哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究
概要
我们提出了利用 hela 细胞研究哺乳动物细胞周期中双链 rna 结合蛋白激酶 rna 激活 (pkr) 的 rna 相互作用的实验方法。该方法利用甲醛对交联 rna-pkr 复合物和免疫沉淀进行富集 pkr 约束 rna。这些 rna 可以通过高通量测序或 qrt-pcr 进一步分析。
Abstract
蛋白激酶 rna 激活 (pkr) 是先天免疫反应蛋白的成员, 并能识别病毒 rna 的双链二级结构。当与病毒双链 rna (dsrna) 结合时, pkr 经历二聚化和随后的自磷酸化。磷酸化 pPKR (ppkr) 变得活跃, 并诱导真核起始因子 2 (eif2α) 的α亚基磷酸化, 以抑制全局平移。越来越多的证据表明, pkr 可以在生理条件下激活, 例如在细胞周期中或在各种压力条件下, 没有感染。然而, 由于缺乏捕获和分析 pkr 相互作用 dsrna 的标准化实验方法, 我们对 pkr rna 激活剂的理解有限。在这里, 我们提出了一个实验方案, 以专门丰富和分析 pkr 绑定 rna 在细胞周期中使用 hela 细胞。利用甲醛的有效交联活性固定 pkr-rna 复合物, 并通过免疫沉淀分离。然后可以进一步处理 pkr 联合免疫沉淀 rna, 以生成高通量测序库。pkr 相互作用的细胞 dsrna 的一个主要类别是线粒体 rna (mtrna), 它可以通过重链和光链 rna 之间的互补相互作用作为分子间 dsrna 存在。为了研究这些双工 mtrna 的散变性, 我们还提出了一种带特异性 qrt-pcr 的方案。我们的协议是针对 pkr 结合 rna 的分析而优化的, 但它可以很容易地进行修改, 以研究其他 dsrna 结合蛋白的细胞 dsrna 或 rna 相互体。
Introduction
蛋白激酶 rna 激活 (pkr), 也被称为真核起始因子2α激酶 2 (EIF2AK2), 是一种特征良好的蛋白激酶, 传输 rna 提供的信息。它属于真核翻译起始2亚基α (eif2α) 激酶家族和磷酸酯 eif2α在丝氨酸51响应感染,以抑制全球翻译1。在此背景下, pkr 被病毒双链 rna (dsrna) 激活, 为 pkr 二聚和自磷酸化2提供了一个平台。除了 eif2α, pkr 还可以磷酸化 p53, 胰岛素受体底物 1, 抑制剂 b, 和 c-jun n-端子激酶 (jnk) 调节活性的许多信号转导途径 3,4,5, 6。
pkr 最初被确定为一种在脊髓灰质炎病毒感染过程中磷酸化 eif2α的激酶, 通过识别脊髓灰质炎病毒的 dsrna 7,8。pkr 越来越多地被发现在免疫反应之外发挥着多方面的作用, 其异常激活或故障在许多人类疾病中都隐含着。在细胞凋亡过程中经常观察到活着的磷酸化 pPKR (ppkr), 是退行性疾病患者的共同特征, 尤其是亨廷顿病、帕金森症和阿尔茨海默病等神经退行性疾病9 10,11,12, 13.此外, pkr 在代谢应激和热休克等各种压力条件下被激活, 14、15、16、17。另一方面, pkr 的抑制导致细胞增殖甚至恶性转化的增加 18,19。pPKR 功能在正常大脑功能和细胞周期中也很重要, 因为在 m 期20、21、22 期间, ppkr 水平升高。在这种情况下, ppkr 抑制全局转换, 并为正确的细胞分裂20所需的关键有丝分裂信号系统提供提示。此外, 长时间激活 pkr 导致 g2m 相细胞周期停止在中国仓鼠卵巢 23.因此, pkr 磷酸化由负反馈回路调节, 以确保在 mg1 过渡期间快速失活21。
