Role of RNA-Binding Proteins in MAPK Signal Transduction Pathway

Mitogen-activated protein kinases (MAPKs), which are found in all eukaryotes, are signal transducing enzymes playing a central role in diverse biological processes, such as cell proliferation, sexual differentiation, and apoptosis. The MAPK signaling pathway plays a key role in the regulation of gene expression through the phosphorylation of transcription factors. Recent studies have identified several RNA-binding proteins (RBPs) as regulators of MAPK signaling because these RBPs bind to the mRNAs encoding the components of the MAPK pathway and regulate the stability of their transcripts. Moreover, RBPs also serve as targets of MAPKs because MAPK phosphorylate and regulate the ability of RBPs to bind and stabilize target mRNAs, thus controlling various cellular functions. In this review, we present evidence for the significance of the MAPK signaling in the regulation of RBPs and their target mRNAs, which provides additional information about the regulatory mechanism underlying gene expression. We further present evidence for the clinical importance of the posttranscriptional regulation of mRNA stability and its implications for drug discovery

Role of the Small GTPase Rho3 in Golgi/Endosome Trafficking through Functional Interaction with Adaptin in Fission Yeast

BACKGROUND: We had previously identified the mutant allele of apm1(+) that encodes a homolog of the mammalian µ1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex, and we demonstrated the role of Apm1 in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we isolated rho3(+), which encodes a Rho-family small GTPase, an important regulator of exocystosis, as a multicopy-suppressor of the temperature-sensitive growth of the apm1-1 mutant cells. Overexpression of Rho3 suppressed the Cl(-) sensitivity and immunosuppressant sensitivity of the apm1-1 mutant cells. Overexpression of Rho3 also suppressed the fragmentation of vacuoles, and the accumulation of v-SNARE Syb1 in Golgi/endosomes and partially suppressed the defective secretion associated with apm1-deletion cells. Notably, electron microscopic observation of the rho3-deletion cells revealed the accumulation of abnormal Golgi-like structures, vacuole fragmentation, and accumulation of secretory vesicles; these phenotypes were very similar to those of the apm1-deletion cells. Furthermore, the rho3-deletion cells and apm1-deletion cells showed very similar phenotypic characteristics, including the sensitivity to the immunosuppressant FK506, the cell wall-damaging agent micafungin, Cl(-), and valproic acid. Green fluorescent protein (GFP)-Rho3 was localized at Golgi/endosomes as well as the plasma membrane and division site. Finally, Rho3 was shown to form a complex with Apm1 as well as with other subunits of the clathrin-associated AP-1 complex in a GTP- and effector domain-dependent manner. CONCLUSIONS/SIGNIFICANCE: Taken together, our findings reveal a novel role of Rho3 in the regulation of Golgi/endosome trafficking and suggest that clathrin-associated adaptor protein-1 and Rho3 co-ordinate in intracellular transport in fission yeast. To the best of our knowledge, this study provides the first evidence of a direct link between the small GTPase Rho and the clathrin-associated adaptor protein-1 in membrane trafficking

Rho3 suppressed various phenotypes associated with Apm1-deletion cells.

<p>(A) Rho3 suppressed the defective localization of GFP-Syb1 in Δ<i>apm1</i> cells. Wild-type cells (wt) and Apm1-deletion (Δ<i>apm1</i>) expressing chromosome-bone GFP-Syb1 cells transformed with pDB248 or the vector containing <i>rho3⁺</i> was cultured in YPD medium at 27°C. The GFP-Syb1 localization was examined under the fluorescence microscope. Bar 10 µm. (B) Rho3 suppressed the defective localization of FM4-64 in Δ<i>apm1</i> cells. Wild-type (wt) and Apm1-deletion cells (Δ<i>apm1</i>) transformed with pDB248 or the vector containing <i>rho3⁺</i> were cultured in YPD medium at 27°C. Cells were collected, labeled with FM4-64 fluorescent dye for 5 min, resuspended in water, and examined by fluorescence microscopy. Bar 10 µm. (C) Wild-type (wt) and Apm1-deletion cells (Δ<i>apm1</i>) transformed with pDB248 or the vector containing <i>rho3⁺</i> cultured in YPD medium at 27°C. Cells were collected, labeled with FM4-64 fluorescent dye for 60 min, resuspended in water, and examined by fluorescence microscopy. Bar 10 µm. (D) Wild-type cells and Δ<i>apm1</i> cells, which were transformed with either the pDB248 vector or the <i>rho3⁺</i>-containing vector, were assayed for acid phosphatase activity as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016842#s2" target="_blank">Materials and Methods</a> section. Values from 3 independent experiments were plotted as means ± S.D.</p

Analysis of acid phosphatase secretion and effect of Rho3 overproduction.

<p>Wild-type cells and Δ<i>apm1</i> cells, which were transformed with either the pDB248 vector or the <i>rho3⁺</i>-containing vector, were assayed for acid phosphatase activity as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016842#s2" target="_blank">Materials and Methods</a> section. Values from 3 independent experiments are expressed as means ± standard deviation.</p

Isolation of Rho3 as a multicopy suppressor of <i>apm1</i> mutant cells.

<p>(A) The <i>apm1</i> mutant cells (<i>apm1-1</i>) were transformed with either the pDB248 multicopy vector, the vector containing <i>apm1⁺</i> or the vector containing <i>rho3⁺</i>. Cells were then streaked onto plates containing YPD, YPD plus 0.2 M MgCl₂, YPD plus 0.5 µg/mL FK506, EMM, or EMM plus 0.5 µg/mL FK506 and then incubated for 4 d at 27°C or for 3 d at 36°C, respectively. (B) Cells transformed with the multicopy vector pDB248, or the genome DNA clones containing <i>rho1⁺</i>, <i>rho2⁺</i>, <i>rho3⁺</i>, <i>rho4⁺</i>, <i>rho5⁺</i>, or <i>cdc42⁺</i> were streaked onto plates containing YPD and incubated for 4 d at 27°C or 3 d at 35°C, respectively.</p

<i>Schizosaccharomyces pombe</i> strains used in this study.

<p><i>Schizosaccharomyces pombe</i> strains used in this study.</p