YB-1 Synthesis Is Regulated by mTOR Signaling Pathway

<div><p>YB-1 is a eukaryotic protein with numerous intra- and extracellular functions based on its ability to interact with RNA, DNA, and many proteins. In spite of achievements in studying its functions, regulation of YB-1 synthesis in the cell remains poorly understood. In the current study Western and Northern blotting were used to determine the amounts of YB-1 and <em>YB-1</em> mRNA in rabbit organs and several cell lines. As found, in the majority of studied eukaryotic cells a considerable proportion of <em>YB-1</em> mRNA was stored in free mRNPs, i.e., was poorly translated. Also, we demonstrated that YB-1 synthesis depended on conditions that determined the rate of cell division. Specific suppression of YB-1 synthesis resulted from inhibition of the mTOR signaling pathway with inhibitor PP242, but not rapamycin. Experiments on reporter constructs showed that dependence of <em>YB-1</em> mRNA translation on activity of the mTOR signaling pathway was dictated by 5′ untranslated regions of this mRNA, irrelatively of the TOP-like sequences at the beginning of 5′ UTR.</p> </div

ATP-Independent Initiation during Cap-Independent Translation of m6A-Modified mRNA

The methylation of adenosine in the N6 position (m6A) is a widely used modification of eukaryotic mRNAs. Its importance for the regulation of mRNA translation was put forward recently, essentially due to the ability of methylated mRNA to be translated in conditions of inhibited cap-dependent translation initiation, e.g., under stress. However, the peculiarities of translation initiation on m6A-modified mRNAs are not fully known. In this study, we used toeprinting and translation in a cell-free system to confirm that m6A-modified mRNAs can be translated in conditions of suppressed cap-dependent translation. We show for the first time that m6A-modified mRNAs display not only decreased elongation, but also a lower efficiency of translation initiation. Additionally, we report relative resistance of m6A-mRNA translation initiation in the absence of ATP and inhibited eIF4A activity. Our novel findings indicate that the scanning of m6A-modified leader sequences is performed by a noncanonical mechanism

Analysis of <i>YB-1</i> mRNA distribution between polysomal and free mRNP fractions in various cell lines. A,

<p>Cells were scraped and lysed. Nuclei and mitochondria were removed by centrifugation, and cytosolic extracts were then spun through a 50% sucrose cushion at 100,000 rpm in a TLA-100 centrifuge (Beckman) for 13 min to separate postpolysomal supernatant from polysomes. Total RNA from postpolysomal supernatant and polysomal fractions (resuspended pellets) were extracted with TRIzol, subjected to agarose gel electrophoresis and Northern blot hybridization to [³²P]-labeled <i>YB-1</i> and <i>GAPDH</i> cDNA. <b>B,</b> Relative radioactivity of the bands was determined using a Packard Cyclone Storage Phosphor System (Packard Instrument Company, Inc.). The sum of relative radioactivity values in free and polysomal mRNP fractions was taken to be 100%.</p

Analysis of <i>YB-1</i> mRNA distribution between polysomal and free mRNP fractions in rabbit organs. A,

<p>Tissue cytosolic extracts were spun through a 50% sucrose cushion at 100,000 rpm in a TLA-100 centrifuge (Beckman) for 13 min to separate postpolysomal supernatant from polysomes. Total RNA from postpolysomal supernatant and polysomal fractions (resuspended pellets) were extracted with TRIzol, subjected to agarose gel electrophoresis and Northern blot hybridization to [³²P]-labeled <i>YB-1</i> and <i>GAPDH</i> cDNA. <b>B,</b> Relative radioactivity of the bands was determined using a Packard Cyclone Storage Phosphor System (Packard Instrument Company, Inc.). The sum of relative radioactivity values in free and polysomal mRNP fractions was taken to be 100%.</p

Dependence of YB-1 synthesis on cell confluence.

<p>NIH3T3 and HEK293 cells of various confluence were [³⁵S]-methionine-labeled for 1 h, harvested and lysed. Cell lysates were counterbalanced by radioactivity (A and C) and used for immunoprecipitation with anti-YB-1 antibody. Proteins bound to antibodies were resolved by acid-urea PAGE, and [³⁵S]-labeled proteins were detected by autoradiography (B and D). Relative radioactivity of the bands was determined using a Packard Cyclone Storage Phosphor System (Packard Instrument Company, Inc.).</p

Renewal of YB-1 amount and YB-1 synthesis after serum starvation release.

<p>3T3 (<b>A</b>) and HEK293(<b>B</b>) cells were serum starved (2 days). Cells were harvested at indicated time intervals after serum addition, lysed and used for Western blot analysis. 3T3(<b>C</b>) and HEK293(<b>D</b>) cells were serum starved (2 days). Control cells, serum starved cells and serum stimulated (6 h) cells were labeled with [³⁵S]-methionine for 2 h, harvested and lysed. Cell lysates were used for immunoprecipitation with anti-YB-1 antibody. Proteins bound to antibodies were resolved by acid-urea PAGE, and [³⁵S]-labeled proteins were detected by autoradiography. Relative radioactivity of the bands was determined using a Packard Cyclone Storage Phosphor System (Packard Instrument Company, Inc.). The level of YB-1 synthesis in cells without serum starvation was taken to be 100%.</p

Assessment of YB-1 synthesis in the cell. A

<p>. HeLa cells were labeled with [³⁵S]-methionine for 2 h, harvested and lysed. Cell lysate was used for immunoprecipitation with preimmune antibody or YB-1 antibody. Proteins bound to antibodies were resolved by acid-urea PAGE, stained with CBB G250, and the [³⁵S]-labeled proteins were detected by autoradiography. Protein with an electrophoretic mobility corresponding to the recombinant YB-1 was cut out from the gel and identified by mass-spectrometry as YB-1. <b>B,</b> Assessment of YB-1 synthesis in cells of various lines. Cells were labeled using [³⁵S]-methionine, harvested and lysed. Cell lysates were counterbalanced by radioactivity and used for immunoprecipitation with anti-YB-1 antibody. Proteins bound to antibodies were resolved by acid-urea PAGE, and the [³⁵S]-labeled proteins were detected by autoradiography.</p

Analysis of YB-1 and <i>YB-1</i> mRNA amounts in the cell.

<p><b>A</b> and <b>B,</b> 15 µg of total protein from lysates of various cell lines (A) or 50 µg of total protein from tissue lysates (B) were analyzed by Western-blotting. <b>C</b> and <b>D,</b> 10 µg of total RNA from lysates of various cell lines (C) or tissue lysates (D) were analyzed by Northern-blotting.</p

Effect of various cell signaling pathway inhibitors on endogenous YB-1 synthesis in the cell.

<p>Untreated (lane 1) or 1 µM PP242-treated (lane 2), or 0.1 µM rapamycin-treated (lane 3), or 0.5 µM wortmannin-treated (lane 4), or 10 µM U0126-treated (lane 5) HeLa cells were labeled with [³⁵S]-methionine for 2 h, harvested and lysed. Cell lysates were counterbalanced by the total protein (<b>A</b>) and used for Western blotting (<b>D</b>) or immunoprecipitation with anti-YB-1 antibody. Proteins bound to antibodies were resolved by acid-urea PAGE, and [³⁵S]-labeled proteins were detected by autoradiography (<b>B</b>). Relative radioactivity of the bands was determined using a Packard Cyclone Storage Phosphor System (Packard Instrument Company, Inc.) (<b>C</b>). The level of YB-1 synthesis in cells without drugs was taken to be 100%.</p