RNA binding specificity of Drosophila muscleblind.

Drosophila muscleblind (Mbl) is an RNA binding protein important in differentiation and development. Mbl is known to have roles in photoreceptor differentiation as well as terminal muscle differentiation (1, 2). In addition to roles in differentiation, Mbl is necessary for early development as Mbl null flies die at late embryo stages or early larval stages (2). Mbl has recently been shown to regulate the alternative splicing of two genes involved in the cytoskeleton, α-actinin and tun (3, 4). The human orthologues of Mbl are the muscleblind-like proteins (MBNL1-3) and these proteins also regulate alternative splicing of transcripts important in development (5, 6). Alternative splicing of the Mbl transcript leads to the production of at least four different Mbl protein isoforms ranging in size from 84 to 316 amino acids. Three Mbl isoforms presumably confer RNA binding through two CX7CX6CX3H zinc fingers; the shortest Mbl isoform contains only one zinc finger. The MBNL proteins have been shown to play an important role in myotonic dystrophy (DM) in humans. There are two subtypes of DM, both involving repeat expansions that result in toxic RNAs. Myotonic dystrophy type 1 (DM1) is caused by an expansion of CTG repeats in the 3′-UTR of the DMPK gene (7-9), while myotonic dystrophy type 2 (DM2) is caused by an expansion of CCTG repeats in the first intron of the ZNF9 gene (10). These two types of expansions appear to function through a common disease mechanism: upon transcription, both the CUG and CCUG repeats fold into double-stranded stem-loops with the helices adopting a primarily A-form structure (11-13). MBNL proteins bind these stem-loop structures leading to the sequestration of these important splicing factors in nuclear foci (14-16). MBNL sequestration results in mis-splicing of various transcripts, several of which have been linked to symptoms observed in DM patients (reviewed in refs 5 and 6). Approximately 20 different human pre-mRNAs have been shown to be mis-spliced in DM (6). The most well-characterized example of mis-splicing in DM is the cardiac troponin T (cTNT) pre-mRNA in which MBNL1 has been shown to promote exclusion of an alternatively spliced exon (17). cTNT is developmentally regulated, and the presence or absence of this exon determines if the expressed protein is the fetal or adult isoform, respectively (18-21). Incorrect cTNT splicing in DM1 patients may be linked to cardiac abnormalities, one of the symptoms of DM1 (5). The binding site for MBNL1 in the cTNT pre-mRNA has been mapped to a region upstream of the skipped exon (17). Recently, we have shown that this section of cTNT RNA folds into a stem-loop with mismatches and adopts a structure similar to the CUG repeat RNA which contains U-U mismatches (22). Yuan and colleagues have also shown that MBNL1 binds a stem-loop containing a pyrimidine-pyrimidine mismatch in an intron near the Tnnt3 fetal exon (23), suggesting MBNL recognizes structured RNA elements in its pre-mRNA targets. Drosophila has been used as a model to investigate several aspects of DM1 pathology. Drosophila overexpressing 162 CUG repeats have nuclear foci that contain localized Mbl and CUG repeats but do not display any abnormal phenotypes (24). Longer, interrupted CUG repeats (480 repeats) result in nuclear foci and cause the flies to have muscle, eye, and other degeneration phenotypes, consistent with the Mbl null flies (1, 25). Overexpression of human MBNL1 can rescue these mutant phenotypes (26), indicating MBNL1 and Mbl are functionally conserved. Since Drosophila has one mbl gene with four protein isoforms (while humans have three muscleblind-like genes, each with multiple isoforms) experimental design and data analysis can be simplified when using Drosophila as a model system. Characterization of the RNA binding properties of Drosophila Mbl will lead to a better understanding of its role in alternative splicing and the role of MBNL in DM. In this study we show that recombinant Mbl (amino acids 1-105) is a highly specific RNA binding protein with a strong preference for structured RNA containing specifically placed guanosines.