Stuart Peltz, Ph.D.

Stuart Peltz, Ph.D.

Professor
Office: 732-235-4790
Lab: 732-235-5447
peltzumdnj.edu

Office:
RWJMS 810
Lab:
RWJMS 813

Proper cellular growth and differentiation is determined by the regulated expression of the genetic material within the cell. Recently, it has become clear that the decay rates of mRNAs are regulated and play an important role in controlling gene expression. It has been my goal to define both cis- and trans-acting factors which regulate mRNA stability and I have chosen to do this in a system capable of high resolution biochemical and genetic analysis, i.e., the yeast Saccharomyces cerevisiae.

Recently, it has become clear that the processes of translation and mRNA decay are intimately linked. Results suggesting this relationship include; a) inhibition of translational elongation can reduce mRNA decay rates; b) ribosome translocation up to or through the a previously identified instability element in the MAT[alpha]1 gene in the yeast Saccharomyces cerevisiae is required for its rapid decay; c) instability elements involved in the rapid decay of a variety of yeast and mammalian mRNAs have been localized to the coding regions of the respective mRNAs; d) instability elements may be rich in rare codons; e) experiments with mammalian cell-free systems indicate that a nuclease activity capable of degrading mammalian mRNAs is associated with polysomes; and e) premature translational termination can enhance mRNA decay rates.

In order to understand how translation and mRNA decay are related, we have been focusing on trying to understand how prematurely terminating translation promotes rapid decay of an mRNA, a process we have called nonsense-mediated mRNA decay. Nonsense mutations can accelerate the decay rate of an mRNA 10- to 20-fold. Our studies show that, in yeast, rapid mRNA decay induced by premature nonsense codons is position-dependent, requires mRNA sequences downstream of the nonsense mutation and is dependent on at multiple trans-acting factors that have been defined by mutational analysis. The objectives of our work are to understand: a) the role of the cis-acting sequences involved in nonsense-mediated mRNA decay and b) to identify the trans-acting factors involved in this decay pathway.

Studies and Results

Cis-acting Elements Involved in Regulating Nonsense-mediated mRNA Decay. The Nonsense Mediated mRNA Decay (NMD) pathway functions by checking whether translation termination has occurred prematurely and subsequently degrading the aberrant mRNAs. In the yeast Saccharomyces cerevisiae it has been proposed that a complex scans 3' of the premature termination codon and searches for a specific sequence called the downstream element (DSE). Recognition of the DSE by the surveillance complex identifies the transcript as aberrant and promotes its rapid decay. Our results establish key events that govern and modulate the activity of the surveillance complex. The results suggest that translation termination is an important event for assembly of the surveillance complex. Neither the activity of the initiation ternary complex after premature translation termination has occurred, nor the elongation phase of translation, are essential for the activity of the NMD pathway. Once assembled, the surveillance complex is active for searching and recognizing a DSE for approximately 200 nts 3' of the stop codon. Importantly, we have also identified a stabilizer sequence (STE) in the GCN4 leader region that inactivates the NMD pathway. Inactivation of the NMD pathway, as a consequence of either the DSE being too far from a stop codon or the presence of the stabilizer element, can be circumvented by inserting sequences containing a new translation initiation/termination cycle immediately 5' of the DSE. Further, the results indicate that the stabilizing elements functions in the context of the GCN4 transcript to inactivate the NMD pathway.

Multiple mof Alleles Affect NMD and Programmed Frameshifting. Recent results have shown that strains harboring the mof2-1, mof4-1, mof5-1 and mof8-1 alleles, previously demonstrated to increase the efficiency of programmed -1 ribosomal frameshifting, decrease the activity of the NMD pathway. The effect of the mof2-1 allele on NMD demonstrated that the wild-type MOF2 gene is identical to the SUI1 gene. Studies on the mof2-1 allele of the SUI1 gene indicate that in addition to its role in recognition of the AUG codon during translation initiation and maintenance of the appropriate reading frame during translation elongation, the Mof2 protein plays a role in the NMD pathway. The Mof2p/Sui1p is conserved throughout nature and the human homologue of the Mof2p/Sui1p functions in yeast cells to activate NMD. These results suggest that factors involved in NMD are general modulators that act in several aspects of translation and mRNA turnover. These results strongly support the idea that translation and mRNA turnover are intimately related processes.

