Proper cellular growth and differentiation is determined by the regulated expression of the. genetic material within the cell. The control of gene expression has been studied intensively and has been demonstrated to be controlled at multiple points from transcriptional initiation to protein stability. Recently, it has become clear that the decay rates of mRNAs vary by as much as fifty-fold, are regulated and play an important role in controlling gene expression. It has been my goal to define both the cis- and trans-acting factors which regulate mRNA stability. Work from many laboratories has demonstrated the requirement for translation in the degradation of mRNAs. Understanding the role of translation in mRNA decay is a major challenge for this field. One clear example of this relationship is the observation that nonsense mutations accelerate the decay rates of mRNAs 10- to 20-fold. Our studies on the nonsense-mediated mRNA decay pathway have demonstrated that: 1) the transcript of yeast PGK1 gene is rapidly degraded by premature nonsense codons in a position-dependent manner, 2) such decay requires specific sequences downstream of the nonsense mutation (preliminary evidence suggests that translational re-initiation is important), 3) there may be a sequence element in the PGK1 transcript that, when translated, renders the PGK1 transcript resistant to the nonsense-mediated mRNA decay pathway and 4) there is at least one trans-acting factor involved in the pathway (pUPF1). The objectives of this proposal are to: A) identify the boundaries of the cis-acting sequences, including both the downstream and resistance elements, and determine their functional regions by mutagenesis; B) identify genes and mutations that i) suppress the effects of the upfl mutation and ii) are involved in inactivating the nonsense-mediated mRNA decay pathway when the resistance element is translated; and C) develop an in vitro mRNA decay system that reflects the nonsense-mediated mRNA decay pathway observed in vivo. Once an in vitro mRNA decay system is established, we will utilize this system to identify the rate limiting RNA cleavage site, investigate the biochemical mechanisms of the nonsense-mediated mRNA decay pathway and identify the role of the proteins that we will be identifying by the genetic screens described above.
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