Desthiobiotin-链亲和素亲和介导纯化间皮瘤细胞 RNA 相互作用蛋白的研究
Summary
Desthiobiotin 标记的合成 25-核苷酸 RNA 寡核苷酸, 其中含有腺嘌呤丰富的元素 (是) 的主题, 允许特定的结合胞浆是结合蛋白。
Abstract
体外RNA 下拉仍然主要用于确定 rna 结合蛋白识别特定 rna 结构和图案的协议的第一步。在这个 rna 下拉协议, 商业上合成的 rna 探针被标记以修改的形式生物素, desthiobiotin 在 RNA 链的 3 ‘ 末端, 可逆地束缚到链亲和素并且因而允许蛋白质洗脱在更加生理上条件。rna desthiobiotin 是固定的, 通过与链亲和素的相互作用, 磁性珠子, 这是用来拉下来的蛋白质, 特别是与 RNA 的利益。非变性和活性蛋白从间皮瘤细胞胞浆的比例被用作蛋白质的来源。此处描述的方法可用于检测已知的 rna 结合蛋白与含有一系列兴趣序列的25核苷酸 (nt) 长 RNA 探针之间的相互作用。这对于完成使用报告向量法实现的 RNA 分子中稳定或不稳定元素的功能表征是有用的。
Introduction
基因表达和基因产物的最终水平可以通过影响 mrna 稳定性和 mrna 翻译率1来严格调节。这些转录后调控机制是通过非编码 rna 和/或 rna 结合蛋白 (限制性商业惯例) 与靶向 mRNA 的相互作用而施加的。它通常是 3 ‘ 未翻译的 mRNA 区域 (3 ‘ UTR-属于基因组2) 的非编码部分, 其中包含特定的cis-管理元素 (), 由反演作用因素 (如 miRNA 或限制性商业惯例) 识别。3. 最好研究的cis元素在 3 ‘ UTR, 是腺嘌呤丰富的元素 (是) 的主题, 这是公认的特定的 AU 结合蛋白 (AUBP), 反过来, 诱导 mRNA 退化/deadenylation (是介导的衰变) 或mRNA 稳定化4。
3 ‘ UTR 的 calretinin mRNA (CALB2) 的大小是 573 bp 长, 包含一个假定的 AUUUA pentamer, 如 AREsite2 的预测, 生物信息学工具 5.与假定的存在相一致的是主题, pmirGLO 向量-报告分析显示了这个元素在 CALB2 mRNA6中的稳定作用。最后, 用体外RNA 下拉法确定了通过 AUBP 稳定 calretinin mRNA 的方法。
由于所有非编码 rna 与蛋白质7相互作用,体外rna 下拉是确定 rna interactors 以帮助破译分子机制的一种很好的方法和首选的检测手段。在这个 rna 下拉法, 商业上被合成的 rna 探针, 被标记以被改进的生物素 (desthiobiotin) 的形式在 RNA 链的 3 ‘ 末端被使用了。rna desthiobiotin 是固定的通过与链亲和素的相互作用的磁性珠子, 这是用来拉下来的蛋白质, 特别是与绑定 RNA 的利益。非变性和活性蛋白从间皮瘤细胞胞浆的比例被用作蛋白质的来源。这种 RNA 束缚的蛋白质是洗脱从磁性珠子, 运行通过 12% SDS 页凝胶, 转移到膜, 并探讨了不同的抗体。
在标准的链亲和素-生物素亲和纯化过程中, 需要苛刻的变性条件来破坏强不可逆转的生物素-链亲和素键洗脱绑定蛋白 8, 这可能导致离解蛋白质复合物。与生物素不同的是, desthiobiotin 可逆地结合到链亲和素, 并以缓冲的生物素溶液进行竞争性置换, 允许蛋白质的温和洗脱, 避免自然生物素化分子的分离 9,表明该技术是理想的分离本地蛋白质复合体在当地条件下。
体外约束条件和严格性是由盐浓度、还原剂和洗涤剂百分比决定的, 应与细胞上下文中存在的情况相近, 以便识别真正的体内交互作用。在此之前所实现的绑定条件已被证明是适当的, 以确定虚宾作为一种金结合蛋白10。这种方法可以节省时间, 因为优化适当的绑定条件可能会耗费时间和挑战性。此外, 该方法可作为任何 RNA 下拉实验的启动协议, 通过改变盐和洗涤剂的浓度, 改变甘油的百分比, 添加其他盐, 可以逐步优化。此外, 我们证明, 即使是一个短的25核苷酸 RNA 探针窝藏 pentamer 是-主题可以用来证明与特定 AUBP 的互动。
Protocol
Representative Results
Discussion
3 ‘ UTRs 属于非编码基因组3, 所有非编码 rna 可以与蛋白质相互作用, 以发挥其功能7。当哺乳动物基因组被发现被普遍转录并产生长非编码 rna 16 的很大一部分时, 新的证据表明这些长非编码 rna 在调节基因表达时的作用与染色质重塑复合体17。这种知识是利用 RNA 下拉法得到的。因此, 要开始破译特定 RNA 的 interactors, 第一步可能?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了瑞士国家科学基金会 Sinergia 赠款 CRSII3 147697, 基金会《应用 Krebsforschung 和苏黎世 Krebsliga 的支持。
Materials
| NE-PER Nuclear and cytoplasmic extraction kit | Thermo Fisher Scientific | 78833 | |
| Pierce Protease Inhibitor Tablets, EDTA-free | Thermo Fisher Scientific | A32965 | |
| Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | 23225 | |
| Pierce Magnetic RNA-protein Pull-Down Kit | Thermo Fisher Scientific | 20164 | Includes magnetic beads and therefore the kit need to be stored at 4 °C |
| Pierce RNA 3’ End Desthiobiotinylation Kit | Thermo Fisher Scientific | 20163 | The kit is a part of the "Pierce Magnetic RNA-protein Pull-Down Kit", and needs to be stored at -20 °C unlike the pull-down kit (4 °C). |
| DynaMag-2, magnetic stand | Thermo Fisher Scientific | 12321D | |
| Anti – HuR (MOUSE) monoclonal antibody | Thermo Fisher Scientific | – | The antibody is included in the Pierce Magnetic RNA-protein Pull-Down Kit – 1:1000 in 5% BSA 1x TTBS – rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS |
