van Hoof Lab
Gene expression is a complex process that all life forms need to carry out in a precisely controlled fashion. The degradation of mRNA is one of the last steps in this process and serves important roles in this process. For example degradation rates of individual mRNAs can be regulated and affect mRNA abundance, and thus how much of each protein is produced by translation. mRNA decay also plays an important role in maintaining the overall fidelity of gene expression by preferentially degrading aberrant mRNAs that are made by mistakes during mRNA processing reactions.
One example of aberrant mRNAs are those that lack a stop codon. We concentrate many of our studies on these mRNAs because they are extremely rapidly degraded, but they are degraded by the same enzymes that degrade other mRNAs. We hope that if we understand nonstop mRNA decay in detail, that this will also help us understand how normal mRNAs are degraded.
We have proposed a model for the degradation of nonstop mRNAs (van Hoof et al., 2002). In this model mRNAs are recognized because a ribosome is stalled at the very 3′ end of an mRNA. In in vitro translation extracts such stalled ribosome-mRNA complexes are stable. Ski7 probably functions to recognize such a stalled ribosome, and to recruit the exosome. This recruitment results in degradation of the mRNA by the exosome. Much of the research in the van Hoof lab is aimed at testing this model, using yeast genetics, molecular biology and biochemistry tools.
Although in most of our experiments we use artificial reporter genes that make nonstop mRNAs, there are also endogenous sources of nonstop mRNAs. One source of nonstop mRNAs is premature cleavage and poly-adenylation (van Hoof et al., 2002). Most of the time, eukaryotic mRNAs are poly-adenylated somewhere 3′ of the stop codon, but an estimated 10% of the time the poly-adenylation machinery makes a mistake, and puts on the poly(A) tail somewhere in the coding region. For some exceptional genes, such as CBP1 and RNA14, up to about half of the mRNA may be prematurely polyadenylated. Another way to generate a nonstop mRNA is by endonucleotylic cleavage within the mRNA.
For the usual reasons we use yeast to understand the metabolism of nonstop mRNAs. However, nonstop mRNAs are also made in human cells and nonstop mRNA decay may contribute to human disease. For example, in most humans there is only one stop codon in the APRT (adenine phosphoribosyltransferase) gene. In patients where this stop codon is mutated to any other codon, the APRT mRNA is very rapidly degraded and less APRT protein is made (Taniguchi, 1998). This can cause kidney failure. A similar condition has been found for the GPR54 (G protein-coupled receptor 54) gene. If the only stop codon in this gene is mutated, the mRNA is rapidly degraded, and the encoded protein is not made. As a result, these patients fail to develop normally during puberty and are sterile. Interestingly, If the nonstop version of the GPR54 gene is overexpressed, the encoded protein is partially functional (Seminara, 2003). This suggests that these patients might benefit from therapies that inhibit nonstop mRNA decay.