FACULTY - SECONDARY FACULTY - ANDREA LADD - RESEARCH DESCRIPTION

Andrea Ladd, Ph.D.

Assistant Staff

Department of Cell Biology
Cleveland Clinic Foundation

Research:
Pre-mRNA alternative splicing regulation
in vertebrate heart and muscle

When a precursor messenger RNA (pre-mRNA) is first transcribed from a gene, it undergoes extensive processing in the nucleus prior to export to the cytoplasm. Introns are removed and exons are pasted together in a process called splicing. Many genes undergo alternative splicing, in which pre-mRNA molecules produced from the same gene are spliced differently to give rise to more than one mature mRNA species. The coding region is often affected, so alternative splicing leads to the production not just of different mRNAs but also different proteins from a single gene. Alternative splicing is highly regulated to prevent the wrong gene products from being produced in the wrong place or at the wrong time. Alternative splicing patterns can be regulated according to cell type, developmental stage, sex, or response to external stimuli.

Alternative splicing is now estimated to affect about three quarters of all human genes, and the misregulation of alternative splicing has been linked to several diseases including muscular dystrophy and heart disease. Rather than pursue the mechanism of how alternative splicing is regulated at the level of a single pre-mRNA molecule, we strive to understand what role alternative splicing regulatory programs play in developmental or disease processes. The regulation of gene expression during development has been intensely studied at the level of transcription, but no one has really looked at the role of alternative splicing in development, so this is a really exciting but understudied field.

Our laboratory uses a combination of molecular biology and embryology approaches and techniques. The two model systems that we use are the chick embryo and the transgenic mouse. Chick embryos are excellent for experiments that require embryo manipulations, whereas mice provide a malleable genetic system. Both are vertebrate systems that model striated muscle development in humans. Working with both of these systems also allows us to look for conservation of alternative splicing regulation, with the idea that alternative splicing regulatory programs that are critical for development will be under greater selection pressure and thus highly conserved.

The role of alternative splicing regulation during early heart development

In the United States, more than 32,000 infants are born with heart defects each year. Heart defects are among the most common birth defects, and are the leading cause of birth defect-related deaths. The key to predicting and preventing heart defects is to understand how cardiac morphogenesis is regulated. The regulation of gene expression at the level of transcription has been well studied during cardiogenesis, but little or no attention has been paid to the role of alternative splicing. Members of the CUG-BP and ETR3-like factor (CELF) and muscleblind-like (MBNL) families of RNA binding proteins have been implicated in alternative splicing regulation in the developing vertebrate heart. CELF and MBNL proteins are expressed in the early heart and have been shown to bind to RNA and promote or repress alternative splice site usage in an antagonistic manner.

One research area in the lab is to investigate the role of CELF/MBNL-mediated alternative splicing programs during cardiac morphogenesis using the chick embryo as a model system. This involves characterizing CELF and MBNL gene expression during embryogenesis, developing profiles of alternative splicing during early heart development, identifying targets of CELF/MBNL regulation, conducting CELF/MBNL knockdown experiments in whole embryo or explant cultures, and determining the functional significance of specific CELF/MBNL-mediated alternative splicing events using oligo-based methods to manipulate splice site choices in culture.

CELF-mediated alternative splicing in heart disease

In the heart, there are reports of abnormal alternative splicing patterns associated with heart disease in human patients (Saba et al., 1996; Weng et al., 2005). We and others have shown that targeted disruption of alternative splicing regulatory programs in the mouse heart causes cardiomyopathy and heart failure (Ladd et al., 2005; Fu et al., 2005; Ding et al., 2004). When CELF activity is disrupted in the hearts of transgenic mice (MHC-CELFΔ), the mice develop severe cardiomyopathy, characterized by specific alternative splicing changes, cardiac dysfunction, and premature death (Ladd et al., 2005 Mol Cell Biol 25: 6267-6278). Interestingly, the penetrance of the phenotype is greater in females than in males despite similar levels of dominant negative protein expression. MHC-CELFΔ males are not only less likely to develop hypertrophy and die young, but are also less likely to exhibit defects in CELF-mediated alternative splicing, suggesting there is a sex-specific modulation of splicing activity.

Another research area follows up the work with the MHC-CELFΔ mice to investigate the role of CELF-mediated alternative splicing in cardiomyopathies. This includes a screen of alternative splicing events in mouse models of cardiac disease and injury, using existing transgenic models in which CELF activity is repressed (MHC-CELFΔ) or elevated (MCKCUG-BP; Ho et al., 2005 14:1539-47) in heart to assess the effects of CELF-mediated alternative splicing on response to cardiac injury, and investigating sex-specific alternative splicing in the heart and whether it contributes to sex differences in cardiac disease outcomes.

CELF-mediated alternative splicing in skeletal muscle

Another area of research in the lab deals with alternative splicing during skeletal muscle development. Many of the developmental transcription programs that control gene expression in heart muscle also participate in skeletal muscle development. Projects will include screening alternative splicing events during skeletal muscle development in chicken and mouse, as well as generating and characterizing transgenic mice that express a dominant negative CELF protein in skeletal muscle (Myo-CELFΔ). The success of MHC-CELFΔ mice (see above) demonstrated the feasibility of the dominant negative approach in vivo. Increased CELF activity and misregulation of alternative splicing of CELF targets have been implicated in the pathogenesis of myotonic dystrophy, a common adult onset form of muscular dystrophy. If Myo-CELFΔ mice are viable, they will be crossed with a mouse model of myotonic dystrophy (Mankodi et al., 2000) to determine whether repressing CELF activity can alleviate or prevent symptoms of the disease.