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Biomedical subjects

Akio Yamashita

Publications and source records attributed to Akio Yamashita.

12 recordsLinked to original sources
PubMed2007

Distant N- and C-terminal domains are required for intrinsic kinase activity of SMG-1, a critical component of nonsense-mediated mRNA decay.

Phosphatidylinositol 3-kinase-related kinases (PIKKs) consisting of SMG-1, ATM, ATR, DNA-PKcs, and mTOR are a family of proteins involved in the surveillance of gene expression in eukaryotic cells. They are involved in mechanisms responsible for genome stability, mRNA quality, and translation. They share a large N-terminal domain and a C-terminal FATC domain in addition to the unique serine/threonine protein kinase (PIKK) domain that is different from classical protein kinases. However, structure-function relationships of PIKKs remain unclear. Here we have focused on one of the PIKK members, SMG-1, which is involved in RNA surveillance, termed nonsense-mediated mRNA decay (NMD), to analyze the roles of conserved and SMG-1-specific sequences on the intrinsic kinase activity. Analyses of sets of point and deletion mutants of SMG-1 in a purified system and intact cells revealed that the long N-terminal region and the conserved leucine in the FATC domain were essential for SMG-1 kinase activity. However, the conserved tryptophan in the TOR SMG-1 (TS) homology domain and the FATC domain was not. In addition, the long insertion region between PIKK and FATC domains was not essential for SMG-1 kinase activity. These results indicated an unexpected feature of SMG-1, i.e. that distantly located N- and C-terminal sequences were essential for the intrinsic kinase activity.

Cell Line↗
PubMed2006

Specific inhibition of nonsense-mediated mRNA decay components, SMG-1 or Upf1, rescues the phenotype of Ullrich disease fibroblasts.

Nonsense-mediated mRNA decay (NMD) is an mRNA quality-control mechanism that degrades aberrant mRNAs containing premature translation termination codons (PTCs). The essential proteins for NMD include SMG-1, a protein kinase, and Upf1, a substrate of SMG-1 with RNA helicase activity. In this study, we evaluated the effects of NMD inhibition by siRNA-mediated knockdown of SMG-1 or Upf1 on the phenotype of Ullrich disease, an autosomal recessive congenital muscular dystrophy. The patient studied showed a homozygous frameshift mutation with a PTC in the collagen VI alpha2 gene, which encodes a truncated but partially functional protein. The patient's fibroblasts showed a nearly complete loss of the triple-helical collagen VI protein and functional defects in the extracellular matrix (ECM) due to the crucial deficiency of the collagen VI alpha2 protein. We have shown that siRNA-mediated knockdown of SMG-1 or Upf1 causes the up-regulation of the mutant triple-helical collagen VI, resulting in the formation of partially functional ECM. We suggest that the inhibition of NMD may be useful as a therapeutic approach to treat some human genetic diseases exacerbated by NMD.

Adult↗
PubMed2006

Binding of a novel SMG-1-Upf1-eRF1-eRF3 complex (SURF) to the exon junction complex triggers Upf1 phosphorylation and nonsense-mediated mRNA decay.

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that degrades mRNA containing premature termination codons (PTCs). In mammalian cells, recognition of PTCs requires translation and depends on the presence on the mRNA with the splicing-dependent exon junction complex (EJC). While it is known that a key event in the triggering of NMD is phosphorylation of the trans-acting factor, Upf1, by SMG-1, the relationship between Upf1 phosphorylation and PTC recognition remains undetermined. Here we show that SMG-1 binds to the mRNA-associated components of the EJC, Upf2, Upf3b, eIF4A3, Magoh, and Y14. Further, we describe a novel complex that contains the NMD factors SMG-1 and Upf1, and the translation termination release factors eRF1 and eRF3 (SURF). Importantly, an association between SURF and the EJC is required for SMG-1-mediated Upf1 phosphorylation and NMD. Thus, the SMG-1-mediated phosphorylation of Upf1 occurs on the association of SURF with EJC, which provides the link between the EJC and recognition of PTCs and triggers NMD.

Codon, Nonsense↗
PubMed2005

Concerted action of poly(A) nucleases and decapping enzyme in mammalian mRNA turnover.

