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• W1990507930 abstract "Muscleblind-like-1 (MBNL1) is a splicing regulatory factor controlling the fetal-to-adult alternative splicing transitions during vertebrate muscle development. Its capture by nuclear CUG expansions is one major cause for type 1 myotonic dystrophy (DM1). Alternative splicing produces MBNL1 isoforms that differ by the presence or absence of the exonic regions 3, 5, and 7. To understand better their respective roles and the consequences of the deregulation of their expression in DM1, here we studied the respective roles of MBNL1 alternative and constitutive exons. By combining genetics, molecular and cellular approaches, we found that (i) the exon 5 and 6 regions are both needed to control the nuclear localization of MBNL1; (ii) the exon 3 region strongly enhances the affinity of MBNL1 for its pre-mRNA target sites; (iii) the exon 3 and 6 regions are both required for the splicing regulatory activity, and this function is not enhanced by an exclusive nuclear localization of MBNL1; and finally (iv) the exon 7 region enhances MBNL1-MBNL1 dimerization properties. Consequently, the abnormally high inclusion of the exon 5 and 7 regions in DM1 is expected to enhance the potential of MBNL1 of being sequestered with nuclear CUG expansions, which provides new insight into DM1 pathophysiology. Muscleblind-like-1 (MBNL1) is a splicing regulatory factor controlling the fetal-to-adult alternative splicing transitions during vertebrate muscle development. Its capture by nuclear CUG expansions is one major cause for type 1 myotonic dystrophy (DM1). Alternative splicing produces MBNL1 isoforms that differ by the presence or absence of the exonic regions 3, 5, and 7. To understand better their respective roles and the consequences of the deregulation of their expression in DM1, here we studied the respective roles of MBNL1 alternative and constitutive exons. By combining genetics, molecular and cellular approaches, we found that (i) the exon 5 and 6 regions are both needed to control the nuclear localization of MBNL1; (ii) the exon 3 region strongly enhances the affinity of MBNL1 for its pre-mRNA target sites; (iii) the exon 3 and 6 regions are both required for the splicing regulatory activity, and this function is not enhanced by an exclusive nuclear localization of MBNL1; and finally (iv) the exon 7 region enhances MBNL1-MBNL1 dimerization properties. Consequently, the abnormally high inclusion of the exon 5 and 7 regions in DM1 is expected to enhance the potential of MBNL1 of being sequestered with nuclear CUG expansions, which provides new insight into DM1 pathophysiology. IntroductionSplicing of pre-mRNA is a key post-transcriptional step in eukaryotic gene expression. A vast majority of vertebrate pre-mRNAs is alternatively spliced, allowing the production of several protein isoforms from transcripts of a given gene (1Black D.L. Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1945) Google Scholar). The regulation of alternative splicing plays a major role in cell differentiation and in development and depends on the expression and activity of numerous splicing regulatory factors that are expressed differentially during development, according to the type of tissue. Defects in these alternative-splicing processes can contribute to pathogenesis, as demonstrated for a growing number of diseases, including neuromuscular diseases such as myotonic dystrophy type 1 (DM1) 9The abbreviations used are: DM1, myotonic dystrophy type 1; 3AT, 3-amino-1,2,4-triazole; BisTris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; hcTNT, human cardiac troponin T; IR, insulin receptor; MBNL1, Muscleblind-like 1. (2Ranum L.P. Cooper T.A. Annu. Rev. Neurosci. 