尽管 pkr 功能范围广泛, 但由于缺乏能够激活 pkr 的标准化高吞吐量实验方法来捕获和识别 dsrna, 我们对 pkr 激活的理解有限。先前的研究表明, pkr 可以与由两个倒置 alu 重复 (iralus)20、24形成的 dsrna 相互作用, 但存在额外的细胞 dsrna 的可能性, 这些细胞 dsrna 可以在细胞周期内或以下激活 pkr。人类细胞的应激条件尚未探索。识别 rna 结合蛋白 (rbp) rna 相互体的常规方法是利用紫外光将 rna-rbp 复合物交联 25、26、27。最近的一项研究将这种紫外交联方法应用于小鼠系统, 并确定小核子蛋白 rna 可以调节 pkr 激活在代谢应激16。利用甲醛的高交联效率, 提出了一种在 hela 细胞28细胞周期中识别 pkr 相互作用 rna 的替代方法。在其他 dsRBPs (如 staufen 和 drosha29、30、31) 的研究中也采用了类似的方法。我们发现 pkr 可以与各种类型的非编码 rna 相互作用, 如短插层核元件 (sine)、长穿插核元素 (line)、内源性逆转录病毒元素 (erv), 甚至α-卫星 rna。此外, 我们还表明 pkr 可以与线粒体 rna (mtrna) 相互作用, 线粒体 rna 通过重链和光链 rna28之间的互补相互作用形成分子间 dsrna。最近的一份出版物进一步支持了我们的数据, 即一些 mtrna 以双工形式存在, 并可以激活 dsrna 传感器, 如黑色素瘤差异相关的蛋白质 5, 以诱导干扰素32。更重要的是, mtrna 的表达和亚细胞定位在细胞周期和各种压力源中被调节, 这可能对它们调节 pkr 激活28的能力很重要。
在本文中, 我们提出了一个详细的协议, 最近开发的甲醛交联和免疫沉淀 (fCLIP) 方法来捕获和分析 pkr 相互作用的 rna 在细胞周期。我们演示了用胸苷和诺加唑制备细胞周期抑制样本的方法。然后, 我们提出了分离 pkr 绑定 rna 的 fclip 过程, 以及一种准备高吞吐量测序库以识别这些 rna 的方法。此外, 我们还描述了使用 qrt-pcr 分析 pkr 绑定 rna 的详细过程。具体而言, 我们提出了一个字符串特定的逆转录过程来分析 mtrna 的字符串。所述协议针对 hara 细胞和 pkr 进行了优化, 但可轻松修改细胞周期样本、fclip 和特异性 qrt-pcr 分析等关键步骤, 以研究细胞 dsrna 或识别其他 dsRNAs 的 rna 干扰体。
Protocol
1. 溶液和细胞制备 解决方案准备 对于细胞培养培养基, 在杜尔贝科改良鹰培养基 (dmem) 的500毫升中加入50毫升的胎儿牛血清 (fbs), 制备平细胞培养培养基。注: 抗生素可以添加到细胞培养基中, 但我们不使用抗生素。 对于0.1% 的甲醛, 在1x 磷酸盐缓冲盐水 (pbs) 中溶解 4% (w/v), 在热板上加热并稀释, 通过添加 1x pbs 使30% 毫升的甲醛。注意: 在烟罩中执行所有步骤, 并注意不要?…
Representative Results
图 1显示了在细胞周期的 s 或 m 阶段逮捕 hela 细胞的过程示意图。对于 m 相抑制样本, 我们可以在显微镜下清楚地显示圆形细胞 (图 2a)。为了检查细胞周期抑制的效率, 可以使用流式细胞仪分析细胞的核含量 (图 2b)。图 3显示了免疫沉淀效率测试的代表性数据, d7f7 抗体显示出优于免疫沉淀 pkr 的能力。免疫沉淀效率的这种差…
Discussion
图 1显示了制备 s 或 m 相抑制样本的过程。为了在 s 期的细胞中, 我们使用了胸苷双块法, 我们用胸苷对细胞进行了两次处理, 中间释放了 9小时, 以确保较高的逮捕效率 (图 1 a)。对于 m 期停止, 我们用胸苷治疗细胞一次, 然后释放 9小时, 然后应用诺康唑在启动基中阻断细胞 (图 1b)。准备细胞周期样本的一个关键步骤是在第一个胸苷?…
開示
The authors have nothing to disclose.
