CSCE 496/896 Project Idea: mRNA Degradation in Yeast

Due date for project write-ups: Dec. 11

First of all, the yeast genome has about 6,400 genes. The genes are of course transcribed into mRNA (and processed by the addition of a 5' cap and a 3' polyAAA tail) and those mRNA are of course translated into proteins. When the mRNA are through being translated, they need to be degraded. Normally, this happens by the mRNA being somehow recognized by the translation machinery and targeted for degradation. First, the PolyAAA tail is removed and then the cap is removed by DCP1. Then the mRNA is degraded 5' to 3' extremely quickly by XRN1, which is an exoribonuclease. However, there is an alternate decay pathway that focuses on abbarent mRNA. These mRNA are ``born'' with a premature stop codon upstream of the normal stop codon. These errors (premature stop codons) can result from errors in the DNA, errors in transcription or errors in splicing. This pathway that recognizes these mRNA and degrades them is called ``nonsense-mediated mRNA decay'' (NMD). There are 3 proteins in this pathway that work together to recognize error-containing mRNA and degrade them using the usual proteins (XRN1 and DCP1). These proteins are known as UPF1, UPF2 and UPF3. If any one of these is removed, the pathway doesn't work and anything that it was supposed to degrade just builds up in the cytoplasm of the cell. Recently, there has been a discovery made by Lelivelt, et al., 1998 (http://144.92.19.47/default.htm) that there are also normal/wildtype mRNA that are degraded by this pathway also. Since we, as a field, previously thought that NMD was a pathway dedicated to getting errors out of the cell, this came as a considerable shock. Lelivelt discovered using an AFFY metrix screen, that approximately 350 of the 6,400 yeast mRNA are degraded by this pathway. One of the normal/wildtype mRNAs that is degraded by this pathway is called PPR1. PPR1 is a transcriptional activator that regulates many of the URA genes (Uracil biosynthetic pathway) in yeast. Since PPR1 regulates many genes, if NMD ceases to function, anything that PPR1 regulates will also build up in the cell. These types of mRNA's are called ``indirect'' targets. Thus, some mRNA are actively sought out by the pathway to be degraded and others are only effected because their regulators are targets of NMD.

PROJECT IDEAS:

1. One idea for a project could be to find what sequence PPR1 protein binds to in DNA (the entire yeast genome is sequenced and readily available on the web) (http://genome-www.stanford.edu/Saccharomyces) and see how many genes can be found that have that binding site. This could be accomplished by downloading the 5' UTRs of all of the yeast genes off of the Cold Spring Harbor Promoter database (http://cgsigma.cshl.org/jian/) and running the PPR1 consensus sequence against all of them. Then, of the genes that could be found, those could be checked in the Lelivelt Database to see if any are elevated in an NMD-background.

2. ATS1 is a suppressor of Beta-tubulin mutations in yeast. It could possibly be a GTP exchange factor based on work that has been done in the Atkin Lab at the University of Nebraska-Lincoln. No one currently knows what transcriptional factor(s) regulates ATS1. It would be useful to know this so that the pathway in which it belongs could be further characterized. The 5' UTR of ATS1 is available on The Saccharomyces Genome Database. (http://genome-www.stanford.edu/Saccharomyces) It could be scanned for which transcriptional activator binding sites it has using the known data from literature. Also, the recognition sequences of all of the characterized transcriptional activators are on the Cold Spring Harbor Promoter Database. (http://cgsigma.cshl.org/jian/)

3. There is a theory that ATS1 is a GTP exchange factor. Another project is to define the domains of ATS1. This could be done by running the protein sequence of ATS1 against known GTP exchange factors and seeing if any common areas can be found. Also, it works (theoretically) in the rearrangement of microtubules and with the cell cycle morphogenesis checkpoint. It could also be run against known proteins that work in these areas to see if homologies can be found. Since this protein is found in yeast, it will be easier to examine than if it were in another organism since much of yeast cell structure and function is already known and posted on the web.