MicroRNAs (miRNAs) are incorporated into miRNP complexes and regulate protein expression post-transcriptionally through binding to 3'-untranslated regions of target mRNAs. Here we describe a ...recapitulation of let-7 miRNA-mediated translational repression in a cell-free system, which was established with extracts prepared from HEK293F cells overexpressing miRNA pathway components. In this system, both the cap and poly(A) tail are required for the translational repression, and let-7 directs the deadenylation of target mRNAs. Our work suggests that let-7 miRNPs containing Argonaute and GW182 impair the synergistic enhancement of translation by the 5'-cap and 3'-poly(A) tail, resulting in translational repression.
In mammalian cells, microRNAs (miRNAs) are incorporated into miRNA-induced silencing complexes (miRISCs), which regulate protein expression post-transcriptionally through binding to 3'-untranslated ...regions of target mRNAs. Argonaute2 (Ago2), a key component of the miRISC, recruits GW182, a component of the processing body (GW/P-body), to the target mRNAs. To elucidate the function of GW182 in an miRNA-mediated translational repression, we analyzed Argonaute-binding sites in GW182. We found that human GW182 contains three binding sites for Ago2, within the amino-terminal glycine tryptophan (GW/WG)-repeated region that is characteristic of the GW182 family proteins. We also found that the first and second Ago2-binding site is conserved within the amino-terminal half of TNRC6B, which is a paralog of GW182. Each of the Ago-binding sites is alone sufficient to bind Ago2. Furthermore, we demonstrated that multiple Argonaute proteins were connected via the GW182 protein. A GW182 fragment containing the Ago2-binding region partially relieved let-7-mediated repression of protein synthesis in a mammalian cell-free system. Coincidentally, let-7-directed target mRNA deadenylation was delayed. Together, these results strongly suggested that the interactions of GW182 with Argonautes may induce the formation of large complexes containing miRNA target mRNAs, and may be critical for miRNA-mediated translational repression.
GW182 family proteins are a key component of microRNA–protein complex eliciting translational repression and/or degradation of microRNA‐targets. The microRNAs in complex with Argonaute proteins bind ...to target mRNAs, and GW182 proteins are recruited by association with Argonaute proteins. The GW182 protein acts as a scaffold that links the Argonaute protein to silencing machineries including the CCR4–NOT complex which accelerates deadenylation and inhibits translation. The carboxyl‐terminal effector domain of GW182 protein, also called the silencing domain, has been shown to bind to the subunits of the CCR4–NOT complex, the CNOT1 and the CNOT9. Here we show that a small region within the amino‐terminal Argonaute‐binding domain of human GW182/TNRC6A can associate with the CCR4–NOT complex. This region resides between the two Argonaute‐binding sites and contains reiterated GW/WG‐motifs. Alanine mutation experiments showed that multiple tryptophan residues are required for the association with the CCR4–NOT complex. Furthermore, co‐expression and immunoprecipitation assays suggested that the CNOT9 subunit of the CCR4–NOT complex is a possible binding partner of this region. Our work, taken together with previous studies, indicates that the human GW182 protein contains multiple binding interfaces to the CCR4–NOT complex.
We show that a small region within the amino‐terminal Ago‐binding domain of human GW182/TNRC6A can associate with the CCR4–NOT complex. This region resides between the two Ago‐binding sites and contains reiterated GW/WG‐motifs. Furthermore, we show that the CNOT9 subunit of the CCR4–NOT complex is a possible binding partner of this region.
To study the functions of RNA-binding proteins independent of their RNA-binding activity, tethering methods have been developed, based on the use of the RNA-binding domain of a well-characterized ...RNA-binding protein and its target RNA. Two bacteriophage proteins have mainly been used as tethers: the MS2 coat protein and the lambda N protein. Here we report an alternative system using the Tat (trans-activator) peptide from the bovine immunodeficiency virus (BIV), which binds to BIV–TAR (trans-activation response) RNA. We demonstrate the usefulness of this system by applying it to the analysis of the TNRC6B protein, a component of the microRNA-induced silencing complex.
