Degree Type

Dissertation

Date of Award

2016

Degree Name

Doctor of Philosophy

Department

Biomedical Sciences

Major

Molecular, Cellular and Developmental Biology

First Advisor

Ravindra N. Singh

Abstract

The Survival Motor Neuron (SMN) protein is a multi-functional protein that participates in a wide variety of critical pathways. Low levels of SMN cause spinal muscular atrophy (SMA), the most common genetic cause of infant mortality. While the role of SMN in the assembly of small nuclear ribonucleoproteins (snRNPs) has been well characterized, many of its other diverse functions have not been thoroughly explored. Here, we examine the critical role of SMN in the growth and development of male mammalian sex organs. We show that low levels of SMN in a mild mouse model of SMA cause impaired testis development, degenerated seminiferous tubules, reduced sperm count, and low fertility. Underscoring an increased requirement for SMN expression, wild type testis showed extremely high levels of SMN protein compared to other tissues. The testis phenotype is linked to increased apoptosis in seminiferous tubules and extreme perturbations in the testis transcriptome. We examine the RNA binding function of SMN by Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to identify RNA sequence and structural motif(s) of SMN. Our results reveal a combination of sequence motifs and structural contexts that drive the specificity of RNA-SMN interactions. Our results of truncation and substitution experiments suggest a requirement for multiple contacts between SMN and RNA to maintain the high affinity. We demonstrate that both affinity and specificity of RNA-SMN interaction are influenced by salt concentrations. To identify in vivo RNA targets of SMN, we performed crosslinking and immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP). HITS-CLIP identified a variety of RNA targets of SMN with an enrichment of mRNAs participating in a number of pathways, including ribosome function and actin cytoskeleton regulation. In order to determine whether expression levels of target RNAs are regulated by SMN, we performed knockdown of SMN levels followed by RNA-Seq. SPON2, LAMB2, and EEF1A2 in particular were all predicted by HITS-CLIP to be bound by SMN and were downregulated upon SMN knockdown, indicating a direct regulatory role for SMN on expression of these genes.

Copyright Owner

Eric William Ottesen

Language

en

File Format

application/pdf

File Size

188 pages

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