Overview of lab projects
- Mechanism of myotonic dystrophy pathogenesis
- Mechanism of cell-specific alternative splicing
- Mechanisms of coordinated splicing regulation
Mechanism of myotonic dystrophy pathogenesis
Myotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy affecting 1 in 8500 people worldwide. It is dominantly inherited and affects multiple organ systems including skeletal and cardiac muscle, the gastrointestinal tract, central nervous system, and endocrine tissues. Two different microsatellite expansions cause DM: a CTG expansion in the 3' untranslated region of the DMPK gene on chromosome 19 causes Type 1 (DM1) and a CCTG expansion in intron 1 of the ZNF9 gene on chromosome 3 causes Type 2 (DM2). Both expansions cause disease by expression of the RNA from the expanded allele. These RNAs accumulate in discrete nuclear foci (Figure 1).
A major pathogenic event in DM is the disruption of regulation of pre-mRNA alternative splicing (reviewed in Faustino and Cooper, 2003). Pre-mRNAs from at least six genes undergo mis-regulated alternative splicing in DM tissues and in all cases, splicing reverts to splicing patterns observed in embryonic tissues. These pre-mRNAs are targets for specific trans-acting factors and splicing of other pre-mRNAs is not affected. The working hypothesis is that the expanded repeat RNA has a trans-dominant effect on a subset of alternatively spliced exons by disrupting the regulatory functions of these proteins (see below). The inappropriate expression of embryonic protein isoforms is responsible for at least two major symptoms of DM: mis-splicing of the muscle specific chloride channel (ClC-1) and insulin receptor (IR) pre-mRNAs correlate with the myotonia and the unusual form of insulin resistance, respectively, observed in individuals with DM (Charlet-B. et al., 2002, Savkur et al, 2001, 2004).
Two families of RNA binding proteins have been implicated in DM pathogenesis: CUG-BP1 and ETR-3 like factors (CELF, also called Brunol) and muscleblind-like proteins (MBNL). Members of both protein families were identified based on their ability to bind CUG repeat RNA. We have shown that both protein families regulate alternative splicing of specific pre-mRNA targets and at least two of the pre-mRNAs mis-regulated in DM1 are targets of regulation by CELF and MBNL proteins (Ho et al., in press, and reviewed in Faustino and Cooper, 2003). Furthermore, MBNL and CELF proteins have antagonistic effects on splicing of both pre-mRNAs. The splicing patterns observed for these pre-mRNAs in DM are consistent with increased CELF activity and/or loss of MBNL activity (Figure 2). The mechanisms by which the expanded repeat RNA induces mis-regulated splicing and ultimately disease are unknown but are likely to involve disruption of CELF and MBNL regulatory activities. Understanding this mechanism is an active area of investigation in the lab. Our current working model is that regulation by MBNL and CELF families is linked as part of a larger regulatory program, most likely involved in regulating splicing of a subset of genes during development. We propose that the expression of expanded repeat RNA either initiates or interferes with a signaling event that then initiates a cascade that affects both CELF and MBNL activities as downstream events.
Our current goals are to: (i) determine the mechanism(s) by which the expression of expanded repeat RNA induces altered functions of CELF and MBNL proteins (what nuclear signaling events are disrupted?); (ii) use bioinformatic, biochemical, and molecular approaches to identify additional pre-mRNA targets of CELF and MBNL proteins whose mis-regulated splicing contributes to severe manifestations of disease such as skeletal muscle dystrophy, cardiac arrhythmias, and central nervous system symptoms; (iii) determine the pathogenic form(s) of the expanded repeat RNA [are the RNA foci pathogenic, protective, or neutral? The repeats form double-stranded RNAs (Figure 2), is this necessary for pathogenesis?]; (iv) use transgenic mouse models that inducibly express CELF proteins or CUG repeat RNA to investigate the mechanisms of pathogenesis and eventually test treatment regimes; (v) use cell lines that inducibly express CUG-repeat RNA to determine the cellular and molecular effects of CUG-repeat RNA expression including on the nuclear functions of CELF and MBNL proteins.