The Yeast HRP1 Gene Binds to the DSE and Interacts with the Upf1 Protein. The results from studies on the sequence requirements for the degradation of nonsense-containing transcripts demonstrated that recognition of the DSE is a critical event in the NMD pathway. We have recently identified the Hrp1 protein as a factor that specifically interacts with a DSE in the PGK1 transcript. The HRP1 gene is an essential gene that is homologous to previously identified mammalian RNA binding proteins (hnRNPA1 and hnRNPD) and contains two RNA recognition motifs. The Hrp1 protein has been shown to shuttle between the nucleus and the cytoplasm. Mutations in HRP1 gene specifically stabilize nonsense-containing mRNAs in vivo. In addition, we have recently found that Hrp1p interacts with the Upf1p. Taken together, these results indicate that the RNA binding protein Hrp1 may be involved in NMD by interacting with the DSE and signaling to the surveillance complex, through at least Upf1p, that an aberrant termination event has occurred.

Characterization of the Gene and Gene Products that Regulate Decapping Activities. Through the efforts of a number of laboratories it is now clear, at least for a subset of mRNAs in mammalian and yeast cells, that poly(A) shortening and/or hydrolysis of the 5' cap structures are important rate determining events in controlling the stability of a given transcript. Based on these results, the goal of one of our projects is the identification and characterization of the genes and gene products that regulate poly(A) shortening and decapping activities in the yeast. Our strategy has been to develop biochemical screens to monitor nuclease activities in cell extracts and then to screen a collection of temperature-sensitive strains for those strains that demonstrate reduced nuclease activities. In addition to this approach, we have also used genetic screens to identify mutations that affect decapping activities. Having isolated the genes, we will then embark on the molecular, biochemical and genetic characterization of the genes and their products. Our task will be to sort out which gene products are part of the decapping or poly(A) shortening enzyme/complex or are direct regulators of these activities versus mutations that indirectly affect them. Furthermore, the mutants that demonstrated decreased decapping or poly(A) shortening activities in cell extracts and increased cellular mRNA abundance phenotypes will be characterized in vivo to precisely determine which step(s) in the decay pathway they affect. The mutants will be characterized to genetically define the complexes involved in the degradation of mRNA, to investigate the interactions between other factors involved in mRNA decay, to determine the order in which the genes function, and investigate how the 5' and 3' ends of the transcripts interact to regulate mRNA decay. The insights gained from these experiments will be evaluated further by molecular and biochemical approaches.

Using genetic, molecular and biochemical approaches, we have identified factors that modulate the decapping activity in yeast cells. A biochemical screen identified two strains that had mutations in the VPS16 gene that led to a reduction of decapping activity in yeast extracts and stabilization of both wild-type and nonsense-containing transcripts. Strains harboring deletions of the VPS16 gene are viable, demonstrate a ts growth phenotype, reduced decapping activity in cell extracts and stabilization of both wild-type and nonsense-containing mRNAs. Further biochemical and genetic analysis demonstrated that the Vps16 protein modulates the activity of the heat shock protein Hsp70. Purification of this factor demonstrated that binding to purified Dcp1 protein inhibited its decapping activity. We are currently investigating the molecular details of how this protein functions to modulate decapping activity in yeast cells.

Development of a Novel Set of Antiviral Agents to Treat AIDS. The effects of two peptidyl-transferase inhibitors, anisomycin and sparsomycin, on ribosomal frameshifting efficiencies and the propagation of yeast double-stranded RNA (dsRNA) viruses were examined. At sub-lethal doses in yeast cells these drugs specifically alter the efficiency of -1, but not of +1, ribosomal frameshifting. These compounds promote loss of the yeast L-A dsRNA virus which utilizes a programmed -1 ribosomal frameshift to produce its Gag-pol fusion protein. Both of these drugs also change the efficiency of -1 ribosomal frameshifting in yeast and mammalian in vitro translation systems, suggesting that they may have applications to control the propagation of viruses of higher eukaryotes which also utilize this translational regulatory mechanism. Our results offer a new set of antiviral agents which may potentially have a broad range of applications in the clinical, veterinary and agricultural fields.

In collaboration with the Dougherty laboratory, we have utilized a HIV colony assay to monitor the effects of sparsomycin on HIV propagation. A preliminary assay to monitor the effect of sparsomycin on cellular growth rate demonstrated that 400 ng/ml began to inhibit cell growth. Therefore, dosages below this amount were used to monitor the effect of sparsomycin on HIV propagation. Remarkably, the results demonstrated that a sparsomycin concentration of 120 ng/ml reduced the HIV titers from 10,000 I.U/ml to 30. These results clearly demonstrate that at concentrations of sparsomycin that do not affect cellular growth rate, there is a dramatic reduction of the number of HIV particles produced. Consistent with the sparsomycin reducing viral titers, recent results also indicate that the frameshifting efficiency is increased three-fold in the presence of these compounds.