Role of a tyrosine phosphorylation of SMG-9 in binding of SMG-9 to IQGAP and the NMD complex

SMG-9 is a component of the NMD complex, a heterotetramer that also includes SMG-1 and SMG-8 in the complex. SMG-9 was also originally identified as a tyrosine-phosphorylated protein but the role of the phosphorylation is not yet known. In this study, we determined that IQGAP protein, an actin cytoskeleton modifier acts as a binding partner with SMG-9 and this binding is regulated by phosphorylation of SMG-9 at Tyr-41. SMG-9 is co-localized with IQGAP1 as a part of the process of actin enrichment in non-stimulated cells, but not in the EGF-stimulated cells. Furthermore, an increase in the ability of SMG-9 to bind to SMG-8 occurs in response to EGF stimulation. These results suggest that tyrosine phosphorylation of SMG-9 may play a role in the formation of the NMD complex in the cells stimulated by the growth factor.     

Identification and characterization of protein interactions in the mammalian mRNA processing body using a novel two-hybrid assay

Components of the mRNA processing body (P-body) regulate critical steps in mRNA storage, transport, translation and degradation. At the core of the P-body is the decapping complex, which removes the 5′ cap from de-adenylated mRNAs and mediates an irreversible step in mRNA degradation. The assembly of P-bodies in Saccharomyces cerevisiae, Arabidopsis thaliana and Drosophila melanogaster has been previously described. Less is known about the assembly of mammalian P-bodies. To investigate the interactions that occur between components of mammalian P-bodies, we developed a fluorescence-based, two-hybrid assay system. The assay depends on the ability of one P-body component, fused to an exogenous nuclear localization sequence (NLS), to recruit other P-body components to the nucleus. The assay was used to investigate interactions between P-body components Ge-1, DCP2, DCP1, EDC3, RAP55, and RCK. The results of this study show that the modified two-hybrid assay can be used to identify protein interactions that occur in a macromolecular complex. The assay can also be used to efficiently detect protein interaction domains. The results provide important insights into mammalian P-body assembly and demonstrate similarities, and critical differences, between P-body assembly in mammalian cells compared with that of other species.     

Modeling of single-cell oil production under nitrogen-limited and substrate inhibition conditions

Sweet sorghum extract was used as substrate for lipid accumulation by the oleaginous fungus Mortierella isabellina in batch cultures. Various initial sugar (13–91 g/L) and nitrogen (100–785 mg/L) concentrations resulting in various C/N (43–53) ratios were tested. Oil accumulation ranged between 43% and 51% corresponding to oil production from 2.2 to 9.3 g/L. A detailed mathematical model was developed. This model is able to adequately predict biomass growth, lipid accumulation, and sugar and nitrogen consumption. The model assumes that fungus growth is inhibited at high sugar concentrations. A set of kinetic experiments was used for model kinetic parameters estimation, while another set of experiments was used for model validation. The developed model could be generalized for similar systems of lipid accumulation and become a useful tool for reactor design for biofuel production. Bioeng. 2011; 108:1049–1055. © 2010 Wiley Periodicals, Inc.     

Association of CRISP2, CCT8, PEBP1 mRNA abundance in sperm and sire conception rate in Holstein bulls

The objective was to determine the association of mRNA expression of cystine rich secretary protein 2 (CRISP2), chaperonin containing T-complex protein 1, subunit 8 (CCT8), and phosphatidylethanolamine-binding protein 1 (PEBP1), in sperm of Holstein bulls with Sire Conception Rate (SCR) scores between −4 and +4. These proteins were involved in sperm capacitation and sperm-egg fusion. Samples of sperm obtained on a single day from Holstein bulls (N = 34) in a commercial AI centre were used to evaluate relative mRNA expression of CRISP2, CCT8, and PEBP1. The mRNA abundance of CRISP2 was positively correlated (r = 0.88; P < 0.002), CCT8 was negatively correlated (r = −0.87; P < 0.002), and PEBP1 was positively correlated (r = 0.83; P < 0.006) with SCR-scores. The means of CRISP2 mRNA abundance was greater among positive SCR-score bulls (2.5 to 8 fold), the means of CCT8 mRNA abundance was greater among the negative SCR-score bulls (9.5 to 3.5 fold), and the means of PEBP1 mRNA abundance was greater for the positive SCR-score bulls (5.4 to 7.7 fold). In multivariate regression models predicting SCR-scores, mRNA abundance of CCT8 was significantly associated with SCR-score in all models. In the presence of CRISP2 mRNA abundance in the model, the SCR score's predictability of PEBP1 was insignificant. However, in the absence of CRISP2 mRNA abundance in the model, the SCR-score's predictability of PEBP1 was significant. In multivariate regression models, CRISP2 and CCT8 mRNA expression in sperm accounted for 95% of the variance in Holstein bull's SCR-scores. In conclusion, Holstein bulls with greater CRISP2 and lower CCT8 mRNA expression in sperm had higher probabilities of siring calves.     