| Anti – α – tubulin (MOUSE) monoclonal antibody | Santa Cruz | 8035 | 1:1000 in 5% BSA 1x TTBS – rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS |
| Anti – PARP (RABBIT) Polyclonal antibody | Cell Signaling | 9542 | 1:1000 in 5% BSA 1x TTBS – goat anti-rabbit secondary antibody 1:10,000 in 5% BSA 1x TTBS |
| Anti – Mesothelin (MOUSE) Monoclonal Antibody | Rockland | 200-301-A87 | |
| Secondary goat anti-rabbit antibody | Cell Signaling | 7074 | |
| Secondary rabbit anti-mouse antibody | Sigma-Aldrich | A-9044 | |
| RPMI – 1640 medium | Sigma-Aldrich | R8758 | |
| FBS – Filtrated Bovine Serum | Pan Biotech | P40-37500 | |
| Penicillin – streptomycin (100x) | Sigma-Aldrich | P4333 | |
| L-Glutamine solution (200 mM) | Sigma-Aldrich | G7513 | |
| 0.25% Trypsin-EDTA (1x) | Gibco by Life technologies | 25200-056 | |
| Mini-PROTEAN 3 Cell | Bio-Rad | 1653301 | |
| Mini Protean System Glass Plates | Bio-Rad | 1653308 | |
| Mini-PROTEAN Spacer Plates with 1.5 mm integrated spacer | Bio-Rad | 1653312 | |
| Mini-PROTEAN Comb, 10-well, 1.5 mm, 66 µL | Bio-Rad | 1653365 | |
| Mini-PROTEAN Tetra Cell Casting Modules | Bio-Rad | 1658050 | |
| RNaseZap RNase Decontamination Solution | Thermo Fisher Scientific | AM9780 | |
| Rotiphorese gel 30 – aqueous 30% acrylamide and bisacrylamide stock solution at a ration of 37.5:1 | Roth | 3029 | |
| 20% SDS | PanReac AppliChem | A3942 | |
| PVDF transfer membrane | Perkin Elmer | NEF1002 | Need to be activated by incubating in pure methanol for 1 min followed by washing in water for 2 min |
| Trans-Blot SD semi-dry transfer cell | Bio-Rad | 1703940 | |
| Clarity Western ECL Substrate | Bio-Rad | 1705061 | |
| FusionFX Digital Imager | Vilber | – | |
| RNA oligo synthesis | Mycrosynth AG, Switzerland | – | the synthesis scale – 0.04 µmol – HPLC purified |
| Bovine serum albumin | Sigma-Aldrich | A7030 | |
| Hydrochloric Acid (HCl) 6 M | PanReac AppliChem | 182883.1211 | |
| Ultra Pure 1 M Tris, pH 7.5 | Thermo Fisher Scientific | 15567027 | |
| Glycerol | Thermo Fisher Scientific | 17904 | 50% sterile aliquots to be stored at -20°C |
| Pierce Streptavidin magnetic beads | Thermo Fisher Scientific | 88817 | Please note that these magnetic beads have an average diameter of 1 µm (range from 0.5 – 1.5 µm). Furthermore, Dynabeads-M280 Streptavidin, which are beads of homogenouse size 2.8 µm, are also used in RNA-pulldown but be aware that this may affect purification process. |
| TEMED | Sigma-Aldrich | T9281 | |
| Ammonium persulfate | Sigma-Aldrich | A3678 | |
| Coomassie G-250 | Applichem | A3480 | |
| Aluminiumsulfate-(14-18)-hydrate | Sigma-Aldrich | 368458 | |
| ortho-phosphoric acid | Applichem | A0637 |
Referências
- Glisovic, T., Bachorik, J. L., Yong, J., Dreyfuss, G. RNA-binding proteins and post-transcriptional gene regulation. FEBS Letters. 582 (14), 1977-1986 (2008).
