Change in Nutritional Status Modulates the Abundance of Critical Pre-initiation Intermediate Complexes During Translation Initiation \u3cem\u3ein Vivo\u3c/em\u3e

In eukaryotic translation initiation, eIF2∙GTP–Met-tRNAiMet ternary complex (TC) interacts with eIF3–eIF1–eIF5 complex to form the multifactor complex (MFC), while eIF2∙GDP associates with eIF2B for guanine nucleotide exchange. Gcn2p phosphorylates eIF2 to inhibit eIF2B. Here we evaluate the abundance of eIFs and their pre-initiation intermediate complexes in gcn2 deletion mutant grown under different conditions. We show that ribosomes are three times as abundant as eIF1, eIF2 and eIF5, while eIF3 is half as abundant as the latter three and hence, the limiting component in MFC formation. By quantitative immunoprecipitation, we estimate that ∼ 15% of the cellular eIF2 is found in TC during rapid growth in a complex rich medium. Most of the TC is found in MFC, and important, ∼ 40% of the total eIF2 is associated with eIF5 but lacks tRNAiMet. When the gcn2Δ mutant grows less rapidly in a defined complete medium, TC abundance increases threefold without altering the abundance of each individual factor. Interestingly, the TC increase is suppressed by eIF5 overexpression and Gcn2p expression. Thus, eIF2B-catalyzed TC formation appears to be fine-tuned by eIF2 phosphorylation and the novel eIF2/eIF5 complex lacking tRNAiMet

The roles of stress-activated Sty1 and Gcn2 kinases and proto-oncoprotein homologue Int6/eIF3e in responses to endogenous oxidative stress during histidine starvation

In fission yeast, Sty1 and Gcn2 are important protein kinases regulating gene expression in response to amino acid starvation. The translation factor subunit eIF3e/Int6 promotes the Sty1-dependent response by increasing the abundance of Atf1, a transcription factor targeted by Sty1. While Gcn2 promotes expression of amino acid biosynthesis enzymes, the mechanism and function for Sty1 activation and Int6/eIF3e involvement during this nutrient stress is not understood. Here we show that mutants lacking sty1+ or gcn2+ display reduced viabilities during histidine depletion stress in a manner suppressible by the antioxidant, N-acetyl cysteine, suggesting that these protein kinases function to alleviate endogenous oxidative damage generated during nutrient starvation. Int6/eIF3e also promotes cell viability by a mechanism involving stimulation of the Sty1 response to oxidative damage. In further support of these observations, microarray data suggests that, during histidine starvation, int6Δ increases the duration of Sty1-activated gene expression linked to oxidative stress due to the initial attenuation of Sty1-dependent transcription. Moreover, loss of gcn2 induces the expression of a new set of genes not activated in wild-type cells starved for histidine. These genes encode heatshock proteins, redox enzymes and proteins involved in mitochondrial maintenance, in agreement with the idea that oxidative stress is imposed onto gcn2Δ cells. Furthermore, the early Sty1 activation promotes a rapid Gcn2 activation on histidine starvation. These results suggest that Gcn2, Sty1, and Int6/eIF3e are functionally integrated and cooperate to respond to oxidative stress that is generated during histidine starvation

Ubiquitination of stalled ribosome triggers ribosome-associated quality control

Translation arrest by polybasic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. Here we report that ubiquitination of the 40S ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 (or RQT1) is required for RQC. We identify a RQC-trigger (RQT) subcomplex composed of the RNA helicase-family protein Slh1/Rqt2, the ubiquitin-binding protein Cue3/Rqt3, and yKR023W/Rqt4 that is required for RQC. The defects in RQC of the RQT mutants correlate with sensitivity to anisomycin, which stalls ribosome at the rotated form. Cryo-electron microscopy analysis reveals that Hel2-bound ribosome are dominantly the rotated form with hybrid tRNAs. Ribosome profiling reveals that ribosomes stalled at the rotated state with specific pairs of codons at P-A sites serve as RQC substrates. Rqt1 specifically ubiquitinates these arrested ribosomes to target them to the RQT complex, allowing subsequent RQC reactions including dissociation of the stalled ribosome into subunits

