Pre-mRNA processing is an essential feature of gene expression in eukaryotic cells. Nearly 200 mRNA processing proteins have been identified, yet little is known about how these factors assemble into functional complexes. Coordination of gene expression and cell cycle progression is critical for cellular health. Our lab recently discovered that two related mRNA processing proteins, Btf and TRAP150, have critical roles in cell cycle regulation. Depletion of Btf or TRAP150 leads to altered mRNA localization, misregulation of abundance and splicing for cell cycle regulator transcripts, and chromosome misalignment during mitosis. Our overall hypothesis is that processing and cellular distribution of mRNAs encoding critical mitotic regulator proteins are regulated by Btf and TRAP150. To test this hypothesis, we will identify proteins and RNAs that associate with Btf or TRAP150, and we will produce innovative in situ reporter minigenes to elucidate RNA processing and subcellular trafficking functions for Btf and TRAP150, primarily focusing on consequences for transcripts encoding cell cycle regulators. The proposed project will challenge views that all pre-mRNAs undergo equivalent maturation processes by the entire host of mRNA processing factors. We will define novel mRNA sorting mechanisms for transcripts belonging to critical cellular pathways such as `cell cycle' and will determine whether pre-mRNA processing factors generally have higher pre-mRNA substrate selectivity than is currently appreciated. Furthermore, since pre-mRNA processing and mRNA trafficking are understudied mechanisms for the regulation of cell cycle progression, our project has the potential to transform our basic understanding of a fundamental eukaryotic cell function. Over the project period, this project will train 6-8 undergraduate students, 1 Ph.D. student, and 2 M.S. students. Sophomore-level laboratory courses in cell biology and molecular genetics required of all WSU Biology majors (300 students per year) as well as specialized upper level undergraduate/graduate cell biology laboratory courses (8 students per year) will utilize antibodies, expression plasmids, minigene reporter cell lines, other reagents and experimental protocols developed in this project. This project will therefore meet AREA goals by increasing scientific literacy of more than 900 university students through exposure to meritorious research.

Public Health Relevance

The RNA processing factors SON, Btf and TRAP150 ensure proper transcription/splicing regulation of their downstream target transcripts as part of a complex web of regulatory pathways required for maintaining genome integrity in human cells. Misregulation of key cell cycle regulator transcripts disrupts organization of microtubules in the mitotic spindle (Ahn et al., 2011; Sharma, et al., 2011), expression/localization of mitotic passenger proteins and ultimately, chromosome segregation at mitosis. Implications for SON, Btf and/or TRAP150 in regulating subcellular mRNA localization (Varia et al., 2013), cell death response and virus mRNA processing and replication control (Sun et al., 2001; Karlas et al., 2010; Sarras et al., 2010;) pluripotency (Lu et al., 2014) and lung development (McPherson et al., 2009) underscores the public health relevance for understanding functions for these factors in human development and diseases such as HBV/influenza/adenovirus infection and cancer.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Academic Research Enhancement Awards (AREA) (R15)
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Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
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Carter, Anthony D
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Wright State University
Schools of Arts and Sciences
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Varia, Sapna; Cheedu, Divya; Markey, Michael et al. (2017) Alignment of Mitotic Chromosomes in Human Cells Involves SR-Like Splicing Factors Btf and TRAP150. Int J Mol Sci 18:
Kim, Jung-Hyun; Shinde, Deepali N; Reijnders, Margot R F et al. (2016) De Novo Mutations in SON Disrupt RNA Splicing of Genes Essential for Brain Development and Metabolism, Causing an Intellectual-Disability Syndrome. Am J Hum Genet 99:711-719
Battini, Vishnu Priya; Bubulya, Athanasios; Bubulya, Paula A (2015) Accurate splicing of HDAC6 pre-mRNA requires SON. Int J Mol Sci 16:5886-99
Lu, Xinyi; Ng, Huck-Hui; Bubulya, Paula A (2014) The role of SON in splicing, development, and disease. Wiley Interdiscip Rev RNA 5:637-46
Lu, Xinyi; Göke, Jonathan; Sachs, Friedrich et al. (2013) SON connects the splicing-regulatory network with pluripotency in human embryonic stem cells. Nat Cell Biol 15:1141-1152
Varia, Sapna; Potabathula, Divya; Deng, Zhihui et al. (2013) Btf and TRAP150 have distinct roles in regulating subcellular mRNA distribution. Nucleus 4:229-40
Peng, Hong-Juan; Henkels, Karen M; Mahankali, Madhu et al. (2011) The dual effect of Rac2 on phospholipase D2 regulation that explains both the onset and termination of chemotaxis. Mol Cell Biol 31:2227-40
Sharma, Alok; Markey, Michael; Torres-Munoz, Keshia et al. (2011) Son maintains accurate splicing for a subset of human pre-mRNAs. J Cell Sci 124:4286-98
Sharma, Alok; Takata, Hideaki; Shibahara, Kei-ichi et al. (2010) Son is essential for nuclear speckle organization and cell cycle progression. Mol Biol Cell 21:650-63
Tripathi, Vidisha; Ellis, Jonathan D; Shen, Zhen et al. (2010) The nuclear-retained noncoding RNA MALAT1 regulates alternative splicing by modulating SR splicing factor phosphorylation. Mol Cell 39:925-38

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