Acknowledgements
这项工作得到了韩国国家研究基金会通过韩国政府科学和信息通信技术部资助的韩国国家研究基金会 (nrf-2016r1c1b29886) 的支持。
Materials
| 0.5 M EDTA, pH 8.0 | Thermo Fisher Scientific | AM9260G | |
| 1 M Tris, pH 7.0 | Thermo Fisher Scientific | AM9855G | |
| 1 M Tris, pH 8.0 | Thermo Fisher Scientific | AM9855G | |
| 1.7 mL microcentrifuge tube | Axygen | MCT-175-C | |
| 10% Nonidet-p40 (NP-40) | Biosolution | BN015 | |
| 10% Urea-acrylamide gel solution | 7 M (w/v) Urea and 0.5X TBE, stored protected from light at 4 °C | ||
| 10X DNA loading buffer | TaKaRa | 9157 | |
| 15 mL conical tube | SPL | 50015 | |
| 3' adaptor | 5'-rApp NN NNT GGA ATT CTC GGG TGC CAA GG/3ddC/-3' | ||
| 3 M Sodium Acetate pH 5.5 | Thermo Fisher Scientific | AM9740 | |
| 5' adaptor | 5'-GUU CAG AGU UCU ACA GUC CGA CGA UCN NNN-3' | ||
| 5 M NaCl | Thermo Fisher Scientific | AM9760G | |
| 50 mL conical tube | SPL | 50050 | |
| Acid-phenol chloroform, pH 4.5 | Thermo Fisher Scientific | AM9722 | |
| Agencourt AMPure XP | Beckman Coulter | A63881 | Magnetic beads DNA/RNA clean up |
| Antarctic alkaline phosphatase | New England Biolabs | M0289S | |
| Anti-DGCR8 | Made in house | ||
| Anti-PKR (D7F7) | Cell signaling technology | 12297S | |
| Anti-PKR (Milli) | Millipore EMD | 07-151 | |
| ATP (100 mM) | GE Healthcare | GE27-2056-01 | |
| Bromophenol blue sodium salt | Sigma-aldrich | B5525 | |
| Calf intestinal alkaline phosphatase | TaKaRa | 2250A | |
| Cell scraper 25 cm 2-position | Sarstedt | 83.183 | |
| CMV promoter sequence | 5'-CGCAAATGGGCGGTAGGCGTG-3' | ||
| Dulbecco's modified eagle medium | Welgene | LM001-05 | |
| dNTP mixture (2.5 mM) | TaKaRa | 4030 | |
| Ethanol, Absolute, ACS Grade | Alfa-Aesar | A9951 | |
| Fetal bovine serum | Merck | M-TMS-013-BKR | |
| Formamide | Merck | 104008 | |
| Glycine | Bio-basic | GB0235 | |
| GlycoBlue coprecipitant (15 mg/mL) | Thermo Fisher Scientific | AM9516 | |
| Isopropanol | Merck | 8.18766.1000 | |
| NEBNext rRNA Depletion Kit | New England Biolabs | E6318 | rRNA Depletion Kit |
| Nocodazole | Sigma-Aldrich | M1404 | |
| Normal rabbit IgG | Cell signaling technology | 2729S | |
| Paraformaldehyde | Sigma-Aldrich | 6148 | |
| PCR forward primer (RP1) | 5'-AAT GAT ACG GCG ACC ACC GCG ATC TAC ACG TTC AGA GTT CTA CAG TCC GA-3' | ||
| PCR index reverse primer (RPI) | 5'-CAA GCA GAA GAC GGC ATA CGA GAT NNN NNN GTG ACT GGA GTT CCT TGG CAC CCG AGA ATT CCA-3' | ||
| PCR tubes with flat cap, 0.2 mL | Axygen | PCR-02-C | |
| Phosphate bufered saline (PBS) Tablet | TaKaRa | T9181 | |
| Phusion high-fidelity DNA polymerase | New England Biolabs | M0530 | High-fidelity polymerase |
| PlateFuge microcentrifuge with swing-out rotor | Benchmark | c2000 | |
| Polynucleotide kinase (PNK) | TaKaRa | 2021A | |
