ポリＡ分解酵素Ccr４は定常状態でのLRG1 mRNAの翻訳抑制に関与する

筑波大学 (University of Tsukuba)201

ポリＡ分解酵素Ccr４は定常状態でのLRG1 mRNAの翻訳抑制に関与する

筑波大学 (University of Tsukuba)201

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

Cytoplasmic deadenylase Ccr4 is required for translational repression of <i>LRG1</i> mRNA in the stationary phase

<div><p>Ccr4 is a major cytoplasmic deadenylase involved in mRNA poly(A) tail shortening in <i>Saccharomyces cerevisiae</i>. We have previously shown that Ccr4 negatively regulates expression of <i>LRG1</i> mRNA encoding a GTPase-activating protein for the small GTPase Rho1, a component of cell wall integrity pathway, and deletion of <i>LRG1</i> suppresses the temperature-sensitive growth defect of the <i>ccr4Δ</i> mutant. We have also shown that the slow growth of the <i>ccr4Δ</i> mutant is suppressed by deletion of another gene, <i>PBP1</i>, encoding a poly(A)-binding protein (Pab1)-binding protein 1; however, the underlying mechanism still remains unknown. In this study, we investigated how <i>ccr4Δ</i> and <i>pbp1Δ</i> mutations influence on the length of poly(A) tail and <i>LRG1</i> mRNA and protein levels during long-term cultivation. In the log-phase <i>ccr4Δ</i> mutant cells, <i>LRG1</i> poly(A) tail was longer and <i>LRG1</i> mRNA level was higher than those in the log-phase wild-type (WT) cells. Unexpectedly, Lrg1 protein level in the <i>ccr4Δ</i> mutant cells was comparable with that in WT. In the stationary-phase <i>ccr4Δ</i> mutant cells, <i>LRG1</i> poly(A) tail length was still longer and <i>LRG1</i> mRNA level was still higher than those in WT cells. In contrast to the log phase, Lrg1 protein level in the stationary-phase <i>ccr4Δ</i> mutant cells was maintained much higher than that in the stationary-phase WT cells. Consistently, active translating ribosomes still remained abundant in the stationary-phase <i>ccr4Δ</i> mutant cells, whereas they were strongly decreased in the stationary-phase WT cells. Loss of <i>PBP1</i> reduced the <i>LRG1</i> poly(A) tail length as well as <i>LRG1</i> mRNA and protein levels in the stationary-phase <i>ccr4Δ</i> mutant cells. Our results suggest that Ccr4 regulates not only <i>LRG1</i> mRNA level through poly(A) shortening but also the translation of <i>LRG1</i> mRNA, and that Pbp1 is involved in the Ccr4-mediated regulation of mRNA stability and translation.</p></div

The other target mRNAs of Puf5 including <i>MCM2</i>, <i>MCM4</i>, <i>MCM7</i>, and <i>ELM1</i> showed the expression patterns similar to that of <i>LRG1</i>.

<p>(A) Protein expressions for products of Puf5 target mRNAs in WT, <i>ccr4Δ</i>, and <i>ccr4Δ pbp1Δ</i> mutant cells. The WT, <i>ccr4Δ</i>, and <i>ccr4Δ pbp1Δ</i> mutant cells harboring the plasmid pRS314-3FLAG-LRG1 were grown at 28°C from the log phase to the stationary phase in SC-Trp media. The cells were collected at the indicated times, and cell extracts were prepared for immunoblotting with anti-Flag (3xFlag-Lrg1), anti-Mcm2, anti Mcm4, anti-Mcm7, anti-Elm1, and anti-Pgk1 antibodies. Pgk1 was used as the loading control. (B) The Lrg1 protein level in WT, <i>puf5Δ</i> mutant in the stationary phase. WT and <i>puf5Δ</i> mutant strains harboring the plasmid pRS314-3FLAG-LRG1 were grown at 28°C from the log phase to the stationary phase in SC-Trp media. The cells were collected at the indicated times, and cell extracts were prepared for immunoblotting with anti-Flag (3xFlag-Lrg1) and anti-Pgk1 antibodies. Pgk1 was used as the loading control.</p

Overexpression of <i>LRG1</i> was toxic to the <i>ccr4Δ</i> mutant but not to the <i>ccr4Δ pbp1Δ</i> mutant at high temperature.

<p>The WT, <i>ccr4Δ</i>, and <i>ccr4Δ pbp1Δ</i> mutant strains harboring the plasmid YEplac195-LRG1 were grown at 28°C to the mid log phase. The same optical densities of cells were spotted onto SC-Ura plates and then incubated at 25°C or 37°C for 3 days.</p

<i>LRG1</i> mRNA and protein levels were increased in the stationary-phase <i>ccr4Δ</i> mutant.

