ABSTRACT
In eukaryotes, termination of messenger RNA (mRNA) translation is mediated by the release factors eRF1 and eRF3. Using Saccharomyces cerevisiae as a model organism, we have identified a member of the DEAD-box protein (DBP) family, the DEAD-box RNA helicase and mRNA export factor Dbp5, as a player in translation termination. Dbp5 interacts genetically with both release factors and the polyadenlyate-binding protein Pab1. A physical interaction was specifically detected with eRF1. Moreover, we show that the helicase activity of Dbp5 is required for efficient stop-codon recognition, and intact Dbp5 is essential for recruitment of eRF3 into termination complexes. Therefore, Dbp5 controls the eRF3-eRF1 interaction and thus eRF3-mediated downstream events.
Subject(s)
DEAD-box RNA Helicases/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , Peptide Chain Termination, Translational , RNA Helicases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Codon, Terminator , DEAD-box RNA Helicases/genetics , Mutation , Nucleocytoplasmic Transport Proteins/genetics , Peptide Termination Factors/genetics , Peptide Termination Factors/metabolism , Poly(A)-Binding Proteins/genetics , Poly(A)-Binding Proteins/metabolism , Polyribosomes/metabolism , RNA Helicases/genetics , RNA Stability , RNA, Fungal/metabolism , RNA, Messenger/metabolism , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
A major challenge in current molecular biology is to understand how sequential steps in gene expression are coupled. Recently, much attention has been focused on the linkage of transcription, processing, and mRNA export. Here we describe the cytoplasmic rearrangement for shuttling mRNA binding proteins in Saccharomyces cerevisiae during translation. While the bulk of Hrp1p, Nab2p, or Mex67p is not associated with polysome containing mRNAs, significant amounts of the serine/arginine (SR)-type shuttling mRNA binding proteins Npl3p, Gbp2p, and Hrb1p remain associated with the mRNA-protein complex during translation. Interestingly, a prolonged association of Npl3p with polysome containing mRNAs results in translational defects, indicating that Npl3p can function as a negative translational regulator. Consistent with this idea, a mutation in NPL3 that slows down translation suppresses growth defects caused by the presence of translation inhibitors or a mutation in eIF5A. Moreover, using sucrose density gradient analysis, we provide evidence that the import receptor Mtr10p, but not the SR protein kinase Sky1p, is involved in the timely regulated release of Npl3p from polysome-associated mRNAs. Together, these data shed light onto the transformation of an exporting to a translating mRNP.
Subject(s)
Heterogeneous-Nuclear Ribonucleoproteins/physiology , Nuclear Proteins/physiology , Nucleocytoplasmic Transport Proteins/chemistry , Protein Biosynthesis , RNA-Binding Proteins/physiology , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/metabolism , Biological Transport , Blotting, Northern , Centrifugation, Density Gradient , Codon, Nonsense , Cycloheximide/pharmacology , Cytoplasm/metabolism , Gene Deletion , Green Fluorescent Proteins/metabolism , Microscopy, Fluorescence , Mutation , Nucleic Acid Hybridization , Plasmids/metabolism , Poly A/chemistry , Poly(A)-Binding Proteins , Polyribosomes/chemistry , Protein Serine-Threonine Kinases/physiology , Protein Synthesis Inhibitors/pharmacology , RNA/metabolism , RNA, Messenger/metabolism , RNA, Ribosomal/chemistry , RNA, Ribosomal/metabolism , RNA-Binding Proteins/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Ribosomes/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Sucrose/pharmacology , Temperature , Time Factors , Transcription, GeneticABSTRACT
Important progress in understanding messenger RNA export from the nucleus could be achieved by increasing the list of proteins that are involved in this process. Here, we present the identification of Gbp2 as a novel shuttling RNA-binding protein in Saccharomyces cerevisiae. Nuclear import of Gbp2 is dependent on the receptor Mtr10 and the serine/arginine-specific protein kinase Sky1. Deletion of the genes encoding both of these proteins or disruption of two of the arginine/serine repeats each shifts the steady-state localization of Gbp2 to the cytoplasm. Interestingly, deletion of MTR10 only also causes an increase in poly(A)(+) RNA binding by Gbp2, suggesting a role of Mtr10 in the dissociation of Gbp2 from mRNA in the cytoplasm. The nuclear export of Gbp2 is always coupled to mRNA export and is dependent on continuous RNA polymerase II transcription and mRNA-export factors. Although GBP2 is not essential for normal cell growth, overexpression of this gene is toxic and causes a nuclear retention of bulk poly(A)(+) RNA. Together, our findings clearly show an involvement of Gbp2 in mRNA transport. In addition, as a non-essential protein, Gbp2 also has the interesting potential to be spatially or temporally regulated.