Regulation of RNA splicing is the primary mechanism responsible for generating the proteome diversity and phenotypic complexity of humans. This post-transcriptional regulatory mechanism is particularly prominent in neuronal cells and the increasing number of neurodegenerative disorders that are associated with splicing dysfunction underscores its biological relevance. The study of the survival motor neuron (SMN) protein provides a unique opportunity to address the basic biology of splicing regulation and the role of RNA dysfunction in human disease. Reduced SMN levels cause spinal muscular atrophy (SMA)-a common inherited neuromuscular disorder characterized by motor neuron degeneration. SMN has a well-established function in the assembly of small nuclear ribonucleoproteins (snRNPs), which are the essential components of the splicing machinery. In SMA mice, the degree of snRNP assembly impairment correlates with disease severity and causes an uneven rather than uniform decrease in the levels of snRNPs, resulting in the alteration of the snRNP profile of tissues. Moreover, restoration of normal snRNP levels coincides with phenotypic correction in animal models of disease. Despite these advances, how defective SMN function in snRNP biogenesis selectively affects motor neurons is unknown. This project will investigate our hypothesis that SMN functions to endow distinct cell types with unique snRNP profiles for the purpose of splicing regulation and that alterations in this process triggered by SMN deficiency cause splicing defects in mRNAs critical for motor neuron biology. Building on the results of our preliminary studies, in Aim 1 we will analyze SMN role in establishing distinct snRNP profiles in different cell types as well as mouse tissues during development. The relevance for splicing regulation of these cell type-specific snRNP profiles will be studied in Aims 2 and 3.
In Aim 2, we will investigate the consequences of SMN depletion on RNA splicing using microarray analyses and cellular model systems with regulated knockdown of SMN. Based on our ability to generate large numbers of motor neurons differentiated from mouse embryonic stem (ES) cells with normal and reduced levels of SMN, we will identify mRNAs affected by SMN deficiency in the cell type relevant to SMA. Through comparative analyses using other types of post-mitotic neurons as well as primary motor neurons from SMA mice, we will define the set of mRNAs whose expression or alternative splicing is selectively affected in motor neurons. In order to establish a mechanistic link between SMN control of snRNP biogenesis and splicing regulation, the cause-effect relationship between SMN-dependent alterations in the snRNP profile and splicing changes will be analyzed in Aim 3. Finally, in Aim 4, the functional role of selected genes and alternative splicing isoforms identified above will be studied using knockdown and over-expression experiments in both normal and SMN- deficient ES cell-derived motor neurons. This approach should identify mRNAs whose SMN-dependent expression or alternative splicing is critical for motor neuron survival and function.
The biomedical relevance of understanding the mechanisms and regulation of RNA processing is highlighted by the growing list of human genetic disorders associated with defects in RNA metabolism. This project is designed to define the normal role of the spinal muscular atrophy (SMA) protein in the post-transcriptional control of gene expression as well as to identify genes whose altered expression may contribute to degeneration of SMN-deficient motor neurons, the neuronal cells selectively affected in SMA. These studies should provide insights into the basic mechanisms of RNA regulation and the molecular defects underlying SMA pathogenesis with the potential of identifying new candidate targets for therapeutic development.
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|Li, Darrick K; Tisdale, Sarah; Lotti, Francesco et al. (2014) SMN control of RNP assembly: from post-transcriptional gene regulation to motor neuron disease. Semin Cell Dev Biol 32:22-9|
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|Tisdale, Sarah; Lotti, Francesco; Saieva, Luciano et al. (2013) SMN is essential for the biogenesis of U7 small nuclear ribonucleoprotein and 3'-end formation of histone mRNAs. Cell Rep 5:1187-95|
|Li, Darrick K; Tisdale, Sarah; Espinoza-Derout, Jorge et al. (2013) A cell system for phenotypic screening of modifiers of SMN2 gene expression and function. PLoS One 8:e71965|
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