Semaphorin-plexin signalling genes associated with human breast tumourigenesis

Introduction

Gene expression profiling has enabled us to demonstrate the heterogeneity of breast cancers. The potential of a tumour to grow and metastasise is partly dependant on its ability to initiate angiogenesis or growth and remodelling of new blood vessels, usually from a pre-existing vascular network, to ensure delivery of oxygen, nutrients, and growth factors to rapidly dividing transformed cells along with access to the systemic circulation. Cell-cell signalling of semaphorin ligands through interaction with their plexin receptors is important for the homeostasis and morphogenesis of many tissues and has been widely studied for a role in neural connectivity, cancer, cell migration and immune responses. This study investigated the role of four semaphorin/plexin signalling genes in human breast cancers in vivo and in vitro.

Materials and methods

mRNA was extracted from formalin fixed paraffin embedded archival breast invasive ductal carcinoma tissue samples of progressive grades (grades I-III) and compared to tissue from benign tumours. Gene expression profiles were determined by microarray using the Affymetrix GeneChip® Human Genome U133 Plus 2.0 Arrays and validated by Q-PCR using a Corbett RotorGene 6000. Following validation, the gene expression profile of the identified targets was correlated with those of the human breast cancer cell lines MCF-7 and MDA-MD-231.

Results

The array data revealed that 888 genes were found to be significantly (p ≤ 0.05) differentially expressed between grades I and II tumours and 563 genes between grade III and benign tumours. From these genes, we identified four genes involved in semaphorin-plexin signalling including SEMA4D which has previously been identified as being involved in increased angiogenesis in breast cancers, and three other genes, SEMA4F, PLXNA2 and PLXNA3, which in the literature were associated with tumourigenesis, but not directly in breast tumourigenesis. The microarray analysis revealed that SEMA4D was significantly (P = 0.0347) down-regulated in the grade III tumours compared to benign tumours; SEMA4F, was significantly (P = 0.0159) down-regulated between grades I and II tumours; PLXNA2 was significantly (P = 0.036) down-regulated between grade III and benign tumours and PLXNA3 significantly (P = 0.042) up-regulated between grades I and II tumours. Gene expression of SEMA4D was validated using Q-PCR, demonstrating the same expression profile in both data sets. When the sample set was increased to incorporate more cases, SEMA4D continued to follow the same expression profile, including statistical significance for the differences observed and small standard deviations. In vitro the same pattern was present where expression for SEMA4D was significantly higher in MDA-MB-231 cells when compared to MCF-7 cells. The expression of SEMA4F, PLXNA2 and PLXNA3 could not be validated using Q-PCR, however in vitro analysis of these three genes revealed that both SEMA4F and PLXNA3 followed the microarray trend in expression, although they did not reach significance. In contrast, PLXNA2 demonstrated statistical significance and was in concordance with the literature.

Discussion

We, and others, have proposed SEMA4D to be a gene with a potentially protective effect in benign tumours that contributes to tumour growth and metastatic suppression. Previous data supports a role for SEMA4F as a tumour suppressor in the peripheral nervous system but our data seems to indicate that the gene is involved in tumour progression in breast cancer. Our in vitro analysis of PLXNA2 revealed that the gene has higher expression in more aggressive breast cancer cell types. Finally, our in vitro analysis on PLXNA3 also suggest that this gene may have some form of growth suppressive role in breast cancer, in addition to a similar role for the gene previously reported in ovarian cancer. From the data obtained in this study, SEMA4D may have a role in more aggressive and potentially metastatic breast tumours.

Conclusions

Semaphorins and their receptors, the plexins, have been implicated in numerous aspects of neural development, however their expression in many other epithelial tissues suggests that the semaphorin-plexin signalling system also contributes to blood vessel growth and development. These findings warrant further investigation of the role of semaphorins and plexins and their role in normal and tumour-induced angiogenesis in vivo and in vitro. This may represent a new front of attack in anti-angiogenic therapies of breast and other cancers.     

Obesity resistance and increased hepatic expression of catabolism-related mRNAs in Cnot3+/- mice

Obesity is a life-threatening factor and is often associated with dysregulation of gene expression. Here, we show that the CNOT3 subunit of the CCR4-NOT deadenylase complex is critical to metabolic regulation. Cnot3(+/-) mice are lean with hepatic and adipose tissues containing reduced levels of lipids, and show increased metabolic rates and enhanced glucose tolerance. Cnot3(+/-) mice remain lean and sensitive to insulin even on a high-fat diet. Furthermore, introduction of Cnot3 haplodeficiency in ob/ob mice ameliorated the obese phenotype. Hepatic expression of most mRNAs is not altered in Cnot3(+/-) vis-à-vis wild-type mice. However, the levels of specific mRNAs, such as those coding for energy metabolism-related PDK4 and IGFBP1, are increased in Cnot3(+/-) hepatocytes, having poly(A) tails that are longer than those seen in control cells. We provide evidence that CNOT3 is involved in recruitment of the CCR4-NOT deadenylase to the 3' end of specific mRNAs. Finally, as CNOT3 levels in the liver and white adipose tissues decrease upon fasting, we propose that CNOT3 responds to feeding conditions to regulate deadenylation-specific mRNAs and energy metabolism.