YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules

<p>Abstract</p> <p>Background</p> <p>YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs.</p> <p>Results</p> <p>We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly <it>in vitro</it>. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes.</p> <p>Conclusion</p> <p>These results suggest that YB-1 may regulate microtubule assembly <it>in vivo </it>and that its interaction with tubulin may contribute to the control of mRNA translation.</p

Formation of Amyloid-Like Fibrils by Y-Box Binding Protein 1 (YB-1) Is Mediated by Its Cold Shock Domain and Modulated by Disordered Terminal Domains

YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of DNA- and mRNA-dependent events in the cell. It consists of three structurally different domains: its central cold shock domain has the structure of a β-barrel, while the flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 is capable of forming elongated fibrils under high ionic strength conditions. Here we report that it is the cold shock domain that is responsible for formation of YB-1 fibrils, while the terminal domains differentially modulate this process depending on salt conditions. We demonstrate that YB-1 fibrils have amyloid-like features, including affinity for specific dyes and a typical X-ray diffraction pattern, and that in contrast to most of amyloids, they disassemble under nearly physiological conditions

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

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 <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 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