The constitutive transport element (CTE) of the type D retroviruses promotes nuclear export of unspliced viral RNAs apparently by recruiting host factor(s) required for export of cellular messenger RNAs. Here, we report the identification of TAP as the cellular factor that specifically binds to wild-type CTE but not to export-deficient CTE mutants. Microinjection experiments performed in Xenopus oocytes demonstrate that TAP directly stimulates CTE-dependent export. Furthermore, TAP overcomes the mRNA export block caused by the presence of saturating amounts of CTE RNA. Thus, TAP, like its yeast homolog Mex67p, is a bona fide mRNA nuclear export mediator. TAP is the second cellular RNA binding protein shown to be directly involved in the export of its target RNA.
The response of eukaryotic cells to heat shock and other forms of stress occurs at both transcriptional and post-transcriptional levels. We used in situ hybridization to determine whether stress affected the subcellular distribution of poly(A]^ RNA in Saccharomyces cerevisiae. Following induction of stress by either heat shock (42°C) or addition of a high concentration of ethanol (10%), the nucleocytoplasmic export of most poly(A)^ RNA was blocked. In situ hybridization indicated that heat-inducible SSA4 and SSAl mRNAs were exported from nuclei under these same conditions. On the other hand, both GALl and URA3 transcripts expressed from the SSA4 promoter accumulated in nuclei following heat shock. Sequences within either the 5' 1600 or the 3' 500 nucleotides of SSA4 mRNA were sufficient to direct GALl mRNA to the cytoplasm during stress. The export of SSA4 mRNA following stress required functional nuclear pore complexes, as SSA4 mRNA accumulated in nuclei following heat shock of cells containing temperature-sensitive nucleoporins. However, the selective export of SSA4 mRNA was maintained in heat-shocked cells carrying temperature-sensitive alleles of RNAl, PRP20, or an inducible dominant-negative allele of GSPl, the S. cerevisiae homolog of RAN/TC4. The results reported here suggest that there is selective export of mRNA in yeast.
Ran/TC4, a Ras-like GTP-binding protein, and its nucleotide exchanger, RCC1, have been implicated in control of protein movement into the nucleus and cytoplasmic accumulation of mRNA. Saccharomyces cerevisiae contains two homologues of the mammalian Ran/TC4, encoded by the GSPI and GSP2 genes. We have constructed yeast strains that overproduce either wild-type Gspl or a form of Gspl with glycine-21 converted to valine (Gspl-G21V), which we show stabilizes the GTP-bound form. Cells producing Gspl-G21V have defects in localization of nuclear proteins; nuclear proteins accumulate in the cytoplasm following galactose induction of Gspl-G21V. Similarly, cells producing Gspl-G21V retain poly(A)+ RNA in their nuclei. These findings suggest that hydrolysis of GTP by Ran/TC4 is necessary for proper import of proteins into the nucleus and appearance of poly(A)+ RNA in the cytoplasm.Movement of proteins and RNAs across the nuclear envelope via the nuclear pores is rapid, specific, and highly regulated. Proteins destined for the nucleoplasm often contain short stretches of amino acids, termed nuclear localization sequences (NLSs), that are sufficient to direct them to the nucleus (reviewed in ref.
The Rev-RRE system and the CTE direct intron-containing RNAs to distinct export pathways. Although previous data have suggested that Rev uses the same export pathway as uracil-rich small nuclear RNAs and 5S ribosomal RNA, the CTE seems to interact with evolutionarily conserved factors that are essential for cellular mRNA export.
We reported previously that heat or ethanol shock in Saccharomyces cerevisiae leads to nuclear retention of most poly(A) + RNA but heat shock mRNAs (encoding Hsp70 proteins Ssa1p and Ssa4p) are efficiently exported in a process that is independent of the small GTPase Ran/Gsp1p, which is essential for most nucleocytoplasmic transport. To gain further insights into proteins essential or nonessential for export of heat shock mRNAs, in situ hybridization analyses to detect mRNA and pulse-labeling of proteins were used to examine several yeast mutant strains for their ability to export heat shock mRNAs following stress. Rip1p is a 42-kD protein associated with nuclear pore complexes and contains nucleoporin-like repeat sequences. It is dispensable for growth of yeast cells under normal conditions, but we report that it is essential for the export of heat shock mRNAs following stress. When SSA4 mRNA was induced from a GAL promoter in the absence of stress, it was efficiently exported in a strain lacking RIP1, indicating that Rip1p is required for export of heat shock mRNAs only following stress. Npl3p, a key mediator of export of poly(A) + RNA, was not required for heat shock mRNA export, whereas Rss1p/Gle1p, a NES-containing factor essential for poly(A) + RNA export, was also required for export of heat shock mRNAs after stress. High-level expression of the HIV-1 Rev protein, but not of Rev mutants, led to a partial block in export of heat shock mRNAs following stress. The data suggest a model wherein the requirement for Npl3p defines the mRNA export pathway, the requirement for Rip1p defines a pathway used for export of heat shock mRNAs after stress, and additional factors, including Rss1p/Gle1p and several nucleoporins (Rat7p/Nup159p, Rat2p/Nup120p, and Nup145p/Rat10p), are required in both pathways.[Key Words: RNA export; heat shock; RIP1; hnRNP; RSS1/GLE1; Rev]Received July 1, 1997; revised version accepted August 28, 1997.A distinguishing feature of eukaryotic cells is the nucleus, a distinct subcellular compartment separated from the cytoplasm by the double-membraned nuclear envelope. Embedded within the nuclear envelope are nuclear pore complexes (NPCs) that serve as the only known channels for transport between the nucleus and the cytoplasm (for review, see Davis 1995;Panté and Aebi 1996). Transport of macromolecules through NPCs is signal-mediated, saturable, and energy dependent. Considerable progress has been made in recent years in identifying (1) receptor molecules that recognize nuclear localization signals (NLSs) within karyophilic proteins and mediate interactions between these proteins and NPCs, (2) a small Ras-like GTPase (Ran in metazoan cells and Gsp1p/Gsp2p in Saccharomyces cerevisiae) and its accessory proteins, which play a central role in nuclear protein import, and (3) distinct components of NPCs required for nuclear protein import (for review, see Gö rlich and
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