The destruction of mRNA is a key event in eukaryotic gene expression, playing a crucial role in early animal development, cellular growth , proliferation and adaptation to stress. Dedicated mRNA stability pathways ensure that aberrant transcripts containing premature stop codons are eliminated and the abundance of key transcripts such as those coding for cytokines, interleukins and proto-oncogenes are tightly controlled. A critical, regulated step in these pathways is the removal of the 5'terminal cap structure by Dcp2, which sentences an mRNA for destruction by exposing the 5'monophosphate of the mRNA body to 5'to 3' exoribonucleases. The activity of Dcp2 is stimulated by a variety of pathway specific co-activators through mechanisms that are not well understood. Using budding yeast as a model system, we will combine biochemical , biophysical and genetic methods to determine how the essential activator Dcp1 regulates the catalytic activity of Dcp2.
In specific aim 1, crystallographic studies of the Dcp1/Dcp2 complex with non- hydrolyzable substrate will guide kinetic and genetic analyses of mutants to dissect the chemical step of decapping;
in specific aim 2, we will use NMR spectroscopy to determine if Dcp1 enhances the catalytic activity of Dcp2 by shifting a conformational equilibrium between inactive and active forms;
in specific aim 3, we will determine the crystal structure of the ternary complex between Dcp1/Dcp2 with the Enhancer of decapping protein family member , Edc1, to define the mechanism of stimulation by co-activators. These integrated studies will shed light on a critical step in several mRNA stability pathways that affects the abundance of thousands of human genes.

Public Health Relevance

The expression of thousands of human genes is regulated by the coordinated destruction of messenger RNA (mRNA). Mutations in protein factors that control this process are observed in human diseases, including several cancers and genetic disorders. We seek to understand molecular mechanisms controlling mRNA decapping: the penultimate, irreversible step committing an mRNA to destruction. The biochemical and structural details provided by these studies may pave the way to treat inherited genetic disorders and cancer.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function B Study Section (MSFB)
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Preusch, Peter C
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University of California San Francisco
Schools of Pharmacy
San Francisco
United States
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Mugridge, Jeffrey S; Tibble, Ryan W; Ziemniak, Marcin et al. (2018) Structure of the activated Edc1-Dcp1-Dcp2-Edc3 mRNA decapping complex with substrate analog poised for catalysis. Nat Commun 9:1152
Paquette, David R; Mugridge, Jeffrey S; Weinberg, David E et al. (2018) Application of a Schizosaccharomyces pombe Edc1-fused Dcp1-Dcp2 decapping enzyme for transcription start site mapping. RNA 24:251-257
Paquette, David R; Tibble, Ryan W; Daifuku, Tristan S et al. (2018) Control of mRNA decapping by autoinhibition. Nucleic Acids Res 46:6318-6329
Ziemniak, Marcin; Mugridge, Jeffrey S; Kowalska, Joanna et al. (2016) Two-headed tetraphosphate cap analogs are inhibitors of the Dcp1/2 RNA decapping complex. RNA 22:518-29
Mugridge, Jeffrey S; Ziemniak, Marcin; Jemielity, Jacek et al. (2016) Structural basis of mRNA-cap recognition by Dcp1-Dcp2. Nat Struct Mol Biol 23:987-994
Aglietti, Robin A; Floor, Stephen N; McClendon, Chris L et al. (2013) Active site conformational dynamics are coupled to catalysis in the mRNA decapping enzyme Dcp2. Structure 21:1571-80
Mugridge, Jeffrey S; Gross, John D (2013) Judge, jury, and executioner: DXO functions as a decapping enzyme and exoribonuclease in pre-mRNA quality control. Mol Cell 50:2-4
Fraser, James S; Gross, John D; Krogan, Nevan J (2013) From systems to structure: bridging networks and mechanism. Mol Cell 49:222-31
Floor, Stephen N; Borja, Mark S; Gross, John D (2012) Interdomain dynamics and coactivation of the mRNA decapping enzyme Dcp2 are mediated by a gatekeeper tryptophan. Proc Natl Acad Sci U S A 109:2872-7
Borja, Mark S; Piotukh, Kirill; Freund, Christian et al. (2011) Dcp1 links coactivators of mRNA decapping to Dcp2 by proline recognition. RNA 17:278-90

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