<p>(A) The growth curves of WT, <i>ccr4Δ</i>, and <i>ccr4Δ pbp1Δ</i> cells in SC-Trp media. The strains harboring the plasmid pRS314-3FLAG-LRG1 were pre-cultured overnight and then transferred into fresh SC-Trp media to grow for 5 days at 28°C. The cell cultures were taken at the indicated times to measure A600 nm. (B) The <i>LRG1</i> poly(A) tail lengths in WT, <i>ccr4Δ</i>, <i>ccr4Δ pbp1Δ</i>, and <i>ccr4Δ pbp1Δ pan2Δ</i> mutant cells in the log phase (4 h) and the stationary phase (48 h). The strains were grown in YPD media from the log phase to the stationary phase at 28°C. The cells were collected at indicated time points for RNA isolation. The <i>LRG1</i> poly(A) tail was amplified using the poly(A) tail length kit. The average poly(A) tail lengths were determined by sequencing. (C) Expression of <i>LRG1</i> mRNA in WT, <i>ccr4Δ</i>, and <i>ccr4Δ pbp1Δ</i> mutants. The strains harboring the plasmid pRS314-3FLAG-LRG1 were grown at 28°C from the log phase to the stationary phase in SC-Trp media. The cells were collected at the indicated times for RNA isolation. The <i>LRG1</i> mRNA levels were quantified by qRT-PCR analysis, and the relative mRNA levels were calculated using delta delta Ct method normalized to <i>SCR1</i> reference gene. The data show mean ± SEM (n = 4) of fold change of <i>LRG1</i> mRNA from WT cells at 4 h of culture. *P < 0.05, **P < 0.01 as determined by Tukey’s test. (D) Expression of Lrg1 protein in WT, <i>ccr4Δ</i>, and <i>ccr4Δ pbp1Δ</i> mutants. The strains harboring the plasmid pRS314-3FLAG-LRG1 were grown at 28°C from the log phase to the stationary phase in SC-Trp media. The cells were collected at the indicated times, and cell extracts were prepared for immunoblotting with anti-Flag (3xFlag-Lrg1) and anti-Pgk1 antibodies. The intensities of 3xFlag-Lrg1 signals were measured and normalized to the Pgk1 signals. The values are plotted as the fold change from WT cells at 4 h of culture. The data show mean ± SEM (n = 3). (E) The deadenylase activity of Ccr4 is required for the regulation of <i>LRG1</i> expression. The plasmid YCplac33-CCR4 or plasmid YCplac33-CCR4-D713A or empty vector was transformed into the <i>ccr4Δ</i> mutant cells harboring plasmid pRS314-3FLAG-LRG1. Transformants were grown at 28°C from the log phase to the stationary phase in SC-Trp-Ura media. The cells were collected at the indicated times, and cell extracts were prepared for immunoblotting with anti-Flag (3xFlag-Lrg1) and anti-Pgk1 antibodies. Pgk1 was used as the loading control.</p

Active translating polysomes were still abundant in the stationary-phase <i>ccr4Δ</i> mutant.

<p>(A) Growth curves of WT, <i>ccr4Δ</i>, and <i>ccr4Δ pbp1Δ</i> cells in YPD media. The strains were pre-cultured overnight and then transferred into fresh YPD media to grow for 5 days at 28°C. The cell cultures were taken at the indicated times to measure A600 nm. (B) The WT, <i>ccr4Δ</i>, and <i>ccr4Δ pbp1Δ</i> mutant cells went into the stationary phase after 48 h of culture in YPD media. The strains were pre-cultured overnight in YPD media and then transferred into fresh YPD media to grow for 5 days at 28°C. The cultures were taken at the indicated times to measure glucose concentration. The ethanol concentrations were measured after glucose in the media had been depleted. (C) Polysome analyses of WT, <i>ccr4Δ</i>, <i>ccr4Δ pbp1Δ</i>, and <i>ccr4Δ pbp1Δ pan2Δ</i> mutant cells in the log phase (4 h). The strains were pre-cultured overnight in YPD media and then transferred into fresh YPD media to grow for 4 h at 28°C. The cells were collected and cell lysates were prepared for polysome analysis as described in material and methods. (D) Polysome analyses of WT, <i>ccr4Δ</i>, <i>ccr4Δ pbp1Δ</i>, and <i>ccr4Δ pbp1Δ pan2Δ</i> mutant cells in the stationary phase (72 h) in YPD. The strains were pre-cultured overnight in YPD media and then transferred into fresh media to grow for 72 h at 28°C. The cells were collected and cell lysates were prepared for polysome analysis as described in material and methods. (E) Polysome analyses and <i>LRG1</i> mRNA levels of WT, <i>ccr4Δ</i>, <i>ccr4Δ pbp1Δ</i>, and <i>ccr4Δ pbp1Δ pan2Δ</i> mutant cells in the stationary phase (72 h) in SC-Trp media. The strains were pre-cultured overnight in SC-Trp media and then transferred into fresh media to grow for 72 h at 28°C. The cells were collected and cell lysates were prepared for polysome analysis as described in material and methods. The same volumes of RNA isolated from each of polysome fractions were subjected to RT-PCR to synthesize cDNAs. The <i>LRG1</i> cDNA was amplified using Taq polymerase. The data show the relative amounts of <i>LRG1</i> cDNA from the polysome fractions of the strains. We obtained similar results in two independent experiments and show a representative.</p