Sequestration of MBNL Proteins

The three human MBNL paralogues are homologues of Drosophila mus-cleblind (mbl), which is required for Drosophila photoreceptor and muscle differentiation (Artero et al. 1998; Begemann et al. 1997). MBNL1, MBNL2, and MBNL3 are located on chromosomes 3, 13, and X, respectively (Fardaei et al. 2002; Miller et al. 2000). MBNL1 was identified based on its ability to bind double-stranded CUG-repeat RNA in HeLa cell nuclear extracts (Miller et al. 2000). All three MBNL proteins colocalize with expanded CUG and CCUG RNA nuclear foci in cultured cells as detected by immunofluorescence (Fardaei et al. 2002; Mankodi et al. 2001; Miller et al. 2000). In addition, MBNL can bind to expanded (up to 50) CCUG, CUG, and CAG repeats as detected by a yeast three-hybrid assay (Kino et al. 2004). Both MBNL1 and MBNL2 are expressed in skeletal muscle and heart, two tissues that are prominently affected in DM (Kanadia et al. 2003b). MBNL3 expression is restricted to the placenta in the adult mice and is more widely expressed in the embryo (Fardaei et al. 2002; Miller et al. 2000).

The observations that muscleblind proteins colocalize with the expanded CUG and CCUG repeats strongly suggests that loss of MBNL function due to sequestration on CUG-repeat RNA plays a major role in DM pathogenesis (Dansithong et al. 2005; Fardaei et al. 2002; Jiang et al. 2004; Mankodi et al. 2001; Miller et al. 2000). Results from Mbnl knockout mice strongly support this hypothesis (Kanadia et al. 2003a). Targeted deletion of exon 3 in mice (MBNL1ae/ae) to eliminate the MBNL1 isoforms that bind expanded CUG/CCUG RNA repeats resulted in myotonia, cataracts, and RNA splicing defects that are striking characteristics of DM.

Histological analysis of muscle revealed increased central nuclei and splitting of myofibers. MBNL1AE/AE mice showed abnormal retention of the TNNT3 fetal exon and CLCN-1 exon7a in skeletal muscle and TNNT2 exon 5 in heart consistent with splicing changes seen in individuals with DM1 and without changes in steady state levels of CUG-BP1 (Kanadia et al. 2003a).

The MBNL family was recently identified as direct regulators of alternative splicing (Ho et al. 2004). Specifically, MBNL proteins regulate splicing of TNNT2 exon 5 and IR exon 11 via direct binding to adjacent intronic elements (Ho et al. 2004). MBNL and CELF proteins have antagonistic effects on the splicing patterns of these two pre-mRNAs, however, MBNL and CELF proteins bind to different sites within the pre-mRNAs indicating that the antagonism is not due to a competition for a common binding site. In addition, regulation by CELF and MBNL appeared to be completely independent, as TNNT2 minigenes containing mutant CUG-BP1 binding sites still responded to MBNL1, and vice versa (Ho et al. 2004).

The patterns of misregulation for TNNT2, IR, and CLCN-1 are consistent with increased CUG-BP1 activity and with decreased MBNL1 activity. Whether the splicing effects in DM are due primarily to loss of MBNL activity or a gain of CELF protein activity remains an open question, with evidence supporting both. Several pieces of evidence, some of which was noted above, support a role for MBNL depletion. First, MBNL proteins colocalize with CUG- and CCUG-repeat RNA foci, which is consistent with a loss of function due to sequestration (Fardaei et al. 2002; Mankodi et al. 2001; Miller et al. 2000). Second, misregulated splicing patterns and striking phenotypic similarities to DM are observed in Mbnl knockout mice (Kanadia et al. 2003a). Third, a recent study showed that loss of MBNL1 function was the critical event in aberrant splicing of IR in DM1 cultured cells (Dansithong et al. 2005). Fourth, of the two pre-mRNAs directly regulated by MBNL proteins (TNNT2 and IR), the splicing patterns of both alternative exons in DM tissues are consistent with a loss of MBNL activity (Ho et al. 2004).

