We are investigating two broad and related areas: developmentally regulated RNA processing and its disruption as the pathogenic mechanism of the microsatellite expansion disease myotonic dystrophy, type 1 (DM1). The investigations of post-transcriptional mechanisms driving physiological changes during postnatal development are complementary to studies of DM1 and provide the basis for an exchange of ideas and collaboration within the lab. There are several potential research projects at any given time; contact Dr. Cooper to explore options.
Mechanism of Myotonic Dystrophy Pathogenesis
Myotonic Dystrophy (DM)
Myotonic dystrophy (DM) is the second most common cause of muscular dystrophy and the most common cause of adult onset muscular dystrophy. There are two forms of DM that are caused by microsatellite expansions in two genes: 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 CNBP gene on chromosome 3 causes Type 2 (DM2). Our focus is on DM1, the more severe form, although the pathogenic mechanisms are similar for both DM1 and DM2. DM1 is dominantly inherited and affects multiple tissues including skeletal muscle (causing 60% mortality), heart (>50% prevalence of arrhythmias and 25% mortality due to sudden cardiac death), central nervous system and gastrointestinal system, among others. DM1 and DM2 expansions cause disease by expression of RNA containing long tracts of CUG or CCUG repeats from the expanded alleles. The RNAs accumulate in discrete nuclear foci (Figure 1) and have the toxic effects described below. For additional information about DM including resources for affected families, see “Myotonic Dystrophy Links” on our Web Resources.
DM1 Pathogenic Mechanisms: Disruption of Developmentally Regulated RNA processing
Pathogenic DMPK alleles contain ~80 to thousands of CTG repeats in the 3’ UTR compared to 5-37 CTG repeats in unaffected individuals. The mRNA expressed from the expanded allele, containing long tracts of CUG repeats (CUGexp RNA), is toxic due to disruption of the functions of at least two families of RNA binding proteins: MBNL and CELF. CUGexp RNA forms a long hairpin structure that binds and sequesters MBNL protein resulting in a loss of function while CELF protein expression is up regulated by an unknown mechanism that involves activated signaling pathways (Figure 2). Members of these families regulate multiple aspects of post-transcriptional gene expression during heart and skeletal muscle postnatal development including alternative splicing, selection of polyadenylation sites, translation, mRNA stability, and mRNA localization. The best-characterized effect of CUGexp RNA is to disrupt splicing regulated by MBNL and/or CELF proteins leading to a failure to transition to adult splicing patterns and the expression of fetal protein isoforms in adult tissues. The inability of fetal isoforms to fulfill the functions required in adult tissues is the molecular basis for disease features such as myotonia however the mechanisms causing muscle wasting and cardiac arrhythmias are unknown and are major areas of investigation in our lab. It was the study of DM1 that revealed the existence of the network of coordinated alternative splicing transitions that occur after birth as tissues remodel from fetal to adult function. This is the second area of investigation in the lab, described below.
DM1 Project Areas
We developed tissue-specific tetracycline-inducible DM1 mouse models to express DMPK RNA containing 960 CUG repeats that reproduce several phenotypic and molecular features of DM1 in heart and skeletal muscle. We also use cell culture models of the disease. Specific projects include:
- Identify the molecular basis for skeletal muscle wasting using the DM1 mouse model, immortalized human DM1 muscle cultures and DM1 patient tissue samples.
- Identify the molecular basis for cardiac arrhythmias using the DM1 mouse model, cell cultures expressing 960 CUG repeat RNA and patient tissue samples.
- Identify proteins associated with foci and non-foci CUGexp RNA to define altered signaling pathways, disease modifiers and potential therapeutic targets.
- Use existing transgenic mouse models that inducibly express CELF proteins and Mbnl1 knock out mice to identify the pathogenic effects of CELF gain of function and MBNL loss of function.
- Determine the mechanisms by which CUGexp RNA activates PKC and identify other signaling pathways with direct relevance to pathogenesis
- Use existing DM1 mouse models for heart and skeletal muscle and cell culture models to test therapeutic approaches including with industrial partners.
