b; Kanadia et al, 2003). In all cases, normal mRNA splice variants are produced, but the normal developmental splicing pattern is disrupted, resulting in expression of fetal protein isoforms that are inappropriate for adult tissues. The insulin resistance and myotonia observed in DM1 correlate with the disruption of splicing of two pre-mRNA targets, IR and ClC-1, respectively (Savkur et al, 2001, 2004; Mankodi et al, 2002; Charlet-B et al, 2002b). The mechanism by which expanded repeats alter the regulation of pre-mRNA alternative splicing is unclear. Two families of RNA-binding proteins have been implicated in DM1 pathogenesis: CUG-BP1 and ETR-3-like factors (CELF) and muscleblind-like (MBNL) proteins (Miller et al, 2000; Fardaei et al, 2001; Ladd et al, 2001). Six CELF (also called BRUNOL) genes have been identified in humans (Good et al, 2000; Ladd et al, 2001, 2004). All six CELF proteins have been shown to regulate pre-mRNA alternative splicing and two (CUG-BP1 and ETR-3/CUG-BP2) have been shown to have cytoplasmic RNA-associated functions (Timchenko et al, 1999; Mukhopadhyay et al, 2003). A functional link has been established between splicing regulation by CELF proteins and DM1 pathogenesis. CUG-BP1 regulates alternative splicing of at least three of the pre-mRNAs (cTNT, IR and ClC-1) that are misregulated in DM striated muscle (Philips et al, 1998; Savkur et al, 2001; Charlet-B et al, 2002b). The splicing patterns observed for all three pre-mRNAs are consistent with increased CUG-BP1 activity and an increase in CUG-BP1 steady-state levels in DM1 striated muscle (Philips et al, 1998; Savkur et al, 2001; Timchenko et al, 2001; Charlet-B et al, 2002b). Furthermore, cTNT minigenes expressed in DM1 muscle cultures or cTNT and IR pre-mRNAs coexpressed with CUG repeat RNA in normal cells reproduce the aberrant splicing patterns observed for endogenous genes in DM cells (Philips et al, 1998; Savkur et al, 2001). The trans-dominant effects of endogenous or coexpressed CUG repeat RNA on cTNT and IR splicing regulation require the intronic CUG-BP1-binding sites, indicating that binding by CUG-BP1 and/or other CELF family members to their cognate intronic regulatory elements is required for induction of aberrant splicing regulation by CUG repeat RNA (Philips et al, 1998; Savkur et al, 2001). The other family of RNA-binding proteins implicated in DM pathogenesis is MBNL, which is homologous to Drosophila mbl proteins required for photoreceptor and muscle differentiation (Begemann et al, 1997; Artero et al, 1998). Three mammalian MBNL genes have been identified: MBNL1 (formally MBNL), MBNL2 (MBLL) and MBNL3 (MBXL), located on chromosomes 3, 13 and X, respectively (Miller et al, 2000; Fardaei et al, 2002). MBNL1 was identified in HeLa cells based on its ability to bind double-stranded CUG repeats (Miller et al, 2000). All three MBNL gene products colocalize with the expanded repeat RNA foci in vivo (Miller et al, 2000; Mankodi et al, 2001; Fardaei et al, 2002). Loss of MBNL function due to sequestration on CUG repeat RNA is proposed to play a role in DM pathogenesis (Miller et al, 2000). Consistent with this proposal, Kanadia et al (2003) demonstrated that a mouse knockout (Mbnl1ΔE3/ΔE3) of specific Mbnl1-encoded isoforms reproduces the myotonia, cataracts and misregulation of splicing observed in DM1 and DM2. Although the loss of MBNL1 function reproduces the abnormal splicing pattern observed for ClC-1 and cTNT, it is not clear if the phenotype is caused by direct or indirect effects on splicing. We tested whether MBNL family members bind and regulate splicing of pre-mRNAs that are misregulated in DM1 tissues. Our results demonstrate that all three MBNL family members are novel splicing regulators that act antagonistically to CELF proteins on the three pre-mRNAs tested: human and chicken cTNT, and human IR. Similar