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Biochemistry Department



Gallie Research Group


Welcome to the Gallie Research Group

Research

Mechanisms regulating the translation of cellular and viral mRNAs: Since the elucidation of the basic translation process in the 1950's and 1960's, translation was thought to occur in a linear fashion. Not until the late 1980's was genetic evidence from yeast studies presented suggesting that the poly(A) tail at the 3' terminus of messenger RNAs may be required for translation initiation which occurs at the 5' terminus. The model presented at that time invoked an interaction between the protein that binds to the poly(A) tail, i.e., the poly(A) binding protein (PABP), with the 60S ribosomal subunit. We demonstrated that the poly(A) tail was functionally co-dependent on the 5' cap structure, to enhance translation in plants, animals, and yeast, which suggested that PABP interacted with a protein bound to the 5' cap structure, not with the 60S ribosomal subunit. As a first step to investigate an interaction between PABP and other components of the translational machinery, we cloned the PABP gene and showed that it functionally complements a yeast pab null mutant and that PABP does indeed interact with the cap-binding components of the translation machinery, i.e., the translation initiation factors 4F and 4B. One functional consequence of this interaction is to increase the binding affinity of PABP for the poly(A) tail. The interaction between PABP and factors bound to the 5' cap structure has been verified by other labs and is now the dominant model explaining the interaction between the termini of an mRNA. The interaction between the termini of the mRNA may act as a selection mechanism such that only full-length mRNAs are recruited for translation. The interaction may also be important to optimize the re-initiation of ribosome onto the mRNA. This may also prove to be an optimization of a process that might have occurred prior to the evolution of poly(A) tails. Related to this, we have shown that the efficiency of translation initiation increases with the length of the 3' untranslated region, data suggesting that a longer 3' untranslated region may increase the likelihood that a terminating ribosome may be re-recruited at the 5' end of the message by maintaining the association of the ribosome with that particular mRNA for a longer period of time. Although most mRNAs have a 5' cap structure and a poly(A) tail, there are some exceptions. Some viral mRNAs, such as tobacco etch virus (TEV) are not capped and other such as tobacco mosaic virus (TMV) terminate in a tRNA-like structure instead of a poly(A) tail. The cell-cycle regulated histone mRNAs in animals and in lower plants terminate in a stem-loop structure instead of a poly(A) tail. These exceptions pose the interesting question of how they achieve efficient translation in the absence of a cap or a poly(A) tail when both are necessary to promote efficient translation initiation. We have shown that the 3'-untranslated region of TMV is functionally equivalent to a poly(A) tail in vivo and in vitro. We demonstrated that a single RNA pseudoknot structure contained within the TMV 3'-untranslated region was responsible for the translational regulation and that this RNA pseudoknot structure was conserved in many virus related to TMV. The 3'-untranslated region of other viruses that do not terminate in a poly(A) tail also enhanced translation, data suggesting that this may be a common theme in many plant RNA viruses. We have shown that the 3' terminal stem-loop structure from cell-cycle regulated histone mRNAs functioned to increase the efficiency of translation initiation in animal cells. Interestingly, it did not function to enhance translation in higher plants which do not have histone mRNAs that terminate in a stem-loop structure. Importantly, whether it be the RNA pseudoknot structure within the 3'-untranslated region of TMV or the 3' terminal stem-loop structure from cell-cycle regulated histone mRNAs, these functional alternatives to a poly(A) tail remained co-dependent on the 5' cap structure for their function as enhancers of translation. Likewise, the 5' leader sequence from the naturally uncapped plant virus, TEV, which promotes translation in a cap-independent manner, was functionally dependent on the presence of the poly(A) tail. We can conclude from these studies that when an alternative to a 5' cap or a poly(A) tail has evolved in an mRNA, a requirement for an interaction between the terminal regulatory elements of the mRNA appears to be a common prerequisite for optimizing translation. In addition to the translational regulation associated with the 3'-untranslated region of TMV, we have shown n a series of papers over the last decade that the TMV 5' leader sequence is a translational enhancer. This was one of the first and most thoroughly investigated examples translational enhancers of which many examples have subsequently been identified. We have continued this exciting research into the mechanism by which the TMV 5' leader sequence functions to enhance translation. We have shown that there is a specific a 102 kDa protein that binds to the TMV 5' leader sequence. We demonstrated that its binding site within the TMV 5' leader sequence mapped to the same subsequence responsible for the translational enhancement associated with the leader. Sequence analysis of the 102 kDa protein, revealed homology to the HSP101/HSP104/ClpB family of heat shock proteins and its expression in yeast complemented a thermotolerance defect caused by a deletion of the HSP104 gene. Up to a 50-fold increase in the translation of W-luc but not luc mRNA was observed in yeast expressing the tobacco HSP101 whereas W failed to enhance translation in the absence of HSP101. Therefore, HSP101 and W comprise a two-component translational regulatory mechanism that can be