The experiments described in this proposal are designed to provide insight into the mechanism and regulation of alternative pre-mRNA splicing (AS) in normal cellular processes as well as into its misregulation in different pathological conditions. Studies are proposed to investigate the roles of the RNA/DNA binding protein TLS/FUS (TLS) in the neurodegenerative disease amyotrophic lateral sclerosis (ALS), of hnRNP proteins in cancer, specifically glioblastoma (GBM), of core splicing factors mutated in myelodysplastic syndromes (MDS), and of AS in differentiation of human embryonic stem cells (hESCs). The following Specific Aims are proposed. 1. TLS/FUS and ALS. Ongoing studies establishing that the MECP2 gene, which encodes methyl CpG binding protein 2, is a TLS target deregulated in the presence of ALS mutant proteins will be continued, as a model for how TLS regulates AS and gene expression generally and how this goes awry in ALS. With N. Shneider, the disease relevance of MECP2 misregulation will be investigated by examining MECP2 expression in ALS mouse models and derived ESC lines differentiated into motor neurons. Finally, additional genes that display similar disruptions in expression as observed with MECP2 will be identified and characterized. 2. hnRNPs A1/A2 and PTB: overexpression in GBM and mechanism of regulation. Experiments examining the roles of hnRNP A1/A2 and PTB in GBM will be continued. PTB/A1/A2-regulated splicing targets recently identified will be investigated with respect to how AS contributes to two key cancer phenotypes, loss of tumor suppressor activity and gain of proliferation-promoting function. How changes in PTB/A1/A2 levels affect their binding to sites in pre-mRNA targets will be analyzed to test the hypothesis that reducing levels of these hnRNPs affects occupancy of different sites in distinct ways. 3. SF3B1 and SRSF2 mutations in MDS. Experiments are proposed to address both the molecular mechanisms and functional significance of SF3B1 and SRSF2 mutations in MDS and related malignancies. Possible splicing-related defects of the mutant proteins will be examined using in vitro assays. With A. Raza and S. Mukherjee, phenotypes induced by mutant proteins will be determined. Analysis of RNA-seq data from MDS patients to detect splicing defects, notably intron retention, will be continued, and the possibiliy that this involves U11/U12 introns examined. The hypothesis that mutant SF3B1 and SRSF2 contribute to MDS by inducing R-loop formation and genomic rearrangements will be tested. The significance of a putative gene fusion already detected will be determined. 4. AS regulation in hESC differentiation. Results obtained by RNA-seq analysis of AS changes accompanying hESC differentiation will be extended to detect additional AS events specific to hESCs. The biological significance and underlying mechanisms of identified hESC-specific AS events, including in transcripts encoding Tcf3, a transcription factor previously implicated in the maintenance of ESC pluripotency, and Tra2?, a splicing factor known to regulate tissue-specific AS, will be investigated, as will the functions of the relevant protein isoforms.
The experiments described in this proposal are designed to increase our understanding of the mechanism and regulation of alternative pre-mRNA splicing, and how this process is linked to other cellular events. Previous studies have revealed that changes in splicing occur during development and disease, and our studies will provide considerable new insight into these changes, how they are regulated, and how they can become deregulated in disease.
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|Conlon, Erin G; Lu, Lei; Sharma, Aarti et al. (2016) The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains. Elife 5:|
|Zhang, Jian; Lieu, Yen K; Ali, Abdullah M et al. (2015) Disease-associated mutation in SRSF2 misregulates splicing by altering RNA-binding affinities. Proc Natl Acad Sci U S A 112:E4726-34|
|Coady, Tristan H; Manley, James L (2015) ALS mutations in TLS/FUS disrupt target gene expression. Genes Dev 29:1696-706|
|Zhou, Xuexia; Wu, Wenwu; Li, Huang et al. (2014) Transcriptome analysis of alternative splicing events regulated by SRSF10 reveals position-dependent splicing modulation. Nucleic Acids Res 42:4019-30|
|Li, Huang; Wang, Zhijia; Zhou, Xuexia et al. (2013) Far upstream element-binding protein 1 and RNA secondary structure both mediate second-step splicing repression. Proc Natl Acad Sci U S A 110:E2687-95|
|Manley, James L (2013) SELEX to identify protein-binding sites on RNA. Cold Spring Harb Protoc 2013:156-63|
|Zhang, Jian; Manley, James L (2013) Misregulation of pre-mRNA alternative splicing in cancer. Cancer Discov 3:1228-37|
|Kanehiro, Yuichi; Todo, Kagefumi; Negishi, Misaki et al. (2012) Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1. Proc Natl Acad Sci U S A 109:1216-21|
|Tan, Adelene Y; Riley, Todd R; Coady, Tristan et al. (2012) TLS/FUS (translocated in liposarcoma/fused in sarcoma) regulates target gene transcription via single-stranded DNA response elements. Proc Natl Acad Sci U S A 109:6030-5|
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