In mammalian cells, the enzymatic pathways involved in cytoplasmic mRNA decay are incompletely defined. In this study, we have used two approaches to disrupt activities of deadenylating and/or decapping enzymes to monitor effects on mRNA decay kinetics and trap decay intermediates. Our results show that deadenylation is the key first step that triggers decay of both wild-type stable and nonsense codon-containing unstable beta-globin mRNAs in mouse NIH3T3 fibroblasts. PAN2 and CCR4 are the major poly(A) nucleases active in cytoplasmic deadenylation that have biphasic kinetics, with PAN2 initiating deadenylation followed by CCR4-mediated poly(A) shortening. DCP2-mediated decapping takes place after deadenylation and may serve as a backup mechanism for triggering mRNA decay when initial deadenylation by PAN2 is compromised. Our findings reveal a functional link between deadenylation and decapping and help to define in vivo pathways for mammalian cytoplasmic mRNA decay.

Animals↗
PubMed2005

The role of SMG-1 in nonsense-mediated mRNA decay.

SMG-1, a member of the PIKK (phosphoinositide 3-kinase related kinases) family, plays a critical role in the mRNA quality control system termed nonsense-mediated mRNA decay (NMD). NMD protects the cells from the accumulation of aberrant mRNAs with premature termination codons (PTCs) that encode nonfunctional or potentially harmful truncated proteins. SMG-1 directly phosphorylates Upf1, another key component of NMD, and this phosphorylation occurs upon recognition of PTC on post-spliced mRNA during the initial round of translation. At present, a variety of tools are available that can specifically suppress NMD, and it is possible to examine the contribution of NMD in a variety of physiological and pathological conditions.

Animals↗
PubMed2005

Ischemic changes in evoked spinal cord potentials during profound hypothermic circulatory arrest in thoracic aortic surgery.

PURPOSE: We examined the changes in evoked spinal cord potentials (ESCP) during profound hypothermic circulatory arrest to estimate the safe ischemic time. METHODS: We monitored ESCPs during surgery for descending thoracic or thoracoabdominal aneurysms in five patients. Evoked spinal cord potential recordings were obtained before cooling (baseline), then every few minutes during circulatory arrest, and at the end of the operation. RESULTS: After circulatory arrest, the amplitude of ESCPs decreased with time. We calculated the simple linear regression between the amplitude of ESCPs and the circulatory arrest time by the least-squares method, and found a highly linear relationship between amplitude and arrest time in all five patients. The time until disappearance of ESCPs was estimated as 50.7 +/- 20.4 min (95% level of confidence). CONCLUSION: When an ESCP disappeared, ischemic spinal cord injury had occurred. This demonstrates the potential value of estimating the time of disappearance of ESCPs to prevent ischemic spinal cord injury during descending thoracic aortic surgery.

Aged↗
PubMed2004

UNR, a new partner of poly(A)-binding protein, plays a key role in translationally coupled mRNA turnover mediated by the c-fos major coding-region determinant.

Messenger RNA decay mediated by the c-fos major protein coding-region determinant of instability (mCRD) is a useful system for studying translationally coupled mRNA turnover. Among the five mCRD-associated proteins identified previously, UNR was found to be an mCRD-binding protein and also a PABP-interacting protein. Interaction between UNR and PABP is necessary for the full destabilization function of the mCRD. By testing different classes of mammalian poly(A) nucleases, we identified CCR4 as a poly(A) nuclease involved in the mCRD-mediated rapid deadenylation in vivo and also associated with UNR. Blocking either translation initiation or elongation greatly impeded poly(A) shortening and mRNA decay mediated by the mCRD, demonstrating that the deadenylation step is coupled to ongoing translation of the message. These findings suggest a model in which the mCRD/UNR complex serves as a "landing/assembly" platform for formation of a deadenylation/decay mRNA-protein complex on an mCRD-containing transcript. The complex is dormant prior to translation. Accelerated deadenylation and decay of the transcript follows ribosome transit through the mCRD. This study provides new insights into a mechanism by which interplay between mRNA turnover and translation determines the lifespan of an mCRD-containing mRNA in the cytoplasm.

Animals↗
PubMed2004

Inhibition of nonsense-mediated mRNA decay rescues the phenotype in Ullrich's disease.

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance system that eliminates aberrant mRNAs containing premature translation termination codons (PTCs). We evaluated the role of NMD in of Ullrich's disease. The patient has a frameshift mutation with a PTC in the collagen VI alpha2 gene causing the loss of collagen VI and functional defects in extracellular matrix (ECM). The pharmacological block of NMD caused upregulation of the mutant collagen VI alpha2 subunit, resulting in collagen VI assembly and partially functional ECM formation. Our results suggest that NMD inhibitors can be used as a therapeutic tool to rescue some human genetic diseases exacerbated by NMD.