2006; 29: 259-277Crossref PubMed Scopus (374) Google Scholar, 3Lee J.E. Cooper T.A. Biochem. Soc. Trans. 2009; 37: 1281-1286Crossref PubMed Scopus (211) Google Scholar).DM1 is an autosomal disorder caused by an unstable CTG repeat expansion in the 3′-untranslated region (UTR) of the DMPK gene (4Brook J.D. McCurrach M.E. Harley H.G. Buckler A.J. Church D. Aburatani H. Hunter K. Stanton V.P. Thirion J.P. Hudson T. et al.Cell. 1992; 69: 385Abstract Full Text PDF PubMed Scopus (10) Google Scholar, 5Fu Y.H. Pizzuti A. Fenwick Jr., R.G. King J. Rajnarayan S. Dunne P.W. Dubel J. Nasser G.A. Ashizawa T. de Jong P. et al.Science. 1992; 255: 1256-1258Crossref PubMed Scopus (1262) Google Scholar, 6Mahadevan M. Tsilfidis C. Sabourin L. Shutler G. Amemiya C. Jansen G. Neville C. Narang M. Barceló J. O'Hoy K. Science. 1992; 255: 1253-1255Crossref PubMed Scopus (1408) Google Scholar). One of the main etiological hypotheses of DM1 is based on a toxic RNA gain of function, leading to the dysregulation of alternative splicing. Mutant transcripts bearing long-CUG repeats acquire unusual A-form double-stranded RNA structures (7Mooers B.H. Logue J.S. Berglund J.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 16626-16631Crossref PubMed Scopus (129) Google Scholar), accumulate in the nucleus, and lead to small ribonucleoprotein inclusions, named foci (8Fardaei M. Larkin K. Brook J.D. Hamshere M.G. Nucleic Acids Res. 2001; 29: 2766-2771Crossref PubMed Scopus (177) Google Scholar) that sequester RNA-binding proteins such as Muscleblind-like 1 (MBNL1). The alternative splicing of several MBNL1 targets is thus abnormally modified in DM1 patients and in a mouse model in which MBNL1 expression is inactivated (9Lin X. Miller J.W. Mankodi A. Kanadia R.N. Yuan Y. Moxley R.T. Swanson M.S. Thornton C.A. Hum. Mol. Genet. 2006; 15: 2087-2097Crossref PubMed Scopus (382) Google Scholar, 10Kanadia R.N. Johnstone K.A. Mankodi A. Lungu C. Thornton C.A. Esson D. Timmers A.M. Hauswirth W.W. Swanson M.S. Science. 2003; 302: 1978-1980Crossref PubMed Scopus (581) Google Scholar, 11Du H. Cline M.S. Osborne R.J. Tuttle D.L. Clark T.A. Donohue J.P. Hall M.P. Shiue L. Swanson M.S. Thornton C.A. Ares Jr., M. Nat. Struct. Mol. Biol. 2010; 17: 187-193Crossref PubMed Scopus (246) Google Scholar). In consequence, a loss-of-function mechanism has been proposed to contribute to DM1 pathogenesis, which is further supported by an in vivo model showing that MBNL1 inactivation leads to many of the symptoms and molecular defects observed in DM1 (10Kanadia R.N. Johnstone K.A. Mankodi A. Lungu C. Thornton C.A. Esson D. Timmers A.M. Hauswirth W.W. Swanson M.S. Science. 2003; 302: 1978-1980Crossref PubMed Scopus (581) Google Scholar, 11Du H. Cline M.S. Osborne R.J. Tuttle D.L. Clark T.A. Donohue J.P. Hall M.P. Shiue L. Swanson M.S. Thornton C.A. Ares Jr., M. Nat. Struct. Mol. Biol. 2010; 17: 187-193Crossref PubMed Scopus (246) Google Scholar).MBNL1 is the best example of splicing regulatory factors known to be involved in development and splicing deregulation in disease. Originally identified in Drosophila melanogaster, MBNL1 is described as being essential for the terminal differentiation of photoreceptors and muscles (12Begemann G. Paricio N. Artero R. Kiss I. Pérez-Alonso M. Mlodzik M. Development. 1997; 124: 4321-4331Crossref PubMed Google Scholar, 13Artero R. Prokop A. Paricio N. Begemann G. Pueyo I. Mlodzik M. Perez-Alonso M. Baylies M.K. Dev. Biol. 1998; 195: 131-143Crossref PubMed Scopus (127) Google Scholar). In humans and in mice, the splicing factor MBNL1 has been shown to participate in the postnatal remodeling of skeletal muscle and of the developing heart by controlling the developmentally regulated switch of a key set of pre-mRNAs (9Lin X. Miller J.W. Mankodi A. Kanadia R.N. Yuan Y. Moxley R.T. Swanson M.S. Thornton C.A. Hum. Mol. Genet. 