There are also several results that support a role for increased CELF activity. First, CUG-BP1 steady state levels are increased in DM skeletal muscle and heart tissues as well as in DM cell cultures (Dansithong et al. 2005; Savkur et al. 2001; Timchenko et al. 2001a). Second, the splicing patterns of all of the three pre-mRNAs (TNNT2, IR, and CLCN-1) shown to be directly regulated by CUG-BP1 are consistent in DM tissues with increased CUG-BP1 activity (Charlet-B. et al. 2002b; Philips et al. 1998; Savkur et al. 2001). Third, a TNNT2 minigene expressed in DM cell cultures reproduces the splicing pattern observed for the endogenous TNNT2 pre-mRNA in DM tissues (Philips et al. 1998). Similarly, the "DM" splicing pattern for both TNNT2 and IR minigenes can be induced in normal cells by co-expression of a plasmid containing expanded CTG repeats (Ho et al. 2004; Philips et al. 1998; Savkur et al. 2001). Importantly, minigenes that contain mutations in the CUG-BP1 binding site are no longer responsive to CUG-BP1, to the effects in DM cell cultures, or to co-expression of CUG-repeat RNA (Philips et al. 1998; Savkur et al. 2001). These results indicate that the effects of the repeats on splicing require the CUG-BP1 binding site and suggest a direct role for CUG-BP1.

There is also evidence suggesting that sequestration of MBNL proteins is not sufficient to explain the trans-acting effects of CUG-repeat RNA on splicing. First, the mutated TNNT2 minigene that is not responsive to CUG-repeat RNA remains responsive to depletion of MBNL1 using siRNAs (Ho et al. 2004). The finding that a minigene that does not respond to co-expression of CUG-repeat RNA still responds to MBNL1 depletion indicates that the effects of CUG RNA on splicing involves more than MBNL depletion (Ho et al. 2004). Second, recent results indicate that MBNL colocalizes with nuclear RNA foci containing either CUG- or CAG-repeat RNA from transiently transfected plasmids. Expanded CUG-repeat RNA induces splicing changes of TNNT2 and IR minigenes when coexpressed with the minigenes but CAG repeats of equal length and expressed at comparable levels have little effect on splicing (Ho et al. 2005b). When FRAP analysis was used to determine the relative affinity of a MBNL1-GFP fusion protein for CUG- and CAG-repeat RNA foci in vivo; no differences were detected (Ho et al. 2005b). Therefore, CUG and CAG RNA appear to have similar abilities to sequester MBNL while only CUG repeats have a trans-dominant effect on splicing. Finally, MBNL3, also called CHCR (Cys3His CCG1-Required), was identified as an inhibitor of muscle differentiation in C2C12 cells (Squillace et al. 2002). Muscle differentiation defects observed in DM is not consistent with loss of MBNL3 function since MBNL3 sequestration in nuclear foci should favor differentiation. These results suggest that while loss of MBNL activity is likely to play a role in the splicing abnormalities observed in DM, the effects of the repeats on alternative splicing regulation appear to involve a mechanism more complex than sequestration of MBNL alone.

Accumulation of RNA foci is an important hallmark of DM (Davis et al. 1997; Jiang et al. 2004; Liquori et al. 2001; Mankodi et al. 2003; Miller et al. 2000; Taneja et al. 1995). The formation of the foci was recently shown to require MBNL as RNAi-mediated depletion of MBNL in DM1 myoblasts reduced the number of foci by 70% (Dansithong et al. 2005). It is clear that the repeat-containing RNA is pathogenic but it is not clear whether foci contain the pathogenic form of the RNA. The finding that CAG-repeat RNA forms foci, colocalizes with MBNL but does not alter splicing strongly suggest that foci formation and the potential to sequester MBNL alone is not sufficient for misregulated alternative splicing. Additional support for the inconsistency between toxicity and foci formation comes from a recent report showing that expression of 162 CTG repeats in the 3' UTR of a reporter gene formed foci in Drosophila tissues without inducing pathology, suggesting that foci formation was not toxic to Drosophila

(Houseley et al. 2005). Similarly, foci formation by RNAs containing only CUG repeats is not sufficient to induce muscle-differentiation defects in the C2C12 cell line (Amack and Mahadevan 2001). In summary, these results strongly suggest that foci formation alone is not pathogenic.

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