to CELF proteins, MBNL proteins can act as activators or repressors of splicing on different pre-mRNAs. MBNL1 binds a common motif near the human and chicken cTNT alternative exons within intronic regions, which appear to be single stranded. Mutations that prevent binding of MBNL to the human cTNT pre-mRNA dramatically reduce or eliminate responsiveness to MBNL proteins in vivo. CELF and MBNL proteins bind to distinct cis-elements and minigenes containing CELF- or MBNL-binding site mutations were used to demonstrate that regulation by one family does not require responsiveness to the other. We also show that modified cTNT and IR minigenes made nonresponsive to the trans-dominant effects of CUG repeat RNA still respond to MBNL depletion, suggesting that CUG repeat RNA affects splicing by a mechanism more complex than MBNL depletion alone. Results All three MBNL proteins regulate alternative splicing To determine whether MBNL proteins can alter the splicing patterns of pre-mRNAs known to be abnormally regulated in DM1 striated muscle, GFP fusion proteins of all three MBNL proteins were transiently expressed with human and chicken cTNT minigenes in primary chicken skeletal muscle cultures. GFP–MBNL1, 2 and 3 strongly repressed inclusion of both human and chicken cTNT exon 5 in primary chicken skeletal muscle cultures, while expression of GFP to levels comparable to, or greater than, GFP–MBNL fusion proteins had no effect on splicing (Figure 1A and B). In addition, there were no differences in the splicing activity of GFP fusion proteins compared to Xpress epitope-tagged MBNL proteins (data not shown). Therefore, MBNL proteins are directly antagonistic to endogenous CELF activity, which activates cTNT exon inclusion in muscle (Charlet-B et al, 2002a). Figure 1.MBNL1, 2 and 3 regulate splicing of cTNT and IR alternative exons. Human and chicken cTNT and human IR minigenes were expressed with or without each of the three GFP–MBNL fusion proteins or with GFP alone. Duplicate transfections were used for extraction of RNA and protein. Inclusion of cTNT exon 5 or IR exon 11 was assayed by RT–PCR. Percent exon inclusion is calculated as ((mRNA+exon)/(mRNA−exon+mRNA+exon)) × 100. Results are derived from at least three independent experiments. Expression of GFP–MBNL1 (∼72 kDa), GFP–MBNL2 (∼58 kDa), GFP–MBNL3 (∼70 kDa) and EGFP (∼27 kDa) was detected by Western blot analysis using an anti-GFP monoclonal antibody. All three MBNL proteins promote exon 5 skipping of (A) chicken and (B) human cTNT exon 5 in primary skeletal muscle cultures. (C) All three MBNL proteins promote exon 11 inclusion in a human IR minigene in HEK293 cells. (D) MBNL proteins have minimal effects on splicing of exon EN in a clathrin light-chain B minigene in primary skeletal muscle cultures. Download figure Download PowerPoint Another pre-mRNA target that is misregulated in DM striated muscle is the IR (Savkur et al, 2001, 2004). To test whether the MBNL family can also regulate human IR, the three MBNL family members were coexpressed with a human IR minigene. In contrast to the inhibitory effect of MBNL on cTNT splicing, coexpression of MBNL family members with an IR minigene strongly induces exon inclusion, whereas GFP alone had no effect (Figure 1C). To determine whether the MBNL family has a general effect on alternative splicing, all three MBNL proteins were coexpressed with a clathrin light-chain minigene containing the neuron-specific exon EN. The EN alternative exon in this minigene strongly responds to overexpression of the SR family of proteins and htra2-β1, but not CELF proteins (Stamm et al, 1999; Singh et al, 2004; data not shown). Overexpression of GFP–MBNL1, 2 and 3 with the clathrin light-chain minigene had no effect on alternative splicing of exon EN (Figure 1D). MBNL expression also did not affect splicing of an artificial alternative