mRNA decay plays a central role in gene expression, with the half-life of virtually every mRNA controlled by regulatory sequences within the mRNA and their cognate binding proteins. Rapid turnover is a characteristic feature of mRNAs encoding growth factors, transcription factors, cytokines and cell signaling molecules, and the selective modulation of this process is one way of controlling the amount of these proteins. The decay of most mRNAs begins with shortening of the poly(A) tail, removal of the 5'cap and simultaneous 5'-3'and 3'-5'degradation of the mRNA body. With the exception of poly(A) shortening these processes act on nontranslat- ing mRNAs. However, the turnover of a subset of the transcriptome is catalyzed by endonuclease cleavage while mRNAs are engaged by translating ribosomes. The prototypical mRNA endonuclease is PMR1, an en- zyme that was originally identified as an estrogen-induced ribonuclease activity whose appearance on polysomes coincides with the destabilization of serum protein mRNAs. The hallmark of endonuclease- mediated mRNA decay is its selectivity for specific mRNAs. This is determined by the formation of an mRNP complex (termed Complex I) containing PMR1 and its translating substrate mRNA. To join this complex PMR1 must be phosphorylated on a tyrosine residue in the polysome-targeting domain of the protein, and the past funding cycle identified c-Src as the kinase that is responsible for this key activation step. This is the first example of direct involvement of an oncogenic tyrosine kinase in mRNA decay, and it raises the possibility that PMR1-mediated mRNA decay may be a target of c-Src in cancer. Consistent with this, PMR1 binds to the Ena/VASP proteins, which are regulators of the actin cytoskeleton, and cell motility is increased in cells ex- pressing catalytically-active PMR1.
Aim 1 will use tandem affinity chromatography to recover the Complex I mRNP, identify its constituent proteins, and determine their role in mRNP assembly and mRNA decay. This is the first step toward deciphering the 'RNP code'for PMR1-mRNA decay. The SH2 domain containing protein that is the 'gatekeeper'for recruiting PMR1 to the mRNP will be of particular interest, since none of these has known RNA-binding activity.
Aim 2 continues work begun in the last cycle using microarrays to identify PMR1 target mRNAs by their recovery with Complex I. These will be compared to mRNAs that are selectively reduced by increasing expression of PMR1 and selectively increased by its knockdown. These will also be used to identify shared sequence or structural features that together with proteins in Aim 1 define the substrate mRNP. Cell motility is increased in cells expressing active PMR1, and the experiments in Aim 3 will use imag- ing of cell movement, quantitative PCR and immunofluorescence to examine the relationship between motility, PMR1 binding to the Ena/VASP proteins, and its recruitment to Complex I. The long-term goal of this work is to understand the molecular mechanisms of PMR1-mediated mRNA decay, how it is regulated and its role in con- trolling gene expression during development, in response to hormonal stimuli, and in malignancy.
mRNA decay is a key step in gene regulation. PMR1 is an mRNA endonuclease that is activated by the oncogenic tyrosine kinase c-Src to degrade a distinct subset of mRNAs. PMR1-mediated mRNA decay is also associated with increased cell motility, raising the possibility of a link between this form of mRNA decay, the c- Src protooncogene and cancer. This research seeks to identify the components of the PMR1 decay complex, identify the scope of PMR1-mediated decay and relate both of these to the invasive growth of cancer cells.
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