Androstadienes↗
PubMed2003

Loss of von Hippel-Lindau protein causes cell density dependent deregulation of CyclinD1 expression through hypoxia-inducible factor.

Loss of the von Hippel-Lindau gene (VHL) expression ca-uses deregulation of contact inhibition of cell growth, which might be one of the bases of the tumor suppressor function of VHL. Here we show that this function of the VHL gene product (pVHL) depends on cell autonomous events. To identify the target gene of pVHL, which is directly involved in the contact inhibition, we compared the gene expression profile between VHL-deficient renal carcinoma 786-O cells and those infected with an adenovirus vector encoding VHL. In addition to known pVHL-regulated genes, such as vascular endothelial growth factor and carbonic anhydrase, we found cyclinD1 as a new target of pVHL at a high cell density. In VHL-expressing cells (VHL (+) cells), the cyclinD1 mRNA expression level diminishes at a high cell density, while it remains at a relatively high level in VHL-deficient cells (VHL (-) cells). The cyclinD1 expression level was also abnormally high in VHL (-) cells at a high cell density. Consequently, the phosporylation level of the retinoblastoma (Rb) protein remained high in these cells, whereas there was no phosporylated Rb in VHL (+) cells under the contact inhibition. The abnormal expression of cyclinD1 at a high cell density was observed even in VHL (+) cells under the hypoxic state. Moreover, ectopic expression of a HIF mutant resistant to pVHL-mediated proteolysis causes the abnormal cyclinD1 expression in VHL (+) cells. Taken together, these observations indicate that VHL is required for the downregulation of cyclinD1 at a high cell density through HIF.

Carbonic Anhydrases↗
PubMed2003

Phosphorylation of hUPF1 induces formation of mRNA surveillance complexes containing hSMG-5 and hSMG-7.

Eukaryotic mRNAs containing premature termination codons (PTCs) are degraded by a process known as nonsense-mediated mRNA decay (NMD). NMD has been suggested to require the recognition of PTC by an mRNA surveillance complex containing UPF1/SMG-2. In multicellular organisms, UPF1/SMG-2 is a phosphoprotein, and its phosphorylation contributes to NMD. Here we show that phosphorylated hUPF1, the human ortholog of UPF1/SMG-2, forms a complex with human orthologs of the C. elegans NMD proteins SMG-5 and SMG-7. The complex also associates with protein phosphatase 2A (PP2A), resulting in dephosphorylation of hUPF1. Overexpression of hSMG-5 mutants that retain interaction with P-hUPF1 but which cannot induce its dephosphorylation impair NMD, suggesting that NMD requires P-hUPF1 dephosphorylation. We also show that P-hUPF1 forms distinct complexes containing different isoforms of hUPF3A. We propose that sequential phosphorylation and dephosphorylation of hUPF1 by hSMG-1 and PP2A, respectively, contribute to the remodeling of the mRNA surveillance complex.

Amino Acid Sequence↗
PubMedDate not supplied

Evaluation of regional aortic distensibility using color kinesis.

Regional aortic stiffness cannot be evaluated by conventional methods. Regional aortic wall velocity during systole in the descending aorta was evaluated by using transesophageal echocardiography with color kinesis. The authors defined regional aortic distensibility (RAD) by considering pulse pressure, with RAD (microm/s/mm Hg) = (regional aortic wall velocity)/(pulse pressure). RAD was evaluated in 38 patients who had coronary artery disease (CAD) and 10 who did not. RAD decreased depending on aging (partial regression coefficient was -5.39 x 10(-1), p<0.001), and RAD was lower in the CAD group than that in the no-CAD group (p<0.05). In the CAD group, 19 patients had a single fixed plaque (4 calcified and 15 noncalcified plaques). RAD in the calcified plaque was lower than that in the noncalcified plaque (p<0.01), and RAD was lower in the noncalcified plaque than that in the no-plaque region (p<0.05). In noncalcified plaques, the relation between RAD and maximum intimal thickness had a significant correlation, r=0.7, p<0.001. The residual of RAD from the regression line was significantly larger in the calcified plaque than that in the noncalcified plaque (p<0.001). In conclusion, RAD can express increasing regional aortic wall stiffness brought about by arteriosclerosis quantitatively. Color kinesis provides information on characteristic difference between calcified and noncalcified plaque.

Analysis of Variance↗