Purpose To examine the ability of young and elderly individuals to control the timing and force of periodic sequential foot tapping. Subjects and Methods Participants were 10 young (age, 22.1 ± 4.3 ...years) and 10 elderly individuals (74.8 ± 6.7 years) who were healthy and active. The foot tapping task consisted of practice (stimulus-synchronized tapping with visual feedback) and recall trials (self-paced tapping without visual feedback), periodically performed in this order, at 500-, 1,000-, and 2,000-ms target interstimulus-onset intervals, with a target force of 20% maximum voluntary contraction of the ankle plantar-flexor muscle. Results The coefficients of variation of force and intertap interval, used for quantifying the steadiness of the trials, were significantly greater in the elderly than in the young individuals. At the 500-ms interstimulus-onset interval, age-related effects were observed on the normalized mean absolute error of force, which was used to quantify the accuracy of the trials. The coefficients of variation of intertap interval for elderly individuals were significantly greater in the practice than in the recall trials at the 500- and 1,000-ms interstimulus-onset intervals. Conclusion The elderly individuals exhibited greater force and timing variability than the young individuals and showed impaired visuomotor processing during foot tapping sequences.
SecA is an ATP-driven motor for protein translocation in bacteria and plants. Mycobacteria and listeria were recently found to possess two functionally distinct secA genes. In this study, we found ...that Cyanidioschyzon merolae, a unicellular red alga, possessed two distinct secA-homologous genes; one encoded in the cell nucleus and the other in the plastid genome. We found that the plastid-encoded SecA homolog showed significant ATPase activity at low temperature, and that the ATPase activity of the nuclear-encoded SecA homolog showed significant activity at high temperature. We propose that the two SecA homologs play different roles in protein translocation.
Purpose: This is an expanded version of the study “Selecting Technical Vocabulary in the Field of Physical Therapy and Determining its Characteristics”, which previously appeared in this journal. The ...aim of this study is to identify the features of English in the field of physical therapy from the viewpoint of collocational patterns. Method: We examined collocational patterns in the two corpora created for the previous study (RA corpus: 397,874 words, PT text corpus: 546,666 words). The heads of collocation for analysis were combinations of adverb+verb, verb+noun, and adjective+noun. In each head, two-word units which co-occur three times or more were extracted from the two corpora. A mutual information score of the units was calculated to identify whether there are unique collocational patterns which have stronger connections in texts in the physical therapy field. Mutual information shows the expected probability of co-occurrence. The higher score tell us the stronger attraction between the words. Results: In all heads, there are combinations of two words showing high scores in common across the two corpora. Conclusion: Knowledge of collocation is vital to improve reading ability. Learning collocational patterns observed in the two corpora will be an effective strategy to increase the speed of understanding physical therapy texts.
The aim of this study was to investigate the effects of culturing human dermal fibroblasts (HDFs) at different temperatures on cell proliferation. In this study, 5×104 cells/dish of HDFs were plated ...in a 35-mm dish and incubated at 31, 33, 35, 37, and 39°C. After 24, 48, and 72 hours of incubation, the cells were detached and the number of live and dead cells were counted using a hemocytometer. For analysis, the ratio of cell number in each temperature condition divided by the cell number in 37°C incubation at 24 hours was used. The cell viability was also calculated for each condition. For statistical analysis, two-way analysis of variance was used for temperature and time, and one-way analysis of variance was used for cell viability. Bonferroni’s multiple comparison test was performed as post hoc analysis. The results showed significant differences (p＜0.01) for both the main effect and the interaction effect in the two-way ANOVA. The ratio of cells at different incubator temperature settings was higher at both 48 and 72 hours, depending on the higher incubation temperature. No significant difference was observed in cell viability. In conclusion, cell proliferation was lower at 31, 33, and 35°C than at 37°C under the five conditions examined in this study, while cell proliferation was enhanced at 39°C compared to 37°C.
The transmembrane anion transport activity of 43 synthetic molecules based on the structure of marine alkaloid tambjamine were assessed in model phospholipid (POPC) liposomes. The anionophoric ...activity of these molecules showed a parabolic dependence with lipophilicity, with an optimum range for transport efficiency. Using a quantitative structure-transport activity (QSAR) approach it was possible to rationalize these results and to quantify the contribution of lipophilicity to the transport activity of these derivatives. While the optimal value of log
and the curvature of the parabolic dependence is a property of the membrane (and so similar for the different series of substituents) we found that for relatively simple substituents in certain locations on the tambjamine core, hydrophobic interactions clearly dominate, but for others, more specific interactions are present that change the position of the membrane hydrophobicity parabolic envelope.
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