2006; 15: 2087-2097Crossref PubMed Scopus (382) Google Scholar, 14Kalsotra A. Xiao X. Ward A.J. Castle J.C. Johnson J.M. Burge C.B. Cooper T.A. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 20333-20338Crossref PubMed Scopus (356) Google Scholar, 15Terenzi F. Ladd A.N. RNA Biol. 2010; 7: 43-55Crossref PubMed Scopus (43) Google Scholar). MBNL1 binds to pre-mRNAs containing YGCY sequence elements (16Ho T.H. Charlet B.N. Poulos M.G. Singh G. Swanson M.S. Cooper T.A. EMBO J. 2004; 23: 3103-3112Crossref PubMed Scopus (367) Google Scholar, 17Goers E.S. Purcell J. Voelker R.B. Gates D.P. Berglund J.A. Nucleic Acids Res. 2010; 38: 2467-2484Crossref PubMed Scopus (93) Google Scholar) and promotes either the inclusion or the exclusion of alternative exons depending on the 5′ or 3′ localization of cis-regulatory elements (17Goers E.S. Purcell J. Voelker R.B. Gates D.P. Berglund J.A. Nucleic Acids Res. 2010; 38: 2467-2484Crossref PubMed Scopus (93) Google Scholar). For instance, the inclusion of exon 5 in human cardiac troponin T (hcTNT/TNNT2) mRNA is inhibited by MBNL1, whereas the inclusion of exon 11 in insulin receptor (IR/INSR) mRNA is promoted by MBNL1 (16Ho T.H. Charlet B.N. Poulos M.G. Singh G. Swanson M.S. Cooper T.A. EMBO J. 2004; 23: 3103-3112Crossref PubMed Scopus (367) Google Scholar). MBNL1 binds to the polypyrimidine tract of hcTNT intron 4 and competes with the splicing factor U2AF for its binding site, explaining the MBNL1 inhibition of exon 5 inclusion (18Warf M.B. Diegel J.V. von Hippel P.H. Berglund J.A. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9203-9208Crossref PubMed Scopus (99) Google Scholar). Conversely, by binding to elements located 3′ to the alternative exon 11 in IR pre-mRNA (IR/INSR), MBNL1 enhances the inclusion of this exon (17Goers E.S. Purcell J. Voelker R.B. Gates D.P. Berglund J.A. Nucleic Acids Res. 2010; 38: 2467-2484Crossref PubMed Scopus (93) Google Scholar, 19Warf M.B. Berglund J.A. RNA. 2007; 13: 2238-2251Crossref PubMed Scopus (133) Google Scholar, 20Sen S. Talukdar I. Liu Y. Tam J. Reddy S. Webster N.J. J. Biol. Chem. 2010; 285: 25426-25437Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).The MBNL1 gene encompasses 12 exons and the coding sequence is distributed over 10 exons numbered 1–10 (Fig. 1A) (21Fardaei M. Rogers M.T. Thorpe H.M. Larkin K. Hamshere M.G. Harper P.S. Brook J.D. Hum. Mol. Genet. 2002; 11: 805-814Crossref PubMed Scopus (351) Google Scholar, 22Kino Y. Mori D. Oma Y. Takeshita Y. Sasagawa N. Ishiura S. Hum. Mol. Genet. 2004; 13: 495-507Crossref PubMed Scopus (142) Google Scholar, 23Pascual M. Vicente M. Monferrer L. Artero R. Differentiation. 2006; 74: 65-80Crossref PubMed Scopus (175) Google Scholar), some of which (exons 3, 5, 7, and 9) are alternatively spliced. Thus, >10 isoforms of MBNL1 have been reported (21Fardaei M. Rogers M.T. Thorpe H.M. Larkin K. Hamshere M.G. Harper P.S. Brook J.D. Hum. Mol. Genet. 2002; 11: 805-814Crossref PubMed Scopus (351) Google Scholar). The expression of these isoforms is developmentally regulated and altered in DM1. These observations suggest a modulation of MBNL1 protein function through alternative splicing. Indeed, the cassette exons 5 and 7 are included mainly in fetal brain and muscle rather than in adult tissues and are preferentially included in DM1 patients compared with controls (9Lin X. Miller J.W. Mankodi A. Kanadia R.N. Yuan Y. Moxley R.T. Swanson M.S. Thornton C.A. Hum. Mol. Genet. 2006; 15: 2087-2097Crossref PubMed Scopus (382) Google Scholar, 24Dhaenens C.M. Schraen-Maschke S. Tran H. Vingtdeux V. Ghanem D. Leroy O. Delplanque J. Vanbrussel E. Delacourte A. Vermersch P. Maurage C.A. Gruffat H. Sergeant A. Mahadevan M.S. Ishiura S. Buée L. Cooper T.A. Caillet-Boudin M.L. Charlet-Berguerand N. Sablonnière B. Sergeant N. Exp. Neurol. 