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

Role of the RNA-Binding Protein Nrd1 in Stress Granule Formation and Its Implication in the Stress Response in Fission Yeast

We have previously identified the RNA recognition motif (RRM)-type RNA-binding protein Nrd1 as an important regulator of the posttranscriptional expression of myosin in fission yeast. Pmk1 MAPK-dependent phosphorylation negatively regulates the RNA-binding activity of Nrd1. Here, we report the role of Nrd1 in stress-induced RNA granules. Nrd1 can localize to poly(A)-binding protein (Pabp)-positive RNA granules in response to various stress stimuli, including heat shock, arsenite treatment, and oxidative stress. Interestingly, compared with the unphosphorylatable Nrd1, Nrd1DD (phosphorylation-mimic version of Nrd1) translocates more quickly from the cytoplasm to the stress granules in response to various stimuli; this suggests that the phosphorylation of Nrd1 by MAPK enhances its localization to stress-induced cytoplasmic granules. Nrd1 binds to Cpc2 (fission yeast RACK) in a phosphorylation-dependent manner and deletion of Cpc2 affects the formation of Nrd1-positive granules upon arsenite treatment. Moreover, the depletion of Nrd1 leads to a delay in Pabp-positive RNA granule formation, and overexpression of Nrd1 results in an increased size and number of Pabp-positive granules. Interestingly, Nrd1 deletion induced resistance to sustained stresses and enhanced sensitivity to transient stresses. In conclusion, our results indicate that Nrd1 plays a role in stress-induced granule formation, which affects stress resistance in fission yeast

Sip1, a conserved AP-1 accessory protein, is important for Golgi/endosome trafficking in fission yeast.

We had previously identified the mutant allele of apm1(+) that encodes a homolog of the mammalian μ 1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex and demonstrated that the AP-1 complex plays a role in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. Here, we isolated a mutant allele of its4(+)/sip1(+), which encodes a conserved AP-1 accessory protein. The its4-1/sip1-i4 mutants and apm1-deletion cells exhibited similar phenotypes, including sensitivity to the calcineurin inhibitor FK506, Cl(-) and valproic acid as well as various defects in Golgi/endosomal trafficking and cytokinesis. Electron micrographs of sip1-i4 mutants revealed vacuole fragmentation and accumulation of abnormal Golgi-like structures and secretory vesicles. Overexpression of Apm1 suppressed defective membrane trafficking in sip1-i4 mutants. The Sip1-green fluorescent protein (GFP) co-localized with Apm1-mCherry at Golgi/endosomes, and Sip1 physically interacted with each subunit of the AP-1 complex. We found that Sip1 was a Golgi/endosomal protein and the sip1-i4 mutation affected AP-1 localization at Golgi/endosomes, thus indicating that Sip1 recruited the AP-1 complex to endosomal membranes by physically interacting with each subunit of this complex. Furthermore, Sip1 is required for the correct localization of Bgs1/Cps1, 1,3-β-D-glucan synthase to polarized growth sites. Consistently, the sip1-i4 mutants displayed a severe sensitivity to micafungin, a potent inhibitor of 1,3-β-D-glucan synthase. Taken together, our findings reveal a role for Sip1 in the regulation of Golgi/endosome trafficking in coordination with the AP-1 complex, and identified Bgs1, required for cell wall synthesis, as the new cargo of AP-1-dependent trafficking

Restricted expression and photic induction of a novel mouse regulatory factor X4 transcript in the suprachiasmatic nucleus.

The regulatory factor X (RFX) family of transcription factors is characterized by a unique and highly conserved 76-amino acid residue DNA-binding domain. Mammals have five RFX genes, but the physiological functions of their products are unknown, with the exception of RFX5.Here a mouse RFX4 transcript was identified that encodes a peptide of 735 amino acids, including the DNA-binding domain. Its expression was localized in the suprachiasmatic nucleus, the central pacemaker site of the circadian clock. Also, light exposure was found to induce its gene expression in a subjective night-specific manner. Polyclonal antibodies were prepared, and an 80-kDa band was detected in the suprachiasmatic nucleus by Western hybridization. A histochemical study showed a localization of the products in the nucleus. This is the first report on mouse RFX4, which contains the RFX DNA-binding motif. Our investigation may provide clues to the physiological function of RFX4

Identification of genes that express in response to light exposure and express rhythmically in a circadian manner in the mouse suprachiasmatic nucleus

Most biological phenomena,including behavoir and metabolic pathways,are governed by an internal clock system that is circadian (i.e.,with a period of approximately 24 h) and is reset by light exposure from outside.In order to understand the molecular basis of the resetting mechanism of the clock,we attempted to isolate light-inducible transcripts in the suprachiasmatic nucleus,where the master clock resides,using a new gene expression profiling procedure.We identified 87 such transcripts,successfully cloned 60 of them and 17 are protein-coding transcripts registered in the public database that were not known to be light inducible.Induction is subjective night specific in most of the transcripts.Interestingly,6 of the transcripts exhibit rhythmic expression in a circadian manner in the suprachiasmatic nucleus

Mouse dexamethasone-induced RAS protein 1 gene is expressed in a circadian rhythmic manner in the suprachiasmatic nucleus.

We identified the Dexamethasone-induced RAS protein 1 (Dexras1) gene as a cycling gene in the suprachiasmatic nucleus (SCN). Investigation of the whole brain using in situ hybridization demonstrated the localization of the expression of the gene in the SCN, thalamus, piriform cortex and hippocampus. However, rhythmic expression of the gene was observed only in the SCN. The rhythmic change in gene expression during 1 day was approximately five-fold, and the maximum expression was observed during subjective night. Real-time PCR using the SCN, paraventricular nucleus and cortex confirmed these results. Next, we analyzed the expression of the Dexras1 gene in the SCN of cryptochrome (Cry) 1 and 2 double knockout mice. We found that the rhythmic expression disappeared. The results indicate that Dexras1 rhythmicity and levels are dependent upon CRYs. This is the first time that the G protein, which may be involved in the input pathway, has been isolated as a cycling gene in the SCN.\nMouse dexamethasone-induced RAS protein 1 gene is expressed in a circadian rhythmic manner in the suprachiasmatic nucleus.\nTakahashi H, Umeda N, Tsutsumi Y, Fukumura R, Ohkaze H, Sujino M, van der Horst G, Yasui A, Inouye ST, Fujimori A, Ohhata T, Araki R, Abe M.\nTranscriptome Profiling Group, National Institute of Radiological Sciences, Anagawa 4-9-1, Inage-ku, Chiba 263-8555, Japan.\nWe identified the Dexamethasone-induced RAS protein 1 (Dexras1) gene as a cycling gene in the suprachiasmatic nucleus (SCN). Investigation of the whole brain using in situ hybridization demonstrated the localization of the expression of the gene in the SCN, thalamus, piriform cortex and hippocampus. However, rhythmic expression of the gene was observed only in the SCN. The rhythmic change in gene expression during 1 day was approximately five-fold, and the maximum expression was observed during subjective night. Real-time PCR using the SCN, paraventricular nucleus and cortex confirmed these results. Next, we analyzed the expression of the Dexras1 gene in the SCN of cryptochrome (Cry) 1 and 2 double knockout mice. We found that the rhythmic expression disappeared. The results indicate that Dexras1 rhythmicity and levels are dependent upon CRYs. This is the first time that the G protein, which may be involved in the input pathway, has been isolated as a cycling gene in the SCN