| Protease inhibitor cocktail set III | Merck | 535140-1MLCN | |
| Proteinase K, recombinant, PCR Grade | Sigma-Aldrich | 3115879001 | |
| qPCR primer sequence: CO1 Heavy | Forward/Reverse: 5′-GCCATAACCCAATACCAAACG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: CO1 Light | Forward/Reverse: 5′-TTGAGGTTGCGGTCTGTTAG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: CO2 Heavy | Forward/Reverse: 5′-CTAGTCCTGTATGCCCTTTTCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: CO2 Light | Forward/Reverse: 5′-GTAAAGGATGCGTAGGGATGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: CO3 Heavy | Forward/Reverse: 5′-CCTTTTACCACTCCAGCCTAG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: CO3 Light | Forward/Reverse: 5′-CTCCTGATGCGAGTAATACGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: CYTB Heavy | Forward/Reverse: 5′-CAATTATACCCTAGCCAACCCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: CYTB Light | Forward/Reverse: 5′-GGATAGTAATAGGGCAAGGACG -3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: GAPDH | Forward/Reverse: 5′-CAACGACCACTTTGTCAAGC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: ND1 Heavy | Forward/Reverse: 5′-TCAAACTCAAACTACGCCCTG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: ND1 Light | Forward/Reverse: 5′-GTTGTGATAAGGGTGGAGAGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: ND4 Heavy | Forward/Reverse: 5′-CTCACACTCATTCTCAACCCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: ND4 Light | Forward/Reverse: 5′-TGTTTGTCGTAGGCAGATGG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: ND5 Heavy | Forward/Reverse: 5′-CTAGGCCTTCTTACGAGCC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: ND5 Light | Forward/Reverse: 5′-TAGGGAGAGCTGGGTTGTTT-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: ND6 Heavy | Forward/Reverse: 5′-TCATACTCTTTCACCCACAGC-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| qPCR primer sequence: ND6 Light | Forward/Reverse: 5′-TGCTGTGGGTGAAAGAGTATG-3′/5′-CGCAAATGGGCGGTAGGCGTG-3′ | ||
| Random hexamer | Thermo Fisher Scientific | SO142 | |
| Recombinant Dnase I (Rnase-free) (5 U/μL) | TaKaRa | 2270A | |
| Recombinant Rnase inhibitor (40 U/μL) | TaKaRa | 2313A | |
| Ribo-Zero rRNA Removal Kit | Illumina | MRZH116 | rRNA Removal Kit |
| Rotator | FINEPCR, ROTATOR AG | D1.5-32 | |
| RT primer sequence: CO1 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGTTGAGGTTGCGGTCTGTTAG-3′ | ||
| RT primer sequence: CO1 Light | 5′-CGCAAATGGGCGGTAGGCGTGGCCATAACCCAATACCAAACG-3′ | ||
| RT primer sequence: CO2 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGGTAAAGGATGCGTAGGGATGG-3′ | ||
| RT primer sequence: CO2 Light | 5′-CGCAAATGGGCGGTAGGCGTGCTAGTCCTGTATGCCCTTTTCC-3′ | ||
| RT primer sequence: CO3 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGCTCCTGATGCGAGTAATACGG-3′ | ||