2008; 210: 467-478Crossref PubMed Scopus (43) Google Scholar). Exons 1, 2, and 4 are always included and encode the four CCCH zinc finger domains involved in RNA binding (25Teplova M. Patel D.J. Nat. Struct. Mol. Biol. 2008; 15: 1343-1351Crossref PubMed Scopus (123) Google Scholar). Exon 3 is often included and may act as a linker joining the second and third zinc finger domains (22Kino Y. Mori D. Oma Y. Takeshita Y. Sasagawa N. Ishiura S. Hum. Mol. Genet. 2004; 13: 495-507Crossref PubMed Scopus (142) Google Scholar, 25Teplova M. Patel D.J. Nat. Struct. Mol. Biol. 2008; 15: 1343-1351Crossref PubMed Scopus (123) Google Scholar). Although the segment encoded by exon 3 is often included in MBNL1 protein, MBNL1 isoforms without this linker have already been found to have a lower affinity for CUG expansions compared with isoforms that include the exon 3-encoded sequence (22Kino Y. Mori D. Oma Y. Takeshita Y. Sasagawa N. Ishiura S. Hum. Mol. Genet. 2004; 13: 495-507Crossref PubMed Scopus (142) Google Scholar). The presence or absence of the sequence encoded by exon 5 has been suggested to modulate the nuclear localization of MBNL1 (9Lin X. Miller J.W. Mankodi A. Kanadia R.N. Yuan Y. Moxley R.T. Swanson M.S. Thornton C.A. Hum. Mol. Genet. 2006; 15: 2087-2097Crossref PubMed Scopus (382) Google Scholar, 15Terenzi F. Ladd A.N. RNA Biol. 2010; 7: 43-55Crossref PubMed Scopus (43) Google Scholar, 24Dhaenens C.M. Schraen-Maschke S. Tran H. Vingtdeux V. Ghanem D. Leroy O. Delplanque J. Vanbrussel E. Delacourte A. Vermersch P. Maurage C.A. Gruffat H. Sergeant A. Mahadevan M.S. Ishiura S. Buée L. Cooper T.A. Caillet-Boudin M.L. Charlet-Berguerand N. Sablonnière B. Sergeant N. Exp. Neurol. 2008; 210: 467-478Crossref PubMed Scopus (43) Google Scholar). However, the possible involvement of the bordering amino acid sequences encoded by constitutive exons such as exon 6 in the cellular localization of MBNL1 has not been investigated yet, and up to now, no functional role has been attributed to the highly conserved amino acid sequence encoded by exon 7 cassette, which is also mainly included in fetal brain and muscles and in MBNL1 protein from DM1 patients. It is therefore important to determine the relationship, if any, between the exon composition and function of MBNL1 isoforms.In the present study, in an effort to define precisely the respective roles of the amino acid sequences encoded by the alternative cassette exons 3, 5, and 7 and constitutive exon 6 of human MBNL1 protein isoforms, we applied a large variety of in vitro and in cellulo approaches. This allowed us to determine the cellular localization, splicing activity, and RNA binding properties of the eight main normal MBNL1 isoforms as well as of a large number of MBNL1 variant proteins. Experiments were performed using both HeLa cells and human myoblasts that did or did not express CUG expansions. Our results provide a better definition of (i) the respective roles of the amino acid sequences encoded by exons 3 and 5 in the RNA binding property, nuclear localization and splicing regulatory property of MBNL1; (ii) the involvement of the exon 6-encoded sequence in MBNL1 nuclear retention and in cellulo splicing regulatory activity; (iii) the identification of a possible role of the exon 7-encoded sequence in MBNL1 self-dimerization.DISCUSSIONAlternative splicing produces several MBNL1 isoforms, differing mainly in the inclusion or exclusion of the in-frame sequences encoded by exons 3, 5, and 7. The exon 3 region has already been proposed to modulate RNA binding affinity, and here we provide new information as to how the presence or absence of the exon 3 region modifies the binding of MBNL1 to its target