Alan B. Sachs
RNA RegulationThe level of an mRNA's expression is determined by both its degradation and translation initiation rates. We are interested in understanding each of these important post-transcriptional control mechanisms through genetic and biochemical experiments in the yeast Saccharomyces cerevisiae. Our immediate goal is to elucidate the role of the poly(A) tail in these processes. Our laboratory is also developing new technology aimed at increasing the rate of DNA polymorphism mapping in the human genome.
In vivo, poly(A) tails function as a complex with the poly(A)-binding protein (Pab1p). As a result, mutations disrupting Pab1p function should be equivalent to those disrupting poly(A) tail function. We have previously shown that Pab1p is essential for yeast cell viability. Loss of function mutations in Pab1p lead to a block in translation initiation. Our recent studies with a yeast in vitro translation system indicate Pab1p is required for 40S ribosomal subunit binding to mRNA. Current experiments are aimed at understanding how Pab1p bound at the 3' end of the mRNA is stimulating the binding of the ribosomal subunit to the 5' end of the mRNA.
One of the mutations that suppress a deletion of the PAB1 gene lies within the Spb8 protein. Mutations within this protein have two phenotypes that distinguish it from other Pab1p bypass suppressors. First, Spb8p mutations do not lead to alterations in the ratio of large to small ribosomal subunits in the cell. This suggests that the protein may be alleviating the requirement for Pab1p in translation through a different mechanism than most of the other suppressors. Secondly, Spb8p mutations lead to the appearance of aberrant mRNA degradation intermediates. This indicates that mutations in this protein can simultaneously effect the mRNA degradation machinery. A further biochemical and genetic analysis of Spb8p should reveal an explanation for these observations.
Pab1p is also required for the induction of poly(A) tail degradation. Cells containing a loss-of-function mutation in Pab1p have very stable poly(A) tails that do not undergo the Pab1p-dependent cytoplasmic shortening reaction. We have purified to homogeneity the enzyme responsible for Pab1p-dependent poly(A) tail degradation (PAN=poly(A) nuclease) based on its ability to degrade poly(A) only in the presence of Pab1p. The genes encoding the two predominant proteins in the purified preparation have been cloned, and we anticipate that a detailed analysis of them will provide information about the role of poly(A) tail degradation in cytoplasmic mRNA metabolism. Through this analysis we also hope to understand more fully the role of the PAN1 protein, which also co-purifies with the nuclease activity in our preparations but does not appear to be required for enzyme activity.
We are also pursuing a detailed biochemical and genetic characterization of Pab1p. By studying an extensive number of site specific mutations within the protein, we hope to understand which parts of the protein are needed to bind mRNA, to stimulate translation initiation, and to stimulate the poly(A) nuclease. By creating a large set of yeast mutants that either bypass suppress the requirement for Pab1p for cell viability, or that show synthetic lethality with mutated forms of Pab1p, we hope to identify interacting proteins and targets of Pab1p. This biochemical and genetic analysis will provide important information about how a highly conserved family of RNA binding proteins, for which Pab1p is the founding member, can simultaneously interact with RNA and other proteins.
In an unrelated project, our lab is developing technology that will allow for the simultaneous screening of a large number of polymorphic sites within human DNA on a large number of people. Towards this goal we are evaluating the feasibility of creating a two allele polymorphic map of the genome. The advantages of this type of map over the popular simple sequence length polymorphism map is it allows for plus/minus scoring of the polymorphic markers. This type of scoring system will ultimately allow for the development of completely automated genotyping of human DNA.