exon flanked by splice sites from human β-globin intron 1 (data not shown). These results demonstrate that MBNL proteins do not have a general effect on alternative splicing, but rather regulate specific pre-mRNA targets. siRNA-mediated depletion of MBNL1 alters splicing of cTNT and IR To determine whether depletion of endogenous MBNL1 protein could also affect the splicing patterns of known DM pre-mRNA targets in human cells, siRNA constructs were designed to target MBNL1, but not MBNL2 and MBNL3. HeLa cells were chosen because they express MBNL1 (Miller et al, 2000) and are amenable to siRNA-mediated depletion (Elbashir et al, 2001). To confirm the specificity of the effects, two siRNA constructs were designed to target different regions of the MBNL1 mRNA. Independent transient transfection of each siRNA construct resulted in a knockdown of endogenous MBNL1 protein to less than 10–20%, based on comparisons to serial dilutions of the untransfected or mock-transfected lysates (Figure 2A; data not shown). Analysis of MBNL1 depletion by immunofluorescence demonstrated predominantly nuclear expression that was greatly reduced in the majority of cells by each siRNA construct (Figure 2B). In addition, the siRNA constructs silenced effectively the expression of GFP–MBNL1, but not GFP–MBNL2, GFP–MBNL3 or GFP from transiently transfected plasmids, and neither MBNL1 siRNA affected the levels of endogenous MBNL2 protein (data not shown). These results indicate that the siRNAs preferentially silence MBNL1. Figure 2.Endogenous MBNL1 regulates the splicing of human cTNT and IR minigenes. siRNA and minigenes were transfected into HeLa cells. (A) Western blot confirming depletion of endogenous MBNL1 by independent transfection of two different siRNA constructs using the MBNL1 monoclonal (mAb) 3A4, which recognizes two MBNL1 isoforms generated by alternative splicing (∼41 and 42 kDa). GAPDH (∼36 kDa) was used as a loading control. (B) Immunofluorescence using mAb 3A4 to confirm depletion of endogenous protein after independent transfection of each MBNL1 siRNA construct. Scale bar, 10 μm. (C) siRNA-mediated depletion of MBNL1 with two independent constructs reproduces the DM splicing patterns for cTNT and IR minigenes. RT–PCR results are from at least three transfections. GFP siRNA had no effect on splicing of any of the tested minigenes. MBNL1 siRNA had minimal effects on splicing of a rat clathrin light-chain minigene. Download figure Download PowerPoint To determine whether depletion of endogenous MBNL1 affected alternative splicing of cTNT, IR and clathrin light chain, the minigenes were transfected with each siRNA construct. Depletion of MBNL1 promoted exon inclusion in cTNT, exon skipping in IR and only minimal splicing changes in the clathrin light-chain minigene (Figure 2C). These splicing effects were not caused by general activation of the mammalian RNAi machinery because siRNA targeting GFP or luciferase and nonspecific pools of siRNA had minimal effects on splicing of the three minigenes (Figure 2C; data not shown). Furthermore, the alteration in cTNT splicing caused by MBNL1 depletion in HeLa cells can be reversed by expression of GFP–MBNL2 or GFP–MBNL3, but not GFP (data not shown), demonstrating that adding back MBNL isoforms not targeted by MBNL1 siRNA rescues the splicing effects of MBNL1 deficiency. Interestingly, siRNA-mediated depletion of MBNL1 reproduces the splicing pattern observed in DM1 for cTNT (exon inclusion) and IR (exon skipping), and is opposite to the pattern observed when MBNL1 is overexpressed. The overexpression and depletion data indicate that endogenous MBNL1 regulates the alternative splicing of cTNT and IR minigenes, and suggest that MBNL1 regulates these pre-mRNAs via specific cis-regulatory elements. The effects of MBNL on the cTNT and IR alternative exons are the opposite of the splicing