sites in pre-mRNA and to CUG expansions. We show for the first time the crucial role of this region in the splicing regulatory property of MBNL1. The exon 5 region has previously been proposed to play a critical role in the nuclear retention of the MBNL1 protein. Here, we show that this nuclear retention also depends on amino acid sequences encoded by exon 6 and that the exon 6 region is required for splicing regulation. Finally, we have identified the role of the alternative exon 7 region in MBNL1 dimerization and showed also that exon 8 is alternative and not constitutive. We will now discuss the implications of our findings for the normal and pathological activities of the MBNL1 splicing factor.Nuclear Localization of MBNL1 and Splicing ActivityMBNL1 isoforms displayed different nucleocytoplasmic localization patterns in HeLa cells and in human myoblasts. Isoforms lacking the exon 5 region were distributed in both the nucleus and the cytoplasm whereas the subcellular localization of those including the exon 5 region was restricted to the nucleus. Cassette exon 5 therefore probably encodes a region involved in the nuclear localization of MBNL1. This corroborates previously published data (9Lin X. Miller J.W. Mankodi A. Kanadia R.N. Yuan Y. Moxley R.T. Swanson M.S. Thornton C.A. Hum. Mol. Genet. 2006; 15: 2087-2097Crossref PubMed Scopus (382) Google Scholar, 15Terenzi F. Ladd A.N. RNA Biol. 2010; 7: 43-55Crossref PubMed Scopus (43) Google Scholar, 24Dhaenens C.M. Schraen-Maschke S. Tran H. Vingtdeux V. Ghanem D. Leroy O. Delplanque J. Vanbrussel E. Delacourte A. Vermersch P. Maurage C.A. Gruffat H. Sergeant A. Mahadevan M.S. Ishiura S. Buée L. Cooper T.A. Caillet-Boudin M.L. Charlet-Berguerand N. Sablonnière B. Sergeant N. Exp. Neurol. 2008; 210: 467-478Crossref PubMed Scopus (43) Google Scholar) and extends them to human myoblasts.Because all MBNL1 isoforms were located in the nucleus, with or without the inclusion of the alternative exon 5 region, we can rule out the idea that this region is solely driving the nuclear localization of MBNL1. This observation was further confirmed using truncated proteins with or without the exon 5 region and lacking the C-terminal region. Indeed, diffuse staining for the truncated proteins MBNL1ΔCT and MBNL1ΔCT5 was present in both the nucleus and the cytoplasm. The apparent molecular size of these truncated proteins fused to the GFP tag was around 60 kDa, excluding passive diffusion through the nuclear pore complex. Thus, the RNA binding domain composed of four CCCH zinc finger motifs was sufficient to target MBNL1 to the nucleus. Furthermore, this result also suggests that the MBNL1 exon 5 region co-operates with another region to maintain MBNL1 in the nucleus. In line with our hypothesis, we observed that both exons 5 and 6 were required to induce a nuclear localization of MBNL1 exclusively, as seen in Fig. 2C. Interestingly, a new type of highly conserved nuclear localization signal motif (the KRAEK motif) has recently been identified in D. melanogaster Muscleblind-like C and E isoforms (33Fernandez-Costa J.M. Artero R. Mol. Cells. 2010; 30: 65-70Crossref PubMed Scopus (15) Google Scholar). This motif has been shown to be sufficient to target Muscleblind-like to the nucleus of D. melanogaster Schneider 2 cells. Although this motif is conserved in the exon 6 region of human MBNL1, but not in exon 5, our data strongly suggest that additional regions might be required for MBNL1 nuclear retention. Thus, the nuclear retention of MBNL1, according to our results, necessitates a bipartite signal with one motif in the exon 5 and the second in the exon 6, but separately, these motifs are nonfunctional. We suggest that the MBNL1 exon 6 region contains a nuclear localization signal that is either