Optimized protocol for tRNA identification in the ribosomal complexes from human cell lines

Summary: Here, we describe a protocol for tRNA identification in the 60S ribosome-nascent peptide complex co-purified with Nuclear Export Mediator Factor (NEMF), a responsible factor for C-terminal alanine and threonine tailing of the nascent peptide. Our protocol is based on regular reverse transcription followed by quantitative Polymerase chain reaction (PCR). Although this method cannot distinguish between amino acid-charged and uncharged and base-modified and unmodified tRNAs, it is a convenient way to estimate the relative level of tRNA species and thus can be useful for researchers.For complete details on the use and execution of this protocol, please refer to Udagawa et al. (2021)

Transmission of Larger Amplitude Ultrasound with SiC Transistor Pulser for Subharmonic Signal Measurements at Closed Cracks

AbstractWith measurements using the Subharmonic Phased Array for Crack Evaluation system, transmission of large-amplitude ultrasound is required to generate subharmonics near closed cracks. We designed a new pulser using a SiC transistor making larger currents available with low output impedance to generate large-amplitude signals. We report on the efficacies of the pulser in comparison with a conventional pulser. We investigated combinations of excitations from several transducers and the two pulsers by measuring signal amplitude, effective voltage, and electric current for each transducer. By applying larger electric currents, larger excitation amplitudes were produced, even when the capacitance oftransducer was larger

Efficacy of prophylactic splenectomy for proximal advanced gastric cancer invading greater curvature

Abstract Background For proximal gastric cancer invading the greater curvature, concomitant splenectomy is frequently performed to secure the clearance of lymph node metastases. However, prognostic impact of prophylactic splenectomy remains unclear. The aim of this study was to clarify the oncological significance of prophylactic splenectomy for advanced proximal gastric cancer invading the greater curvature. Methods Retrospective review of 108 patients who underwent total or subtotal gastrectomy for advanced proximal gastric cancer involving the greater curvature was performed. Short-term and long-term outcomes were compared between the patients who underwent splenectomy (n = 63) and those who did not (n = 45). Results Patients who underwent splenectomy showed higher amount of blood loss (538 vs. 450 mL, p = 0.016) and morbidity rate (30.2 vs. 13.3, p = 0.041) compared with those who did not undergo splenectomy. In particular, pancreas-related complications were frequently observed among patients who received splenectomy (17.4 vs. 0%, p = 0.003). However, no significant improvement of long-term outcomes were confirmed in the cases with splenectomy (5-year recurrence-free rate, 60.2 vs. 67.3%; p = 0.609 and 5-year overall survival rates, 63.7 vs. 73.6%; p = 0.769). On the other hand, splenectomy was correlated with marginally better survival in patients with Borrmann type 1 or 2 gastric cancer (p = 0.072). Conclusions For advanced proximal gastric cancer involving the greater curvature, prophylactic splenectomy may have no significant prognostic impact despite the increased morbidity rate after surgery. Such surgical procedure should be avoided as long as lymph node involvement is not evident

Specific miRNA Stabilization by Gld2-Catalyzed Monoadenylation

MicroRNAs (miRNAs) are small, noncoding RNAs that inhibit translation and promote mRNA decay. The levels of mature miRNAs are the result of different rates of transcription, processing, and turnover. The noncanonical polymerase Gld2 has been implicated in the stabilization of miR-122, possibly through catalyzing 3′ monoadenylation; however, there is little evidence that this relationship is one of cause and effect. Here, we biochemically characterize Gld2’s involvement in miRNA monoadenylation and its effect on miRNA stability. We find that Gld2 directly monoadenylates and stabilizes specific miRNA populations in human fibroblasts and that sensitivity to monoadenylation-induced stability depends on nucleotides in the miRNA 3′ end. These results establish a mechanism of miRNA stability and resulting posttranscriptional gene regulation