patterns induced by CELF proteins, implying an antagonistic relationship between these protein families. MBNL1 directly binds to introns adjacent to the human and chicken cTNT alternative exons To determine whether the splicing effects of MBNL1 on pre-mRNA targets were direct or indirect, we performed a UV-crosslinking assay using purified recombinant GST–MBNL1 and uniformly labeled in vitro-transcribed segments from the human cTNT gene. The human cTNT minigene contains a 732 nucleotide (nt) cTNT genomic fragment that is necessary and sufficient to respond to MBNL1 overexpression and depletion (Figures 1 and 2C). To identify MBNL1-binding sites within this cTNT pre-mRNA region, uniformly 32P-labeled, in vitro-transcribed RNAs covering the entire region were used for UV-crosslinking binding assays. As shown in Figure 3A, the binding of GST–MBNL1 on human cTNT was mapped to a 41 nt region within the 3′ splice site of exon 5 (compare RNAs C, D, E and F) located between a near-consensus branch point sequence and the 3′ cleavage site of the upstream intron. Scanning mutagenesis identified two MBNL1-binding sites located 18 and 36 nt upstream from exon 5 (Figure 3A). The absence of binding to long intronic segments (RNAs F and C) and RNAs containing nucleotide substitutions (RNAs H, J and M; see below) demonstrate binding specificity. This analysis indicates that, for cTNT, the MBNL1-binding site is distinct from the CUG-BP1-binding site, which is located downstream from the alternative exon (Philips et al, 1998). Figure 3.MBNL1 binds upstream of exon 5 in human cTNT at a site distinct from the CUG-BP1-binding site. (A) Binding of recombinant GST–MBNL1 to uniformly 32P-labeled RNA was assayed by UV crosslinking. Scanning mutagenesis was performed by replacing 6 nt blocks with AUAAUA and identified two binding sites 18 and 36 nt upstream of the alternative exon. The MBNL1-binding sites (M) and the CUG-BP1-binding site (C) are located on opposite sides of exon 5. (+) and (−) indicate binding; (•) indicates a putative branch point adenosine. (B) Four nucleotide substitutions significantly reduce binding of recombinant MBNL1 detected by UV crosslinking. Competition of GST–MBNL1 binding to 32P-labeled RNA G by the indicated picomoles of nonlabeled RNAs G or M shown in A). (C, D) MBNL1-binding site mutations reduce responsiveness to MBNL1, MBNL2 and MBNL3 coexpression but not CUG-BP1 in COSM6 cells. Human cTNT minigenes containing the natural sequence (C) or the four nucleotide substitutions (mutation M in A) in the MBNL1-binding site (D) were coexpressed with GFP or the indicated GFP fusion proteins. Exon inclusion was assayed by RT–PCR. Download figure Download PowerPoint Nucleotide substitutions that disrupt both MBNL1-binding sites were introduced into the human cTNT minigene to test whether MBNL1 binding was required to affect responsiveness to MBNL1 expression in vivo. As the MBNL-binding site is located within the 3′ splice site of intron 4, only four nucleotide substitutions were introduced to reduce the effects of MBNL-binding site mutations on basal splicing efficiency (RNA M, Figure 3A). These substitutions prevented binding of recombinant MBNL1 to an RNA that is otherwise identical to RNA G containing the wild-type sequence (Figure 3B). In addition, nonlabeled RNA M was much less efficient than RNA G in competing binding of MBNL1 to labeled RNA G (Figure 3B). When introduced into the human cTNT minigene, the MBNL1-binding site mutation significantly reduced (MBNL1 and MBNL3) or eliminated (MBNL2) responsiveness to MBNL proteins (Figure 3C and D), demonstrating that loss of MBNL1 binding in vitro directly correlates with decreased responsiveness to MBNL1 in vivo. In contrast, the MBNL1-binding site mutations had little effect on responsiveness to CUG-BP1 (Figures 3C and D). GFP alone had minimal effects on