functional in the presence of the exon 5 region or is regulated by an adjacent upstream sequence. Importantly, we cannot exclude the possibility that post-translational modification around the exon 5 sequence also regulates the subcellular localization of MBNL1.Because splicing occurs in the nucleus, we asked whether there was a relationship between strict nuclear localization and the splicing activity of MBNL1. Surprisingly, with a similar level of expression, we observed no difference in the splicing activity of the different MBNL1 isoforms on hcTNT exon 5 or IR exon 11 splicing, as assayed using minigenes. This result raises the question of the interest of a restrictive nuclear localization. One possibility is that nuclear retention is a cellular mechanism that impedes MBNL1 cytoplasmic activity while maintaining its splicing activity when located in the nucleus. Few studies have reported the potential cytoplasmic functions of MBNL1; judging by its similarity to MBNL2, another member of the MBNL protein family, MBNL1 could be involved in mRNA transport and/or stability (34Adereth Y. Dammai V. Kose N. Li R. Hsu T. Nat. Cell Biol. 2005; 7: 1240-1247Crossref PubMed Scopus (105) Google Scholar). In line with this observation, some recent studies have demonstrated a global change in gene expression in MBNL1 knock-out mice (11Du H. Cline M.S. Osborne R.J. Tuttle D.L. Clark T.A. Donohue J.P. Hall M.P. Shiue L. Swanson M.S. Thornton C.A. Ares Jr., M. Nat. Struct. Mol. Biol. 2010; 17: 187-193Crossref PubMed Scopus (246) Google Scholar, 35Osborne R.J. Lin X. Welle S. Sobczak K. O'Rourke J.R. Swanson M.S. Thornton C.A. Hum. Mol. Genet. 2009; 18: 1471-1481Crossref PubMed Scopus (136) Google Scholar). Stability and translatability may also be regulated by MBNL1 under conditions of stress, in which MBNL1 co-localizes with stress granules (36Onishi H. Kino Y. Morita T. Futai E. Sasagawa N. Ishiura S. J. Neurosci. Res. 2008; 86: 1994-2002Crossref PubMed Scopus (59) Google Scholar). In this latter study, the authors used the isoform MBNL140, which displayed both nuclear and cytoplasmic localization at base line. However, this property cannot be extrapolated to other isoforms of MBNL1, especially to MBNL1 isoforms having the exon 5 and 6 sequences. Additional work is needed to determine whether MBNL1 isoforms located only in the nucleus also co-localize with stress granules under conditions of stress. Accordingly, the function of MBNL1 isoforms lacking exon 3 also remains to be established.MBNL1 Splicing Activity Depends on Both Exon 3- and Exon 6-Encoded RegionsMBNL1 splicing factor can enhance or repress the inclusion of alternative exons (16Ho T.H. Charlet B.N. Poulos M.G. Singh G. Swanson M.S. Cooper T.A. EMBO J. 2004; 23: 3103-3112Crossref PubMed Scopus (367) Google Scholar). Based on our data, when an isoform is active in splicing, it can have a silencing or an enhancing activity on splicing, depending on the targeted splicing site. Our results exclude the possibility that some isoforms may be dedicated to splicing inhibition, whereas others would have splicing activation properties. As suggested previously, MBNL1 splicing activity might rather depend on the localization of its RNA-binding elements (17Goers E.S. Purcell J. Voelker R.B. Gates D.P. Berglund J.A. Nucleic Acids Res. 2010; 38: 2467-2484Crossref PubMed Scopus (93) Google Scholar). Indeed, the MBNL1 exon 3 region is located between the zinc finger 2 and zinc finger 3 motifs and acts as a linker between the zinc finger 1/2 and zinc finger 3/4 domains that structure the MBNL1 RNA binding domain (22Kino Y. Mori D. Oma Y. Takeshita Y. Sasagawa N. Ishiura S. Hum. Mol. Genet. 