In eukaryotes, intracellular signaling events are essential for transmitting information from the external environment to gene expression networks (reviewed in reference 7
). This communication is particularly important for allowing rapid cellular adaption and survival during stress conditions. It is well established that environmental stress results in an evolutionarily conserved global alteration of the nuclear mRNA export pathway (6
). In response to heat shock, transcripts from genes encoding heat shock proteins (hsp) are exported, whereas most non-hsp poly(A+
) mRNAs are retained and accumulate in the nucleus (53
). Together with the coincident increased transcription of heat shock genes, the retention of non-hsp mRNAs results in a coordinated mechanism for the rapid production of the heat shock proteins essential for survival and stress recovery (6
). How signal transduction pathways modulate the mRNA export mechanism is not fully defined.
For mRNA export under normal growth conditions, the tr
anscription and mRNA ex
port (TREX) complex is critical for coupling mRNA biogenesis with messenger ribonucleoprotein particle (mRNP) packaging to allow formation of an export-competent mRNP (1
). The TREX complex includes the essential mRNA-binding protein Yra1 and the export receptor Mex67-Mtr2 in Saccharomyces cerevisiae
(TAP/NXF1-p15/NXT1 in vertebrates) (22
). Recruitment of Mex67-Mtr2 appears to be the penultimate step, which stimulates release of the mature mRNP from chromatin-associated biogenesis factors and the transition to early mRNA export steps (31
). Association of Mex67-Mtr2 with the mRNP is key for export and directly mediates subsequent targeting to nuclear pore complexes (NPCs) in the nuclear envelope and NPC translocation via interactions between Mex67-Mtr2 and NPC proteins (nucleoporins [Nups]) (reviewed in reference 34
). Thus, defining the mechanisms that control interactions between mRNA-binding proteins and Mex67-Mtr2 will likely reveal important regulatory steps.
Several essential mRNA-binding proteins have been implicated in the mRNA export mechanism (32
). In the budding yeast S. cerevisiae
, this includes Npl3, Yra1, and Nab2 (21
). These factors couple mRNA biogenesis steps, such as transcriptional elongation, pre-mRNA splicing, and 3′-end formation with assembly of an export-competent mRNP (4
). Other key S. cerevisiae
components involved in early mRNA export are Mlp1 and Mlp2 (18
). The Mlp proteins associate with the NPC and promote docking of mRNPs to the nuclear envelope. Studies have shown that the poly(A+
)-binding protein Nab2 interacts directly with Mlp1, with loss of the Nab2-Mlp1 interaction enhancing the growth and mRNA export defects of mex67
). This suggests that a Nab2-Mlp1 step is central to efficient mRNA export. Importantly, the Mlp proteins also function in a nuclear quality control mechanism that acts to retain unspliced or aberrantly processed mRNAs in the nucleus (16
). Although the precise role for the Mlp proteins in quality control is unknown, genetic and biochemical evidence suggests that Nab2 and Yra1 are linked to this process.
Many of the factors required for normal, non-hsp mRNA export are also essential for hsp export; specifically, Mex67, the DEAD-box protein Dbp5, and the Dbp5 activator Gle1 (3
). Altering the functions of these proteins results in the impaired export of all mRNAs, consistent with their general roles in the mRNA export process. In contrast, hsp mRNA export is independent of some factors required for normal mRNA export, including the mRNA-binding proteins Yra1 and Npl3 (35
). Furthermore, hsp mRNAs show a requirement for the NPC protein Nup42, whereas this factor is dispensable for efficient mRNA export under normal growth conditions (50
). Given that several studies have found that specific mRNA-binding proteins associated with transcripts whose protein products are functionally linked, regulating the mRNP composition for transcripts might be a mechanism for controlling mRNA export (12
). However, it is not clear how mRNP composition is affected during heat shock or whether other molecular signals differentiate between hsp and non-hsp mRNAs during mRNA export.
The stress induced by heat shock is known to trigger a cascade of intracellular signaling events, including stimulation of the mitogen-activated protein kinase (MAPK) pathways (7
). S. cerevisiae
has five distinct MAP kinase pathways, each of which can be activated by specific extracellular stimuli. The Bck1-Slt2/Mpk1 pathway is specifically initiated by certain forms of cellular stress, including cell wall stress and heat shock (42
). When cells undergo heat shock, Pkc1 initiates the kinase cascade by phosphorylating the MAPK kinase kinase (MAPKKK) (Bck1), which then phosphorylates two redundant MAPKKs (Mkk1 and Mkk2) for phosphorylation of the MAPK (Slt2/Mpk1) (38
). Activated Slt2 phosphorylates both transcriptional activators and repressors, altering the gene expression pattern to allow cell survival (10
). Interestingly, the vertebrate ortholog of the Mlp proteins, designated Tpr1, is a target of a MAP kinase pathway in vertebrate cells (76
). Thus, there is a potential connection between these signaling pathways and mRNA export during heat shock.
In this study, we demonstrate that the essential poly(A+) mRNA-binding protein Nab2 is a target for heat shock-dependent phosphorylation by the MAP kinase Slt2. Unlike wild-type cells, slt2 null (Δ) mutant cells fail to accumulate poly(A+) RNA in the nucleus during heat shock. Further, Nab2 and Mlp1 form intranuclear foci upon heat shock, whereas Mex67 retains a normal cellular distribution. On the basis of a direct physical interaction between Nab2 and Mex67-Mtr2 and their differential association in complexes from heat-shocked cells, we propose a model whereby Slt2 and Mlp1 promote nuclear retention of non-hsp mRNPs by uncoupling the mRNA export receptor, Mex67, from specific mRNA-binding proteins during heat shock. This mechanism allows selectivity for hsp mRNP export during stress and facilitates thermotolerance and rapid recovery.