A Functional Genomic approach to the Cellular Events in Spinal Muscular Atrophy

Chapter 1. Spinal muscular atrophy (SMA) is a common motor neuron disease caused by homozygous loss of the survival motor neuron 1 (SMN1) gene. A nearly identical copy of human SMN2 gene has a C-to-T translationally silent mutation at position +6 of exon 7 and produces exon 7 skipping mRNAs. By using a novel reporter system, we have identified the RNA binding motif protein 4 (RBM4) and Heterogeneous nuclear ribonucleoprotein U-like protein 1 (hnRNPUL1) as up-regulators for full-length SMN protein expression by a genome-wide gain-of-function cDNA screen. These two proteins promoted SMN2 exon 7 inclusion which stimulates full-length SMN protein expression. Disruption of the N-terminal RNA recognition motif (RRM) on RBM4 and deletion of the N-terminal SAP domain on hnRNPUL1 largely abolished their exon 7 inclusion ability. Furthermore, we show that RBM4 binds to hnRNPUL1 and load to SMN2 RNA through TRA2B. A model describing sequential binding with TRA2B is proposed. The combination of wild type TRA2B with mutant hnRNPUL1 or RBM4 dramatically decreased the ratio of exon 7 included transcript while co-transfection wild type hnRNPUL1 with mutant RBM4 showed similar effect. Collectively, our study here not only reveals two unique factors that increase SMN2 exon 7 inclusion but also illustrate a new model of SMN2 RNA splicing mechanism.;We have also used the reporter system reporter system to screen compounds that either increase SMN2 expression, protein stability or modulate correct splicing. Compounds with high SMN2 selectivity and low cytotoxicity were classified into several categories based on structural scaffolds and structure-activity-relationship (SAR) studies. Compounds of interest were further tested in SMA patient fibroblasts. Early passage human fibroblast cell lines from a 3-year-old type I SMA patient with 2 copies of SMN2 and one heterozygous carrier (GM03814) were used to test compound effects on SMN2 in a genomic context. We selected two compound scaffolds to analyze; isoindolinone (TK45), and aminopyrazol (TK24). Both scaffolds showed an increase in gem counts as well as SMN protein levels as assessed by western blot after normalization with β-actin loading control. We also tested compound effect on SMN2 mRNA levels using two primer/probe pairs to distinguish SMN2 exon 7 inclusion or skipping. Interestingly, the compounds tested had different effects on the ratio of full length to alternatively spliced SMN2 mRNA. As expected, HDAC inhibitors increased Δ7 SMN2 but increased full length (fl) SMN2 to an even greater degree, likely because of an increase in TRA2B expression. TK24 and TK45, however, increased Δ7 SMN2 and fl SMN2 to a similar degree. These results suggest these compounds work by different mechanisms compare to HDAC inhibitors. Finally, the effects of the compounds were examed in SMA-iPS derived neurons. Both compounds showed significant increases in SMN expression level.;Chapter 2. Approximately 3,500 mammalian genes are predicted to be secreted or single-pass transmembrane proteins. The function of the majority of these genes is still unknown, and a number of the encoded proteins might find use as new therapeutic agents themselves or as targets for small molecule or antibody drug development. To analyze the physiological activities of the extracellular proteome, we developed a large-scale, high-throughput screening platform. To determine its utility, this library was screened in a high-content cellular assay to identify regulators of human Hippo pathway. We found that the c1qTNF5 promote Yap nuclear localization in high-density culture. Our results also indicate that activation of the Hippo pathway by the c1qTNF5 is sufficient to proliferate human RPE cells and several other primary cells types.