splicing. We conclude that MBNL proteins regulate splicing by binding to the human cTNT pre-mRNA and that regulation by CUG-BP1 does not require the MBNL1-binding site. UV-crosslinking analysis was performed to identify MBNL1-binding site(s) associated with the chicken cTNT alternative exon 5. The genomic segment of chicken cTNT that responds to MBNL expression contains 99 and 142 nt of upstream and downstream introns flanking the alternative exon, respectively. Within the intronic segments are four muscle-specific splicing enhancers (MSEs, Figure 4A) previously shown to be required for enhanced exon inclusion in embryonic striated muscle (Ryan and Cooper, 1996; Cooper, 1998) and required for regulation by all the six CELF family members (Ladd et al, 2001, 2004). RNAs containing MSEs 1–4 or individual MSEs were transcribed in vitro as uniformly 32P-labeled RNAs and used for UV crosslinking. GST–MBNL1 bound strongly to MSE4 and slightly to MSE1 (Figure 4A). In competition studies, nonlabeled MSE1 RNA poorly competed in the binding of GST–MBNL1 to RNA containing MSE1–4, while MSE4 effectively competed in binding (Figure 4B), consistent with the UV-crosslinking results. The absence of competition by MSE2 or MSE3 demonstrates the sequence specificity of MBNL1 binding (Figure 4B). To define the MBNL1-binding site(s) within MSE4, scanning mutagenesis was performed. Two regions required for MBNL1 binding were identified at 94 and 120 nt downstream from the exon (Figure 4C). Alignment of the four MBNL1-binding sites in chicken and human cTNT revealed a common motif of YGCU(U/G)Y (Figure 4D). Figure 4.MBNL1 binds to cis-elements in chicken cTNT intron 5 required for muscle-specific splicing. (A) The chicken cTNT MSE1–4 RNA contains an alternative exon flanked by four MSEs. GST–MBNL1 bound weakly to MSE1 and strongly to MSE4 in UV-crosslinking assays. (B) Competition of GST–MBNL1 binding to labeled chicken cTNT MSE1–4 RNA by nonlabeled MSE RNAs. Picomoles of competitor RNA are indicated. (C) Scanning mutagenesis identified two MBNL1-binding sites within MSE4. (D) Alignment of the four MBNL1-binding motifs in human and chicken cTNT reveals a common motif. Download figure Download PowerPoint CELF protein cis-regulatory elements in cTNT and IR are not required for regulation by MBNL1 The CUG-BP1-binding site located downstream from exon 5 in the human cTNT minigene is required for regulation by all six CELF proteins (Philips et al, 1998; T Ho, unpublished data), and is distinct from the MBNL-binding site mapped in Figure 3. The results shown above demonstrate that CUG-BP1 regulates minigenes in which MBNL1-binding site mutations have greatly reduced or eliminated MBNL responsiveness (Figure 3D). To determine whether MBNL1 can regulate minigenes lacking the CUG-BP1-binding site, GFP–MBNL1 or MBNL1 siRNA was cotransfected with a human cTNT minigene containing mutated CUG-BP1-binding sites. The overexpression and depletion results demonstrate that cTNT minigenes containing the mutant and wild-type CUG-BP1-binding sites are equally responsive to MBNL1 (Figure 5A and B). GFP–MBNL2 and 3 also showed similar regulation of wild-type and mutant human cTNT minigenes (data not shown). Figure 5.Regulation of human cTNT by MBNL1 is independent of CELF regulation. The (A) wild-type cTNT minigene or a (B) mutant cTNT minigene with point mutations that prevent CUG-BP1 binding and regulation were cotransfected with the indicated siRNA constructs, a plasmid expressing a DMPK minigene with 960 CUG repeats (Philips et al, 1998) or a GFP–MBNL1 expression plasmid in HeLa cells. Download figure Download PowerPoint Similarly, for the IR minigene, regulation by CUG-BP1 requires a CUG-BP1-binding site in a 110 nt region located upstream of IR exon 11 (Savkur et al, 2001). A mutant IR minigene lacking the CUG-BP1-binding site was coexpressed with