- Mayr, C. Evolution and biological roles of alternative 3’UTRs. Trends in Cell Biology. 26 (3), 227-237 (2016).
- Matoulkova, E., Michalova, E., Vojtesek, B., Hrstka, R. The role of the 3′ untranslated region in post-transcriptional regulation of protein expression in mammalian cells. RNA Biology. 9 (5), 563-576 (2012).
- von Roretz, C., Di Marco, S., Mazroui, R., Gallouzi, I. E. Turnover of AU-rich-containing mRNAs during stress: a matter of survival. Wiley Interdisciplinary Reviews: RNA. 2 (3), 336-347 (2011).
- Fallmann, J., Sedlyarov, V., Tanzer, A., Kovarik, P., Hofacker, I. L. AREsite2: An enhanced database for the comprehensive investigation of AU/GU/U-rich elements. Nucleic Acids Research. 44, D90-D95 (2016).
- Kresoja-Rakic, J., et al. Posttranscriptional regulation controls calretinin expression in malignant pleural mesothelioma. Frontiers in Genetics. 8 (70), (2017).
- Chu, C., Spitale, R. C., Chang, H. Y. Technologies to probe functions and mechanisms of long noncoding RNAs. Nature Structural & Molecular Biology. 22 (1), 29-35 (2015).
- Diamandis, E. P., Christopoulos, T. K. The biotin-(strept)avidin system: principles and applications in biotechnology. Clinical Chemistry. 37 (5), 625-636 (1991).
- Hirsch, J. D., et al. Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: Uses for protein labeling, detection, and isolation. Analytical Biochemistry. 308 (2), 343-357 (2002).
- . Magnetic bead technology for better assay development Available from: https://assets.thermofisher.com/TFS-Assets/LSG/brochures/1602577-Magnetic-Bead-Technology-Brochure.pdf (2013)
- Usami, N., et al. Establishment and characterization of four malignant pleural mesothelioma cell lines from Japanese patients. Cancer Science. 97 (5), 387-394 (2006).
- Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227 (5259), 680-685 (1970).
- Coulson, R. M., Cleveland, D. W. Ferritin synthesis is controlled by iron-dependent translational derepression and by changes in synthesis/transport of nuclear ferritin RNAs. Proceedings of the National Academy of Sciences of the United States of America. 90 (16), 7613-7617 (1993).
- Leibold, E. A., Munro, H. N. Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5′ untranslated region of ferritin heavy- and light-subunit mRNAs. Proceedings of the National Academy of Sciences of the United States of America. 85 (7), 2171-2175 (1988).
- Srikantan, S., Gorospe, M. HuR function in disease. Frontiers in Bioscience (Landmark Edition). 17, 189-205 (2012).
- Morris, K. V., Mattick, J. S. The rise of regulatory RNA. Nature Reviews: Genetics. 15 (6), 423-437 (2014).
- Rinn, J. L., et al. Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell. 129 (7), 1311-1323 (2007).
- Zhao, J., Sun, B. K., Erwin, J. A., Song, J. J., Lee, J. T. Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome. Science. 322 (5902), 750-756 (2008).
- McHugh, C. A., et al. The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3. Nature. 521 (7551), 232-236 (2015).
