Title: RNA-Dependent Control of Histone Gene Expression by the Spinal Muscular Atrophy Protein SMN
Description: Ribonucleoproteins (RNPs) are RNA-protein complexes that carry out a variety of key cellular functions and are essential for the regulation of gene expression. Small nuclear RNPs (snRNPs) are a class of RNPs that regulate gene expression at the level of RNA processing in the nucleus. These RNPs are subject to complex and highly regulated biogenesis pathways in order to ensure sufficient snRNP levels are present within the cell. snRNPs are required for viability of all eukaryotic cells and the importance of proper snRNP function in vivo is further highlighted by the fact that the fatal motor neuron disease spinal muscular atrophy (SMA) is caused by a genetic deficiency in the ubiquitously expressed survival motor neuron (SMN) protein, an essential component of the snRNP biogenesis machinery. The most well characterized targets of SMN for RNP assembly are the spliceosomal snRNPs, which are critical factors that carry out pre-mRNA splicing. However, SMN is not believed to be solely dedicated to spliceosomal snRNP biogenesis but rather is thought to be a general RNP assembly machine.

Yet, no other RNP targets of the SMN complex had previously been characterized in a conclusive manner. Understanding the cellular targets of SMN-mediated RNP assembly is critical for elucidating basic mechanisms of RNA regulation. Furthermore, despite increased understanding of the molecular function of SMN in spliceosomal snRNP biogenesis and the cellular basis of SMA in animal models, the molecular mechanisms through which loss of SMN function leads to motor neuron disease remain poorly defined. Thus, identifying additional RNP pathways that are dependent on SMN is key to uncover the molecular mechanisms of SMA and may also help in the design of novel therapeutic approaches to this devastating childhood disorder that is currently untreatable. In an effort to expand on the established RNP targets of SMN for assembly, in this dissertation I explore the hypothesis that SMN is required for the biogenesis and function of U7 snRNP and that disruption of this pathway induced by SMN deficiency contributes to motor neuron pathology in SMA.

While structurally analogous to spliceosomal snRNPs, U7 snRNP functions not in splicing but rather in the unique 3’-end processing mechanism of replication-dependent histone mRNAs. Here, I first provide detailed molecular characterization of the in vivo functional requirement of SMN for U7 snRNP biogenesis as well as histone mRNA 3’-end processing and proper histone gene expression. I go on to demonstrate that in a mouse model of SMA U7 snRNP biogenesis and function are severely impaired by SMN deficiency and these defects occur in disease-relevant SMA motor neurons. I then describe the development of a novel molecular strategy to restore U7 snRNP activity in a setting of SMN deficiency in order to investigate the functional consequences of U7 dysfunction in SMA. Finally, I apply this U7 restoration strategy to a mouse model of SMA using AAV9-mediated gene delivery and establish that disrupted U7 activity contributes to select aspects of motor neuron dysfunction in SMA mice. Collectively, my dissertation work provides a significant expansion in our understanding of RNP pathways controlled by SMN and, for the first time, establishes the contribution of an SMN-dependent RNA pathway to SMA pathology in a mouse model of the disease that best recapitulates the human condition both genetically and phenotypically.

The continuation of this work in the future not only may lead to a detailed molecular understanding of the mechanisms of SMA but possibly also to the development of novel therapeutic approaches for this deadly disease that are complementary to SMN upregulation.