The addition of a cap to the 5'end of all eukaryotic mRNAs is the first step in post-transcriptional processing, and its removal is generally thought to irreversibly commit mRNA to decay. In erythroid cells nonsense- containing ?-globin mRNA is cleaved by a cytoplasmic endonuclease to generate decay intermediates that are both stable and capped. Although most capping enzyme is nuclear, we identified a 140 kDa cytoplasmic capping enzyme complex that contains a 5'-monophosphate kinase capable of transforming the 5'end of decapped RNA into a diphosphate capping substrate. Although cytoplasmic capping enzyme is not associated with either P bodies or stress granules evidence for its biological role was demonstrated by the reduced recovery from stress of cells expressing a dominant negative form of this protein. The corollary to cytoplasmic capping is an uncapped transcriptome, evidence of which was recently identified by our lab in mammalian cells and by others in Arabidopsis. These mRNAs were linked to cytoplasmic capping by their increased representation in the uncapped pool following expression of a dominant negative form of capping enzyme.
Aim 1 will use biochemical approaches to identify and characterize the components of the cytoplasmic capping enzyme complex, with particular emphasis on the novel 5'-monophosphate kinase. These findings will guide development of molecular and genetic tools for characterizing the biological function of cytoplasmic capping. Experiments in Aim 2 will characterize the 5'ends of a selected number of the identified re-capping substrates and study dynamic changes in their cap status after interfering with cytoplasmic capping. The 3'ends of these RNAs will also be examined to determine if deadenylation and/or oligouridylylation lead to the accumulation of uncapped mRNAs. The last portion of Aim 2 will combine deep sequencing with the tools developed in Aim 1 to generate a comprehensive picture of the uncapped transcriptome and its relationship to cytoplasmic capping.
Aim 3 will address the biological relevance of cytoplasmic capping as it relates to the cycling of mRNAs between translating and non-translating states. These experiments will examine the impact of altering the size and number of P bodies and interfering with different steps leading to decapping and P body assembly, and examine the relationship of microRNA silencing to the accumulation of uncapped mRNAs and/or their restoration to the translating pool. Lastly, iTRAQ mass spectrometry will be used to determine if altering cytoplasmic capping changes the complexity of the proteome. Cytoplasmic capping has the potential to broadly impact our understanding of normal and disease processes that are linked to post-transcriptional control, including stem cells, embryonic development, cancer and neuroscience.

Public Health Relevance

The endpoint of most gene expression is the production of a protein product, and the regulation of mRNA translation is essential for such diverse processes as development, learning and memory, the cellular response to stress and the development and growth of cancers. Non-translating mRNAs are stored for later use or degraded, and little is known about the state of these stored mRNAs or how they are re-activated. This proposal examines cytoplasmic capping as a new regulatory process with broad implications for post- transcriptional gene regulation, RNA silencing and translational control.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics C Study Section (MGC)
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Bender, Michael T
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Ohio State University
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Kiss, Daniel L; Baez, William D; Huebner, Kay et al. (2018) Loss of fragile histidine triad (Fhit) protein expression alters the translation of cancer-associated mRNAs. BMC Res Notes 11:178
Trotman, Jackson B; Agana, Bernice A; Giltmier, Andrew J et al. (2018) RNA-binding proteins and heat-shock protein 90 are constituents of the cytoplasmic capping enzyme interactome. J Biol Chem 293:16596-16607
Trotman, Jackson B; Schoenberg, Daniel R (2018) RNA Cap Methyltransferase Activity Assay. Bio Protoc 8:
Kiss, Daniel L; Baez, William; Huebner, Kay et al. (2017) Impact of FHIT loss on the translation of cancer-associated mRNAs. Mol Cancer 16:179
Kiss, Daniel L; Waters, Catherine E; Ouda, Iman M et al. (2017) Identification of Fhit as a post-transcriptional effector of Thymidine Kinase 1 expression. Biochim Biophys Acta Gene Regul Mech 1860:374-382
Trotman, Jackson B; Giltmier, Andrew J; Mukherjee, Chandrama et al. (2017) RNA guanine-7 methyltransferase catalyzes the methylation of cytoplasmically recapped RNAs. Nucleic Acids Res 45:10726-10739
Gu, Shan-Qing; Gallego-Perez, Daniel; McClory, Sean P et al. (2016) The human PMR1 endonuclease stimulates cell motility by down regulating miR-200 family microRNAs. Nucleic Acids Res 44:5811-9
Kiss, Daniel L; Oman, Kenji M; Dougherty, Julie A et al. (2016) Cap homeostasis is independent of poly(A) tail length. Nucleic Acids Res 44:304-14
Kiss, Daniel L; Oman, Kenji; Bundschuh, Ralf et al. (2015) Uncapped 5' ends of mRNAs targeted by cytoplasmic capping map to the vicinity of downstream CAGE tags. FEBS Lett 589:279-84
Mukherjee, Chandrama; Bakthavachalu, Baskar; Schoenberg, Daniel R (2014) The cytoplasmic capping complex assembles on adapter protein nck1 bound to the proline-rich C-terminus of Mammalian capping enzyme. PLoS Biol 12:e1001933

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