Tissue development, regeneration and disease, MicroRNAs, Myotonic dystrophy, RNA therapeutics

Overview

The unifying theme of Kalsotra lab's research is to identify post-transcriptional mechanisms that are key for normal tissue development, and when misregulated result in disease. Our major goal is to capitalize on these insights and apply them as therapeutic approaches for tissue regeneration and repair.

We employ tissue-specific knockout/transgenic mouse models, biochemistry, and genome-wide molecular approaches (RNA-seq and iCLIP) to define the post-transcriptional regulatory networks that coordinate developmental and regenerative programs.

1. Alternative splicing regulation in heart and liver development.

Alternative splicing is a process by which a single gene produces multiple polypeptides with potentially different functions. Over 90% of human genes are alternatively spliced giving rise to greater than 100,000 proteins from less than 20,000 genes.

Due to tight spatiotemporal control, alternative splicing not only generates an extremely diverse proteome but also directs its regulated expression in response to a wide range of cues. Importantly, splicing defects are now recognized as central to many human diseases including neurological disorders, muscular dystrophies and cancer.

In this project we are systematically investigating the role of alternative splicing in mammalian heart and liver tissue development. We are also utilizing modified antisense oligonucleotides to investigate the phenotypic consequences of individual mRNA processing events on liver physiology and function.

2. Functional role of alternative splicing in liver regeneration.

The liver carries out a large number of physiological processes, and is therefore, indispensable for organismal survival. Although the human liver possesses an exceptional ability to regenerate, it often fails to repair itself during many liver disorders.

We have recently identified a developmentally regulated splicing network in mice that is redeployed during adult liver regeneration.

The major goal of this project is to delineate the mechanisms that are instrumental in re-initiating the developmental splicing program in response to liver injury. These studies will provide important insights into how quiescent stem cells and hepatic progenitors may be instructed to proliferate or differentiate to combat different liver disorders.

3. MicroRNAs and small molecule therapeutics for myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is an autosomal dominant, multi-systemic neuromuscular disorder that is a common cause of congenital and adult onset muscular dystrophy. The causative mutation is a CTG expansion in the 3’-UTR of DMPK gene resulting in aberrant expression of CUG repeat containing RNA that accumulates in nuclear foci, depletes a splicing regulatory protein, MBNL1, and causes misregulation of developmentally regulated alternative splicing.

From a global screen in a heart-specific and inducible DM1 mouse model, we have recently discovered a specific defect in Mef2 transcriptional program in DM1 that results in loss of expression of its target genes including scores of cardiac microRNAs. We are currently optimizing different therapeutic approaches to correct microRNA misregulation in DM1 cell culture and mouse models.

In collaboration with Steve Zimmerman laboratory, (UIUC), we are also testing the in vivo efficacy of their small molecule inhibitors that can release Mbnl1 from the CUG RNA in a DM1 mouse model.

4. mRNA polyadenylation in heart development and disease.

As transcription terminates, the 3’-end of most eukaryotic mRNAs is cleaved and polyadenylated. Polyadenylation adds a 3’-poly(A) tail that is bound by the poly(A)-binding proteins to regulate mRNA stability, localization and translation.

More than 50% of mammalian genes use alternative poly(A) sites, which affect the mRNA’s accessibility to regulatory factors including microRNAs and RNA binding proteins. While the choice of poly(A) site(s) selection is tightly regulated during development, the key determinative factors affecting this process are largely unknown.

We are currently studying the regulatory programs that control alternative polyadenylation during mouse heart development. We are also characterizing the roles of poly(A) binding proteins in cardiac development and disease.