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University of Michigan (Michigan)
Validating Modulators of SMN Protein Levels from a Genome-Wide RNAi Screen
Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder characterized by muscle wasting due to the degeneration of the motor neurons in the anterior horn of the spinal cord. SMA is incurable and is one of the leading genetic causes of infant death with many children dying before the age of two. Recessive mutations in the SMN1 gene, and a subsequent deficiency of the SMN protein, cause the disease. All patients retain one or more copies of SMN2, an inverted duplication of SMN1. SMN2 pre-mRNA is alternatively spliced to primarily produce a non-functional truncated SMN protein that is rapidly degraded. SMN2 does, however, produce low levels of the functional full length SMN protein.
Given that SMN2 produces some full-length SMN protein, increasing these baseline levels may be a way to ameliorate the SMA phenotype. Understanding genes that modulate SMN protein levels is one approach to developing treatments for SMA. We thus performed a genome-wide RNAi screen using a cell line stably expressing a SMN-luciferase reporter to identify new candidate SMN modulators. We were then able to effectively deplete NEURL2, CAD and U2AF1 using siRNA in SMA patient-derived fibroblasts cells. We observed increased SMN protein levels following depletion of NEURL2, CAD and U2AF1, validating results from the RNAi screen. Next, we decided to investigate splicing as a mechanism behind SMN modulation after a bioinformatics analysis implicated many candidate genes in RNA processing and metabolism. We validated two genes identified from the screen that modulate SMN splicing, PUF60 and U2AF1. Lastly, to test the effect of candidate genes on an indicator of SMN function, we produced GFP-tagged constructs for expression in mammalian cells. We plan to test the effect of expression of genes identified by the screen, including SRSF3 and U2AF1, on SMN protein level and assembly into nuclear bodies known as gems or Cajal bodies.
Last Modified September 19, 2014