This application describes a multidisciplinary collaboration between Dr. Michael Sheets, a developmental biologist who focuses on post-transcriptional regulation of early Xenopus embryogenesis and Dr. Colin Dewey, an expert in biostatistics and bioinformatics who focuses on analyzing gene expression using deep sequencing methodologies. While progress has been achieved in understanding Xenopus laevis development from a transcriptional point of view, significantly less is known about the translation of mRNA transcripts into proteins. We propose to use ribosome profiling to globally measure the translational activity of Xenopus mRNAs across the window of development that extends from oogenesis to gastrulation. Results from the proposed experiments will provide an unprecedented view of the dynamic translational landscape that exists during Xenopus development and this data has numerous applications. First, our experiments will identify biologically relevant Xenopus mRNAs. Transcriptome analysis only identifies the mRNAs present in cells, but it cannot define which mRNAs are translated into protein and therefore biologically relevant. We will use ribosome profiling to identify the actively translated and biologically relevant Xenopus mRNAs. This subset of the mRNA population and especially those that exhibit regulation represent ideal candidates for loss of function studies. Second, our results will identify groups of mRNAs that exhibit similar patterns of translational regulation. These co-regulated mRNAs will provide important starting points for molecular studies that seek to identify common sequence motifs for RNA binding proteins or miRNAs that mediate regulation. Third, ribosome profiling in the presence of the inhibitor harringtonine will allow us to identify the translational initiation site(s) for each Xenpus mRNA in our samples. The position of initiation defines the amino termini of the protein product encoded by an mRNA and therefore globally identifying the initiation sites of all mRNAs defines the N-terminus of the proteome. In addition, identifying the sites of initiation for specific mRNAs can reveal the presence of 5'RNA sequences, called uORFs that often function to regulate translational initiation. Results from the proposed experiments will provide an unprecedented genomic scale analysis of mRNA translation in Xenopus and how the translation of each mRNA changes during development. Our results and the associated methods will be useful for Xenopus researchers and researchers addressing the same questions in other vertebrate embryos.

Public Health Relevance

Proteins are the molecular machines that perform the functions of all living cells. Our research is focused on how proteins are synthesized and how this synthesis changes depending upon the individual needs of specific cells.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21HD076828-02
Application #
8697085
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Coulombe, James N
Project Start
2013-07-05
Project End
2015-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Wisconsin Madison
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Madison
State
WI
Country
United States
Zip Code
53715
Fink, Dustin M; Sun, Miranda R; Heyne, Galen W et al. (2018) Coordinated d-cyclin/Foxd1 activation drives mitogenic activity of the Sonic Hedgehog signaling pathway. Cell Signal 44:1-9
Slosarek, Erin L; Schuh, Amber L; Pustova, Iryna et al. (2018) Pathogenic TFG Mutations Underlying Hereditary Spastic Paraplegia Impair Secretory Protein Trafficking and Axon Fasciculation. Cell Rep 24:2248-2260
Dowdle, Megan E; Imboden, Susanne Blaser; Park, Sookhee et al. (2017) Horizontal Gel Electrophoresis for Enhanced Detection of Protein-RNA Complexes. J Vis Exp :
Sheets, Michael D; Fox, Catherine A; Dowdle, Megan E et al. (2017) Controlling the Messenger: Regulated Translation of Maternal mRNAs in Xenopus laevis Development. Adv Exp Med Biol 953:49-82
Sheets, Michael D (2015) Building the Future: Post-transcriptional Regulation of Cell Fate Decisions Prior to the Xenopus Midblastula Transition. Curr Top Dev Biol 113:233-70