2004; 13: 495-507Crossref PubMed Scopus (142) Google Scholar, 25Teplova M. Patel D.J. Nat. Struct. Mol. Biol. 2008; 15: 1343-1351Crossref PubMed Scopus (123) Google Scholar) and probably modulate its RNA binding affinity. Triple-hybrid assays and gel shift experiments confirmed that the presence of the exon 3 region was essential for MBNL1 efficient binding to its pre-mRNA target. Moreover, we observed that all MBNL1 isoforms that included the exon 3 region (MBNL140, MBNL141, MBNL142, and MBNL143) strongly modified MBNL1 splicing activity with respect to hcTNT exon 5 and IR exon 11, whereas those that lacked exon 3 (MBNL135, MBNL136, MBNL137, and MBNL138) barely did. Also in accordance with the need for an RNA binding domain for MBNL1 activity, experiments performed with a truncated protein revealed that the splicing activity of the exon 3 region was weak but accurate (Fig. 4, MBNL1ΔCT3). The recovery of full splicing activity was observed with proteins that expressed both the exon 3 and exon 6 regions (Fig. 4, MBNL1ΔCT3). This result suggests that MBNL1 splicing activity mainly resides in the exon 6 region. Furthermore, it also suggests that MBNL1 splicing activity likely depends on its affinity for its RNA targets and the presence of both exon 3 and 6 regions.Relevance to DM1 PathologyMyotonic dystrophy type 1 belongs to the triplet repeat disorder family. The unstable CTG expansions are transcribed into RNA and retained in the nucleus. These toxic RNAs have been shown to sequester MBNL1 proteins (8Fardaei M. Larkin K. Brook J.D. Hamshere M.G. Nucleic Acids Res. 2001; 29: 2766-2771Crossref PubMed Scopus (177) Google Scholar, 37Miller J.W. Urbinati C.R. Teng-Umnuay P. Stenberg M.G. Byrne B.J. Thornton C.A. Swanson M.S. EMBO J. 2000; 19: 4439-4448Crossref PubMed Scopus (696) Google Scholar). There are various MBNL1 protein isoforms, which in our study differed in the presence or absence of the exon 3, 5, and 7 regions. All of them co-localized with the CUG repeats expressed in DM1 myoblasts. The MBNL1 RNA binding domain alone (MBNL1ΔCT, MBNL1ΔCT3) was sufficient to mediate MBNL1 binding to the CUG repeats, as previously suggested (19Warf M.B. Berglund J.A. RNA. 2007; 13: 2238-2251Crossref PubMed Scopus (133) Google Scholar), with or without the exon 3 region. Furthermore, using the yeast triple-hybrid system, we observed that the presence of the exon 3 region strongly enhanced the binding affinity of MBNL1 to the CUG repeats. Therefore, MBNL1 isoforms that lack the exon 3 region likely interact with CUG repeats very weakly. We thus suggest that their sequestration is not direct but mediated by the dimerization of MBNL1 with itself. The self-dimerization of MBNL1 has been previously reported and shown to occur via the C-terminal tail of MBNL1 (32Yuan Y. Compton S.A. Sobczak K. Stenberg M.G. Thornton C.A. Griffith J.D. Swanson M.S. Nucleic Acids Res. 2007; 35: 5474-5486Crossref PubMed Scopus (165) Google Scholar). In our study, double-hybrid assays performed with the MBNL1 C-terminal tail revealed that these C-terminal fragments only dimerized with the full-length MBNL1. The presence of exon 7 also improved MBNL1 binding on both CUG repeats and hcTNT mRNA, most probably by promoting MBNL1 dimerization. Interestingly, the expression of MBNL1 protein isoforms, including the exon 7, region is greatly increased with the development of DM1 (9Lin X. Miller J.W. Mankodi A. Kanadia R.N. Yuan Y. Moxley R.T. Swanson M.S. Thornton C.A. Hum. Mol. Genet. 2006; 15: 2087-2097Crossref PubMed Scopus (382) Google Scholar, 24Dhaenens C.M. Schraen-Maschke S. Tran H. Vingtdeux V. Ghanem D. Leroy O. Delplanque J. Vanbrussel E. Delacourte A. Vermersch P. Maurage C.A. Gruffat H. Sergeant A. Mahadevan M.S. Ishiura S. Buée L. Cooper T.A. Caillet-Boudin M.L. Charlet-Berguerand N. Sablonnière B. Sergeant N. Exp. Neurol. 