Coupling of termination, 3' processing, and mRNA export.
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.
Hammell
C M
CM
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Gross
Stefan
S
Zenklusen
Daniel
D
Heath
Catherine V
CV
Stutz
Francoise
F
Moore
Claire
C
Cole
C N
CN
D000483:Alleles; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism; D005434:Flow Cytometry; D005656:Fungal Proteins / Q000378:metabolism; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D028884:Karyopherins / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D016366:Open Reading Frames; D000073718:Pancreatitis-Associated Proteins; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D014158:Transcription, Genetic; D020798:Two-Hybrid System Techniques; D039221:mRNA Cleavage and Polyadenylation Factors; D000097606:Exportin 1 Protein
D005656:Fungal Proteins; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D000073718:Pancreatitis-Associated Proteins; D011062:Polynucleotide Adenylyltransferase; D012313:RNA; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D029701:Saccharomyces cerevisiae Proteins; D039221:mRNA Cleavage and Polyadenylation Factors; D000097606:Exportin 1 Protein; C512637:PCF11 protein, S cerevisiae; C000612141:REG3A protein, human; C512638:RNA14 protein, S cerevisiae; C512635:RNA15 protein, S cerevisiae