RNA Processing Machines in Biology and Disease

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PROJECT SUMMARY / ABSTRACT The goal of this work is to understand the disease-causative roles and basic biology of the human spliceosome. Pre-mRNA splicing is the best-known function of the spliceosome, but this machinery also houses RNA/DNA binding proteins with roles in many steps of gene expression. The lab focuses on the motor neuron disease amyotrophic lateral sclerosis (ALS) and on blood cancers. Greater than one third of ALS-causative genes encode RNA/DNA binding proteins yet their functions are not well understood. Our new research led to the exciting discovery that three of these proteins (FUS, TAF15, MATR3) are essential for expression of a set of antigen presentation genes, which play critical protective roles in the immune system. Remarkably, the three ALS genes are also required for expression of the master transcription control factor of the antigen presentation genes. In ALS, hyperactivation of the immune system is known for its detrimental effects on motor neurons. However, the immune system is emerging as a double-edged sword in ALS as studies indicate that loss of its protective functions also contributes to motor neuron death. At present, little is known about the protective roles. To determine whether loss of the antigen presentation factors due to mutant ALS genes contributes to ALS, human embryonic stem (ES) cells will be CRISPR-edited to harbor ALS-causative mutations in the RNA/DNA binding genes. The ES cells will be differentiated into microglia, which are the immune cells of the central nervous system. Co-culture systems will be used to determine how motor neurons are affected by the mutant microglia. Proteomics, transcriptomics, and functional assays will be used to assess effects on microglia and motor neurons. A critical objective of the work is to determine whether loss of the antigen presentation factors can be extended to other forms of ALS. If so, it raises the exciting possibility that therapies targeting these factors may be efficacious for multiple types of ALS. The goal of our other research project is to determine how mutation of the spliceosomal protein SF3B1 contributes to blood cancers. We CRISPR-edited ES cells to harbor an SF3B1 cancer mutation and will differentiate the ES cells into hematopoietic lineages. Transcriptomics/proteomics and impacts on differentiation will be used to identify changes that may contribute to SF3B1 cancers. We identified a set of mis-splicing signature genes common to different SF3B1 cancers and will determine how their mis- splicing affects differentiation. Mis-splicing of signatures that are candidates for contributing to SF3B1 cancers will be corrected using antisense technology, with the goal of developing the technology as a therapeutic. Finally, we identified two genes that are robustly upregulated only in spliceosome-mutated myelodysplastic syndrome (MDS), which is main type of blood cancer associated with spliceosome mutations. Both upregulated genes play key roles in hematopoiesis. Their potential contribution to MDS will be examined using the hematopoietic differentiation assays. Together, this research will lead to important advances in identifying the roles of defective spliceosomal genes in both ALS and hematological cancers as well as aid in identifying new therapeutic targets.