Primary focus will be on global characterization of late erythroid transcriptome(s) with emphasis on detection of new transcripts and new isoforms of known transcripts, dynamic evolution of the transcriptome during late erythropoiesis, and mechanisms by which an evolutionarily conserved alternative splicing program shapes the transcriptome. Differentiating erythroblasts execute a diverse and dynamic pre-mRNA alternative splicing program that cooperates with the transcriptional program to ensure synthesis of the appropriate stage-specific proteome as cells acquire specialized functional properties. Proper regulation of alternative splicing is extremely relevant to human health, for misregulation is a major contributor to many human diseases yet the erythroid splicing program, its regulation, and its importance in erythroid biology remain poorly understood. This project proposes a global analysis of the stage-specific erythroid transcriptome by RNA-Seq analysis and advanced bioinformatic strategies to address these issues and to generate a wealth of new information of use to many other investigators studying erythroid differentiation and erythroid biology. To explore the hypothesis that a conserved mammalian erythroid alternative splicing program regulates critical erythroid functions, investigators with expertise in erythroid differentiation, alternative splicing regulation, and computational analysis of deep sequencing data have come together to propose three specific research aims.
Aim 1 will define the mouse erythroid stage-specific transcriptome using highly purified FACS-sorted erythroid cells from bone marrow (proerythroblasts as well as basophilic, polychromatic, and orthochromatic erythroblast stages). Advanced computational analysis of RNA-seq data enables comparison among the differentiation stages and between erythroid and non-erythroid cells, to characterize erythroid isoform diversity and stage-specific switches in alternative splicing that imply functional changes in the encoded proteome.
Aim 2 will perform a similar analysis of human erythroblasts that are highly purified by FACS sorting. Comparison of human and mouse data will facilitate the definition of evolutionarily conserved erythroid-specific and dynamic switches in isoform expression that suggests critical erythroid functions, in addition to highlighting isoform differences that exist between mouse and human cells.
Aim 3 proposes a mechanistic study of the conserved alternative splicing events defined in Aims 1 and 2 by using computational and biochemical approaches to analyze cis- regulatory sequences and splicing factor proteins that direct these splicing networks. Ultimately, this work should reveal the splicing regulatory network(s) that orchestrate programmed splicing in differentiating erythroid cells. Long term benefits anticipated from this work include greatly improved insights into regulation of biological processes in erythroid cells by alternative protein isoforms. Moreover, the RNA-Seq data itself may stimulate studies of the transcriptional and post-transcriptional regulation of this transcriptome.

Public Health Relevance

Understanding how tissue-specific alternative splicing is regulated is extremely relevant to human health, because splicing mis-regulation is the underlying cause of a significant number of human genetic diseases. This project will lay the foundations for analysis of alternative splicing networks in that operate during differentiation of erythroblasts into mature red cells. This knowledge will stimulate mechanistic studies of normal red cell function and may contribute to understanding of aberrant red cells due to splicing defects.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Special Emphasis Panel (ZRG1-VH-F (02))
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Bishop, Terry Rogers
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Lawrence Berkeley National Laboratory
Other Domestic Higher Education
United States
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Pimentel, Harold; Parra, Marilyn; Gee, Sherry L et al. (2016) A dynamic intron retention program enriched in RNA processing genes regulates gene expression during terminal erythropoiesis. Nucleic Acids Res 44:838-51
Pimentel, Harold; Sturmfels, Pascal; Bray, Nicolas et al. (2016) The Lair: a resource for exploratory analysis of published RNA-Seq data. BMC Bioinformatics 17:490
Conboy, John G (2016) Developmental regulation of RNA processing by Rbfox proteins. Wiley Interdiscip Rev RNA :
Pimentel, Harold; Parra, Marilyn; Gee, Sherry et al. (2014) A dynamic alternative splicing program regulates gene expression during terminal erythropoiesis. Nucleic Acids Res 42:4031-42
Lovci, Michael T; Ghanem, Dana; Marr, Henry et al. (2013) Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges. Nat Struct Mol Biol 20:1434-42
Trapnell, Cole; Hendrickson, David G; Sauvageau, Martin et al. (2013) Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol 31:46-53