Relevant publications include: PNAS 110, 13570; PNAS 109, 4221; Cell Reports 6, 336; ACS Chem Biol. 12, 2503; Hum. Mol. Genet. 27, 2789and check out the most recent publications.
Mechanisms and Consequences of Coordinated Splicing Regulation
We are using three experimental systems to identify previously unknown roles for coordinated alternative splicing and additional post-transcriptional regulatory mechanisms during periods of physiological change: postnatal heart development, postnatal skeletal muscle development and differentiation of cultured skeletal muscle myoblasts. Cell culture and mouse development provide complementary experimental strategies to develop approaches to test hypotheses relevant to tissue function.
Postnatal development is a period of dynamic transcriptional and post-transcriptional gene regulation driving tissue remodeling from fetal to adult physiology. For example, within two weeks after birth the cardiomyocytes of mouse heart stop proliferating and heart growth occurs by cell hypertrophy, cardiomyocytes transition from glycolytic to oxidative metabolism to match the increased need for energy by a rapidly growing body and the T-tubules and the sarcoplasmic reticulum form to manage Ca+2 handling establishing excitation contraction coupling to synchronize contraction within cardiomyocytes. There is interesting biology during postnatal development that has not been explored at the molecular level and our goal is to identify specific molecular mechanisms determinative for critical aspects of adult tissue function. Reversal of changes associated with postnatal development is common in disease such as cancer and heart failure, supporting relevance of postnatal splicing transitions to pathogenic mechanisms. In addition, the postnatal splicing transitions regulated by CELF and MBNL proteins are disrupted in DM1 and we apply knowledge gained regarding the normal regulatory programs that are disrupted by the disease to understand the physiological impact in DM1.
We used the Genomic and RNA Profiling Core at Baylor College of Medicine to perform RNA-seq during mouse heart and skeletal muscle postnatal development and during C2C12 differentiation and identified extensive transitions in gene expression, alternative splicing, alternative polyadenylation, and alternative promoter usage (Figure 3). The postnatal splicing transitions are complete within the first four weeks after birth. In heart, up to 40 percent are conserved between murine and avian development strongly suggesting that the resulting protein isoform transitions are functionally important. In heart and skeletal muscle postnatal development and muscle differentiation in vitro, most genes that produce protein isoform transitions via alternative splicing do not show substantial changes in mRNA levels indicating that alternative splicing produces protein isoform transitions often without a substantial change in gene total output. In one set of projects we are using CRISPR/Cas9 to generate mice with genomic deletions of alternative exons that show conserved developmental regulation to force expression of endogenous fetal isoforms through development and in adults. The work with mice is combined with cell culture and protein biochemical investigations of high priority genes to determine the functions of the protein isoforms. Since the specific functions of the two isoforms and often the gene itself are unknown in heart or skeletal muscle, the results often lead to identification of previously unknown aspects of adult tissue physiology.In addition, many of these splicing events are altered in DM1 and therefore simultaneously investigate mechanisms contributing to postnatal tissue remodeling and DM1 pathogenesis.
Developmentally Regulated Splicing Project Areas
We are using CRISPR/Cas9 in mouse zygotes to delete alternative exons included during postnatal development. We identified several genes for which failure to express the adult isoform produces a defect in heart and/or skeletal muscle function demonstrating significance to function in vivo. Individual genes provide separate projects that cover different biological processes. This set of projects provides a relatively rapid and transformative approach to determine the functions of postnatal protein isoform transitions in vivo.
We developed a splicing reporter that expresses different fluorescent proteins for different splicing patterns to identify cell subpopulations within cultures and mouse tissues that differ in splicing regulation. The reporter can be used for high throughput screens to identify mechanisms of splicing regulation.
General questions driving projects development include: What are the dominant regulators of the multiple coordinated splicing networks during postnatal development? What mechanisms are used to modify the activities of the splicing regulators during development (protein abundance, nuclear:cytoplasmic distribution, post-translational modifications)? What signaling pathways are activated to modulate the activities of the regulators? What are the functional consequences of the protein isoform transitions that occur during postnatal development?
Additional projects are available and are developed with input from lab members.