recapitulated in yeast. Analysis of HSP101 function in yeast translation mutants suggested that the initiation factor (eIF) 3 and one of the two eIF4G proteins were required for the HSP101-mediated enhancement. The RNA-binding and translational regulatory activities of HSP101 were inactive in respiring cells or in cells subject to nutrient limitation but its thermotolerance function remained unaffected. This is the first identification of a protein required for specific translational enhancement of capped mRNAs, the first report of a translational regulatory function for any heat shock protein, and the first functional distinction between the two eIF4G proteins present in eukaryotes. We have also been interested in how the activity of the translational machinery is regulated and have shown that the activity of the 5' cap structure and its functional interaction with the poly(A) tail is regulated in animal cells by insulin and serum starvation. We also showed that the phosphorylation state of several of the translational initiation factors in wheat are developmentally regulated. Analyzing the impact of heat shock on plant gene expression Thermal stress is an environmental factor that many plants must contend with on a daily basis during the summer. Heat shock has profound effects on protein synthesis and affects transcriptional activity as well as translation. I have had a long term interest in how heat shock impacts the translational process which is an area that has received little attention to date. We previously demonstrated that the leader sequence of an mRNA can play an important role in overcoming the translation repression that normally occurs following a heat shock. Following a heat shock, the communication that we had shown between the 5' cap structure and the poly(A) tail, was disrupted, primarily through the loss in the function of the cap. We demonstrated that heat shock causes dramatic changes in the phosphorylation status of two translation initiation factors, i.e., eIF4B and eIF4A, both of which are associated with the 5' cap structure and that recovery from heat shock was greatly affected when the expression of the heat shock proteins was blocked. In addition to the heat-shock-mediated repression of translation, we observed that the stability of mRNAs increases following a heat shock and correlates with the severity of the stress. We observed this in plants but not in animals, suggesting an important difference in the way that plants respond to heat stress compared to animals. The increase in mRNA stability could be a result of a heat-mediated repression in the mRNA-degradatory machinery and we have observed that the activity of all detectable RNase activities decreased following a heat shock and correlated with the severity of the stress. Plant transformation technology For many years, I have developed new technologies for transforming cells. This began with the first demonstration a decade ago of RNA delivery using electroporation for transient expression studies in plants which was extended to animal cells. Since that time, we have developed procedures for RNA delivery into yeast and have developed transformation procedures for delivering DNA or RNA to aleurone cells of rice, and maize, as well as the transformation of maize endosperm cells. Signaling involved in plant apoptosis I have a long standing interest in seed quality. This lead to the development of the transformation technologies mentioned above. As one outcome from our studies with transformation of developing maize endosperm cells, we had observed that viable cells could be obtained from maize endosperm only up to a certain developmental stage and that following this, the cells had undergone a developmental program of cell death. We observed that cell death initiated during mid-development specifically within the upper central endosperm that then expands outward and downward to engulf the entire. A burst of ethylene synthesis was observed prior to the onset of pcd whereas the application of an inhibitor of ethylene biosynthesis delays the onset of cell death. Moreover, mutations affecting starch biosynthesis such as shrunken1 (sh1) and shrunken2 (sh2) exhibited a premature onset and an accelerated execution of the pcd program during endosperm development which correlated with a significantly higher level of ethylene production in mutant kernals. Maize endosperm DNA underwent internucleosomal cleavage concomitant with the progression of cell death whereas embryo DNA remained intact. Alterations in the onset and progression of maize endosperm DNA degradation by biochemical or genetic manipulation of ethylene levels correlated with corresponding changes in the progression of cell death, suggesting that nuclease activation and cell death may be ethylene-regulated events. We also observed that wheat endosperm undergoes a programmed cell death during its development that shares features with the maize program but differs in some aspects of its execution. Cell death initiated and progressed stochastically in wheat endosperm in contrast to maize where cell death initiates within the upper central endosperm and expands outward. Following a peak of ethylene production during early development, wheat endosperm DNA underwent internucleosomal fragmentation that was detectable from mid to late development. The developmental onset and progression of DNA degradation was regulated by the level of ethylene production or perception. These observations suggest that programmed cell death of the endosperm and regulation of this program by ethylene is not unique to maize but that differences in the execution of the program appear to exist among cereals. We are also examining whether the changes in the phosphorylation status of the translational machinery that we have observed may be an important component of the programmed cell death program.



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