| RT primer sequence: CO3 Light | 5′-CGCAAATGGGCGGTAGGCGTGCCTTTTACCACTCCAGCCTAG-3′ | ||
| RT primer sequence: CYTB Heavy | 5′-CGCAAATGGGCGGTAGGCGTGGGATAGTAATAGGGCAAGGACG-3′ | ||
| RT primer sequence: CYTB Light | 5′-CGCAAATGGGCGGTAGGCGTGCAATTATACCCTAGCCAACCCC-3′ | ||
| RT primer sequence: GAPDH | 5′-CGCAAATGGGCGGTAGGCGTGTGAGCGATGTGGCTCGGCT-3′ | ||
| RT primer sequence: ND1 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGGTTGTGATAAGGGTGGAGAGG-3′ | ||
| RT primer sequence: ND1 Light | 5′-CGCAAATGGGCGGTAGGCGTGTCAAACTCAAACTACGCCCTG-3′ | ||
| RT primer sequence: ND4 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGTGTTTGTCGTAGGCAGATGG-3′ | ||
| RT primer sequence: ND4 Light | 5′-CGCAAATGGGCGGTAGGCGTGCCTCACACTCATTCTCAACCC-3′ | ||
| RT primer sequence: ND5 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGTTTGGGTTGAGGTGATGATG-3′ | ||
| RT primer sequence: ND5 Light | 5′-CGCAAATGGGCGGTAGGCGTGCATTGTCGCATCCACCTTTA-3′ | ||
| RT primer sequence: ND6 Heavy | 5′-CGCAAATGGGCGGTAGGCGTGGGTTGAGGTCTTGGTGAGTG-3′ | ||
| RT primer sequence: ND6 Light | 5′-CGCAAATGGGCGGTAGGCGTGCCCATAATCATACAAAGCCCC-3′ | ||
| Siliconized polypropylene 1.5 mL G-tube | Bio Plas | 4167SLS50 | |
| Sodium dedecyl sulfate | Biosesang | S1010 | |
| Sodium deoxycholate | Sigma-Aldrich | D6750 | |
| SUPERase In Rnase inhibitor | Thermo Fisher Scientific | AM2694 | |
| SuperScript III reverse transcriptase | Thermo Fisher Scientific | 18080093 | Reverse transcriptase for library preparation |
| SuperScript IV reverse transcriptase | Thermo Fisher Scientific | 18090010 | Reverse transcriptase for qRT-PCR |
| SYBR gold nucleic acid gl stain | Thermo Fisher Scientific | S11494 | |
| T4 polynucleotide kinase | New England Biolabs | M0201S | |
| T4 RNA ligase 1 (ssRNA Ligase) | New England Biolabs | M0204 | |
| T4 RNA ligase 2, truncated KQ | New England Biolabs | M0373 | |
| Thermomixer | Eppendorf ThermoMixer C with ThermoTop | ||
| Thymidine | Sigma-Aldrich | T9250 | |
| Tris-borate-EDTA buffer (TBE) | TaKara | T9122 | |
| Triton X-100 | Promega | H5142 | |
| Ultralink Protein A sepharose beads | Thermo Fisher Scientific | 22810 | Protein A beads |
| Ultrasonicator | Bioruptor | ||
| Urea | Bio-basic | UB0148 | |
| Vortex mixer | DAIHAN Scientific | VM-10 | |
| Xylene cyanol | Sigma-Aldrich | X4126 | |
| γ-32P-ATP (10 μCi/μL, 3.3 μM) | PerkinElmer | BLU502A100UC |
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タグ
RNA InteractorsProtein Kinase RNA-ActivatedMammalian Cell CycleCrosslinkingFormaldehydeImmunoprecipitationNeurodegenerative DiseaseParkinson’s DiseaseAlzheimer’s DiseaseDouble-stranded RNARNA-binding ProteinsPost-transcriptional RegulationGene ExpressionCell Cycle ArrestHeLa CellsThymidineNocodazole
