Coupling of Termination, 3′ Processing, and mRNA Export

Abstract

In a screen to identify genes required for mRNA export in Saccharomyces cerevisiae, we isolated an allele of poly(A) polymerase (PAP1) and novel alleles encoding several other 3′ processing factors. Many newly isolated and some previously described mutants (rna14-48, rna14-49, rna14-64, rna15-58, and pcf11-1 strains) are defective in polymerase II (Pol II) termination but, interestingly, retain the ability to polyadenylate these improperly processed transcripts at the nonpermissive temperature. Deletion of the cis-acting sequences required to couple 3′ processing and termination also produces transcripts that fail to exit the nucleus, suggesting that all of these processes (cleavage, termination, and export) are coupled. We also find that several but not all mRNA export mutants produce improperly 3′ processed transcripts at the nonpermissive temperature. 3′ maturation defects in mRNA export mutants include improper Pol II termination and/or the previously characterized hyperpolyadenylation of transcripts. Importantly, not all mRNA export mutants have defects in 3′ processing. The similarity of the phenotypes of some mRNA export mutants and 3′ processing mutants indicates that some factors from each process may mechanistically interact to couple mRNA processing and export. Consistent with this assumption, we present evidence that Xpo1p interacts in vivo with several 3′ processing factors and that the addition of recombinant Xpo1p to in vitro processing reaction mixtures stimulates 3′ maturation. Of the core 3′ processing factors tested (Rna14p, Rna15p, Pcf11p, Hrp1p, Fip1p, and Cft1p), only Hrp1p shuttles. Overexpression of Rat8p/Dbp5p suppresses both 3′ processing and mRNA export defects found in xpo1-1 cells.

We thank members of the Cole, Moore, and Stutz laboratories for discussions and for critical review of this manuscript. We thank Ewan Dunn for consistent discussion and input into this project. We also thank Francoise Wyers, N. Proudfoot, Pamela Silver, Ed Hurt, Marvin Wickens, Steve Elledge, and Erica Golemis for strains and plasmids.

This research was supported in part by grants to C. N. Cole from the National Science Foundation (9983378) and National Institute of General Medical Sciences, National Institutes of Health (GM33998), by a grant to C. A. Moore from the National Institutes of Health (GM41752), and by a grant to F. Stutz from the Swiss National Science Foundation (3100-61378). C. M. Hammell was supported by a training grant (CA09658) from the National Cancer Institute, National Institutes of Health.