GFP–MBNL1, 2 and 3 in HEK293 cells (Figure 6A) or MBNL1 siRNA constructs in HeLa cells (Figure 6B) to determine whether regulation by MBNL proteins requires the CUG-BP1-binding site. The mutant IR minigenes displayed regulation by MBNL proteins, which was comparable to the wild-type IR minigenes (compare Figures 6A and 1C and 6B and 2C). We conclude that regulation of human cTNT and IR by MBNL proteins does not require the CUG-BP1-binding site. Figure 6.Deletion of the human IR CUG-BP1-binding site does not affect regulation by MBNL1. (A) All the three MBNL proteins promote exon 11 inclusion of a mutant human IR minigene lacking the CUG-BP1-binding site in HEK293 cells. (B) RNAi depletion of MBNL1 in HeLa cells using the indicated siRNA constructs promotes exon 11 skipping in a human IR minigene lacking the CUG-BP1-binding site. Download figure Download PowerPoint Mutant cTNT and IR minigenes lacking the CUG-BP1-binding site respond as strongly as nonmutated minigenes to MBNL1 depletion by RNAi (Figures 5B and 6B). However, neither of these minigenes respond to the trans-dominant effects of coexpressed CUG repeat RNA as do the nonmutated minigenes (Philips et al, 1998; Savkur et al, 2001; 960CTG, Figure 5). The RNAi results demonstrate that the mutated cTNT and IR minigenes are ‘competent’ to respond to MBNL1 depletion and yet they do not respond to coexpression of CUG repeat RNA. Therefore, while our results demonstrate that MBNL proteins are alternative splicing regulators of cTNT and IR alternative exons, this result suggests that MBNL depletion by CUG repeat RNA is not sufficient to account for the trans-dominant effect of CUG repeat RNA on splicing (see Discussion). Discussion The MBNL family of proteins regulate alternative splicing In this paper, we show that MBNL proteins function as potent target-specific regulators of pre-mRNA alternative splicing. This finding has particular relevance to the molecular mechanism of DM pathogenesis in which loss of MBNL function due to sequestration on expanded CUG repeat RNA has been proposed to play a major role (Miller et al, 2000; Kanadia et al, 2003). We employed overexpression and siRNA-mediated depletion to demonstrate that all three MBNL genes encode factors that regulate splicing of the three pre-mRNAs that were tested, human cTNT, chicken cTNT and human IR. MBNL1 binds to the introns flanking exon 5 in both chicken and human cTNT pre-mRNAs, and point mutations that eliminate binding in vitro also eliminated or decreased responsiveness of the human cTNT minigene to MBNL1, MBNL2 and MBNL3 coexpression. These results demonstrate that regulation by MBNL protein is mediated via binding the pre-mRNA, and suggest that all three MBNL proteins regulate human cTNT splicing by binding to the same site. Proteins from all three MBNL genes contain two pairs of Cys3His zinc-finger-related motifs with identical spacing between cysteine and histidine residues in fingers 1 and 3 (CX7CX6CX3H) and fingers 2 and 4 (CX7CX4CX3H) (Miller et al, 2000; Fardaei et al, 2002; Squillace et al, 2002). The Cys3His-type zinc-finger is an evolutionarily conserved motif found in a number of proteins that perform diverse RNA-processing functions, and mutation of this motif results in a loss of RNA binding and disrupts protein function (Bai and Tolias, 1996, 1998; Lai et al, 1999; Stoecklin et al, 2002). MBNL1 was described originally as a double-stranded RNA-binding protein and has been shown to bind expanded CUG repeats that form an extended hairpin in vitro (Michalows" @default.
• W2092831145 created "2016-06-24" @default.
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• W2092831145 date "2004-07-15" @default.
• W2092831145 modified "2023-10-15" @default.
• W2092831145 title "Muscleblind proteins regulate alternative splicing" @default.
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