Coordination of 3' mRNA Processing with Export to the Cytoplasm

Dartmouth College, Hanover NH

Investigators

Abstract

Charles Cole MCB 9983378 The production of proteins within complex (eukaryotic) cells requires the synthesis and processing of mRNAs in the cell nucleus followed by their transport to the cytoplasm where protein synthesis takes place. Gene expression can be regulated at most or all of these steps. Evidence is accumulating that RNA synthesis (transcription) and processing are coupled. Preliminary evidence suggests that export of the mRNA from the nucleus is also coupled to other steps in mRNA formation. This research project will determine the mechanism of this coupling. The model to be examined is that one or more 3' processing factors remain associated with the mRNA following cleavage and polyadenylation, and then mediate important interactions between the mRNP and the mRNA export machinery. These processing factors are proposed to exit the nucleus with the mRNP and to shuttle back to the nucleus following their removal from the mRNP. Experiments will be performed using budding yeast, Saccharomyces cerevisiae. Preliminary data indicates that mRNAs accumulate in the nucleus when the signals for 3' processing are mutated or when mutant 3' processing factors are produced. In addition, several genetic screens have linked 3' processing factors to mRNA export. When 3' processing is aberrant, mRNAs are produced with extended 3' untranslated regions and a poly(A) tail. At least one 3' processing factor shuttles between nucleus and cytoplasm. These data are consistent with the idea that mRNA exportability requires formation of correct 3' ends through the normal mechanism. To test this possibility, the effect of forming an mRNA 3' end via a cis-acting, hammerhead ribozyme will be examined. Other studies will determine which 3' processing factors shuttle and whether they exit the nucleus with mRNA. The poly(A) tails on the extended mRNAs will be analyzed for sequences able to mediate nuclear retention of the RNA. Fusions of 3' processing factors to the RNA binding domain of Rev (or MS2 coat protein) will be used to test the ability of processing factors tethered to the mRNA to participate in mRNA export. Additional mutant alleles will be prepared and characterized in an attempt to separate the export and 3' processing functions of specific factors. Other preliminary studies suggest that export factors may participate in 3' processing. Using in vitro extracts, a consistent 2-3 fold stimulation of cleavage and polyadenylation was seen when GST-Xpo1p was added to the extract. This increased the processing efficiency from 25-30% to ~75%. Several in vitro experiments, in collaboration with Dr. Claire Moore, will be performed to determine the basis for the observed stimulation of processing by Xpo1p and to determine whether Ran/Gsp1p is also involved. The ability of other transport factors to stimulate 3' processing will be determined.