2008; 210: 467-478Crossref PubMed Scopus (43) Google Scholar). These isoforms could therefore contribute to the focal localization of all MBNL1 isoforms, with or without the exon 3 region. Altogether, our results suggest that the binding of MBNL1 proteins to the CUG repeats could either be direct or indirect, through the dimerization of MBNL1 with itself. Thus, the MBNL1 missplicing observed in DM1 could enhance its own sequestration in the foci.CONCLUSIONAltogether, our study reveals the importance of the alternative splicing of MBNL1 in the post-transcriptional regulation of the subcellular localization and function of MBNL1. We attempted to understand better the function of the various MBNL1 isoforms. Our results show that (i) the presence of the exon 3 region is essential to ensure a high binding affinity of MBNL1 for pre-mRNA, and thus its splicing activity; (ii) the presence of the exon 5 region ensures a restrictive nuclear localization; (iii) exons 3 and 5 are fully functional only in the presence of the exon 6 region; and (iv) the exon 7 region modulates the dimerization properties of MBNL1. Our data therefore indicate that association of MBNL1 with CUG repeats depends both on RNA-protein interactions and on self-dimerization. During fetal development and in DM1 pathophysiology, both MBNL1 exons 5 and 7 are included. This could contribute to the exclusively nuclear localization of MBNL1 and to its self-dimerization, two key features for the sequestration within foci. However, further studies will be needed for a better understanding of all the molecular consequences of MBNL1 sequestration in DM1. This knowledge is important to define precisely the mechanism for a potential therapeutic development. IntroductionSplicing of pre-mRNA is a key post-transcriptional step in eukaryotic gene expression. A vast majority of vertebrate pre-mRNAs is alternatively spliced, allowing the production of several protein isoforms from transcripts of a given gene (1Black D.L. Annu. Rev. Biochem. 2003; 72: 291-336Crossref PubMed Scopus (1945) Google Scholar). The regulation of alternative splicing plays a major role in cell differentiation and in development and depends on the expression and activity of numerous splicing regulatory factors that are expressed differentially during development, according to the type of tissue. Defects in these alternative-splicing processes can contribute to pathogenesis, as demonstrated for a growing number of diseases, including neuromuscular diseases such as myotonic dystrophy type 1 (DM1) 9The abbreviations used are: DM1, myotonic dystrophy type 1; 3AT, 3-amino-1,2,4-triazole; BisTris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; hcTNT, human cardiac troponin T; IR, insulin receptor; MBNL1, Muscleblind-like 1. (2Ranum L.P. Cooper T.A. Annu. Rev. Neurosci. 2006; 29: 259-277Crossref PubMed Scopus (374) Google Scholar, 3Lee J.E. Cooper T.A. Biochem. Soc. Trans. 2009; 37: 1281-1286Crossref PubMed Scopus (211) Google Scholar).DM1 is an autosomal disorder caused by an unstable CTG repeat expansion in the 3′-untranslated region (UTR) of the DMPK gene (4Brook J.D. McCurrach M.E. Harley H.G. Buckler A.J. Church D. Aburatani H. Hunter K. Stanton V.P. Thirion J.P. Hudson T. et al.Cell. 1992; 69: 385Abstract Full Text PDF PubMed Scopus (10) Google Scholar, 5Fu Y.H. Pizzuti A. Fenwick Jr., R.G. King J. Rajnarayan S. Dunne P.W. Dubel J. Nasser G.A. Ashizawa T. de Jong P. et al.Science. 1992; 255: 1256-1258Crossref PubMed Scopus (1262) Google Scholar, 6Mahadevan M. Tsilfidis C" @default.
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• W1990507930 title "Analysis of Exonic Regions Involved in Nuclear Localization, Splicing Activity, and Dimerization of Muscleblind-like-1 Isoforms" @default.
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