Abstract
Since the advent of high-throughput sequencing, non-coding RNAs have emerged as important players in physiological and pathological conditions at all stages of cellular development. Non-coding RNAs can be divided into two large classes: (1) short RNAs, such as miRNAs, which bind and inhibit translation of mRNA; (2) long noncoding RNAs, a diverse class of molecules with a variety of regulatory roles. This dissertation focuses on the role of miRNAs and lncRNAs in skeletal muscle differentiation. The goal of my project is to (1) establish essentiality of miR-206/-1a family in myogenesis, (2) examine functions of MUNC lncRNA that are independent of MYOD1 and (3) determine the dependency of MUNC lncRNA structure and function.
In my first study, I used in vitro and in vivo miR-206/mir-1a-1/miR-1a-2 triple knockout (KO) models to investigate if this miRNA family is essential for myogenesis, as assumed by the field, and if its lack prevents skeletal muscle differentiation, development, and function. We determined that triple KO C2C12 murine myoblasts can differentiate, create myotubes, but have impaired mitochondrial function in comparison to control cells. In our in vivo model we showed that lack of a single miRNA gene leads to decrease of physical performance, with triple KO animals being the weakest and slowest. Nevertheless, triple KO animals developed skeletal muscles, albeit with thinner muscle fiber size. We also found upregulation of miR-206/-1a targets at mRNA and/or protein levels, indicating that the KO was effective at de-repressing the microRNA targets and observed increased number of PAX7 positive satellite stem cells in the skeletal muscles of the triple KO animals. These results show that miR-206/-1a family is not essential for myogenesis but acts instead as a modulator of optimal differentiation of skeletal myoblasts.
MUNC is an enhancer RNA (eRNA) expressed from an enhancer, distal regulatory region, known to be important for expression of Myod1. In the second project we studied MUNC lncRNA and determined whether it regulated gene expression independent of MYOD1, an important promyogenic transcription factor. First, we tried to establish MUNC functional domains by deletion mutagenesis and overexpressing different combinations of exon/intron fragments. These data in combination with bioinformatically predicted structures were not able to determine any fragment important for the promyogenic functions of MUNC. Overexpression of full length MUNC in Myod1 KO C2C12 cells showed that MUNC induces many myogenic transcripts in the complete absence of MYOD1 protein. Genome wide analysis shows that while many genes are regulated by MUNC in a MYOD1 dependent manner, there are many genes that are activated or repressed by MUNC independent of MYOD1. Thus, although MUNC is an eRNA of Myod1, it is also a trans-acting lncRNA whose sequence, structure and co-operating factors include but are not limited to MYOD1, to regulate many myogenic genes.
The third study aims to determine the structural domains responsible for MUNC functions. In the beginning, we discovered that the spliced MUNC lncRNA is much more potent promyogenic factor than the unspliced (genomic) MUNC RNA. Next using SHAPE-MaP technique we experimentally derived a model of MUNC secondary structures. Based on that, we made a series of deletion mutants in MUNC and determined their effects on the function of the spliced form of the lncRNA. First, we found that CH1 and CH4 domains are the most important for induction of promyogenic factors by MUNC and for the promyogenic phenotype. Second, we established that MUNC needs specific structural domains to interact with the cohesin complex, containing the SMC3 protein, which has been proposed to be a major effector of regulation of gene expression by the lncRNA. Third, we determined the specific domains of MUNC are required to bind to specific genomic sites and for regulating the expression of adjoining genes. We observed that MUNC-SMC3 binding may not be sufficient or required for regulation of gene expression by MUNC. Structure-function studies of lncRNAs is a new area of research that offers not only an understanding of molecular mechanisms of lncRNA function, but also may allow for modulating potential therapeutic targets by specifically designed small molecules.