Mechanism of cell-specific alternative splicing
We are investigating the mechanisms that regulate alternative splicing on several levels. One is the molecular details of how regulatory factors that directly bind the pre-mRNA communicate to the basal splicing machinery and promote inclusion or skipping of a variably spliced region. Another area, described below ("Mechanisms of coordinated splicing regulation"), is to determine how the activities of factors that regulate splicing are modulated during development (using mouse models) and during cellular differentiation (using cell culture models) and to identify the upstream signaling events responsible for their modulation. We will also investigate the regulatory networks that coordinate splicing during development.
To investigate the molecular details of regulation, we work on three families of splicing regulators: CELF, MBNL, and polypyrimidine tract binding protein (PTB). These proteins have been shown to have positive or negative effects on splicing of different alternative exons. One goal is to understand how CELF proteins function as positive regulators of alternative splicing. Our best characterized model system is cardiac troponin T (cTNT), in which a developmentally regulated alternative exon (exon 5) is included in embryonic heart and skeletal muscle and skipped in adult tissues. We have previously identified the intronic elements surrounding cTNT exon 5 that are required for exon inclusion in embryonic striated muscle (Ryan and Cooper, 1996; Philips et al., 1998). The CELF proteins bind to U/G-rich motifs within these elements and directly activate exon inclusion (Ladd et al., 2001, 2004; Charlet-B. et al., 2002). In addition to positive regulation by CELF proteins, cTNT exon inclusion is negatively regulated by MBNL proteins (Ho et al., in press) and PTB (Charlet-B. et al., 2002). It is clear that individual cell-specific alternative splicing decisions are regulated by combinatorial control involving multiple factors that bind to multiple sites and have antagonistic activities. Evidence also indicates that, as in regulation of cell-specific transcription, recruitment (or repression) of the basal machinery requires assembly of a multicomponent complex (Figure 3).
The specific question being addressed is, how does binding of the positive acting CELF splicing regulators recruit or stabilize binding of the basal splicing machinery? CELF interacting proteins will be identified by yeast two-hybrid analysis, co-immunoprecipitation, and Tandem Affinity Purification (TAP). Recombinant CELF proteins switch splicing from exon skipping to exon inclusion in a cell-free splicing assay (Charlet-B. et al., 2002). This provides an excellent experimental system to determine the mechanism for activation. CELF-activated splicing complexes assembled on the pre-mRNA will be isolated using affinity chromatography and the components identified by mass spectrometry. Protein domains that are required for splicing activation in vivo (likely contacts for protein:protein interactions) will be identified to correlate protein interactions with splicing function.
Mechanisms of coordinated splicing regulation
An array of alternative splicing transitions occurs during cellular differentiation and organismal development. Different sets of splicing factors (such as CELF, MBNL, and PTB) are each likely to regulate numerous specific alternative splicing transitions (Figure 4) and this regulation is likely to be coordinated by interacting networks similar to those described for transcriptional regulation.
We are addressing this unexplored "next level" in understanding the regulation of alternative splicing by investigating regulation during development. Our current goals are to address the following questions: What is the level of coordinated regulation during development? What regulatory factors are the dominant determinants of specific developmental splicing changes? How are the activities of the splicing modulators modified during development (protein abundance, nuclear:cytoplasmic distribution, post-translational modifications)? What are the signaling pathways responsible for modulating the activities of the dominant regulators?
We are investigating regulation during development of heart and skeletal muscle tissues in mice and during skeletal muscle differentiation in cell culture. Bioinformatics, splicing microarrays, and RT-PCR analyses are being used to identify alternative splicing events of particular interest such as those that are conserved between species, result in physiologically important protein isoform transitions, and/or are targets of CELF or MBNL regulation. In the long term, we expect to use the global analysis of multiple and varied transitions to identify networks responsible for coordinated regulation. Our initial focus is on networks regulated by CELF and MBNL proteins. Most CELF and MBNL gene products undergo alternative splicing and express different protein isoforms that may exhibit functional differences. There is great potential for cross- and auto-regulation even just among these two families. In addition, recent results indicate that phosphorylation of CELF proteins (and probably MBNL proteins) has the potential for modulating regulatory activities.