方法调查小RNA和核糖体占用的调控作用<em>恶性疟原虫</em
Summary
Human microRNAs translocate from host erythrocytes to Plasmodium falciparum parasites. Here, the techniques used to transfect synthetic microRNAs into host erythrocytes and isolate all RNAs from P. falciparum are described. In addition, this paper will detail a method of polysome isolation in P. falciparum to determine the ribosomal occupancy and translational potential of parasite transcripts.
Abstract
遗传变异负责镰状细胞等位基因（HBS）使红细胞的疟原虫，P.抗感染疟原虫 。这个阻力，这是众所周知的是多因素的分子基础，仍然不完全理解。最近的研究发现，红细胞微RNA，一旦转移进入疟原虫的差异表达，同时影响基因调控和寄生虫生长。这些miRNA后来所示，通过5'RNA融合寄生虫mRNA的谨慎的子集形成了嵌合体RNA转录抑制mRNA的翻译。在这里，中使用的技术来研究基因调控和P的平移潜在的功能作用和假定机制底层红细胞的microRNA 疟原虫，包括改性合成微小RNA转染到宿主红细胞，进行详细说明。最后，一个多核糖梯度法是用来确定TRA的程度nslation这些成绩单。一起，这些技术使我们能够证明红细胞微RNA的失调水平有助于镰状红细胞的细胞内在疟疾电阻。
Introduction
疟疾，造成疟原虫属顶复门寄生虫，是人类最常见的寄生虫病，感染全球大约200万人，每年造成周围60万人死亡1。感染人类的五个疟原虫物种的，最相关的人类疾病是P.疟原虫和P.间日疟原虫 ，由于严重疟疾并发症其广泛分布和潜力。疟原虫的生命周期既需要蚊子和人类的感染。当受感染的蚊子叮咬人类，寄生虫通过血液循环到达肝脏，在首轮的复制发生移动。后从主机肝裂殖子破裂，它们感染附近的红血细胞，发起或者无性或有性复制。复制的无性阶段，持续48小时在体育疟原虫，是本研究的焦点，因为它是最发作的同时在源咏叹调 症状和容易概括体外 。
虽然一些公共卫生措施，包括改善抗疟疾疗法，已经有所减少全球疟疾负担，抗药性疟原虫的持续出现，呈现为疟疾防治工作中的问题。这可能意味着新的治疗方法的一个领域是研究遗传变异如何不同赋予抵抗疟疾。在疟疾流行的地区，各种红细胞多态性的现象相当普遍2,3。这些突变，镰状细胞是也许是最突出，通常与对有症状疟疾感染4发病实质性抗性相关。由它们引起红细胞抵抗疟疾感染的基本机制是不完全的了解。寄生红细胞与血红蛋白突变，受增强吞噬功能，通过增强细胞的刚性和脱水，这与减少入侵体育相关疟原虫 5。 HBC的等位基因也影响蛋白质表达在红细胞表面和与细胞骨架的重塑，进一步抑制寄生虫发育6,7。最后，P.疟原虫生长不佳内纯合镰刀（HBSS）红细胞8,9 体外 ，提示疟疾电阻的固有红细胞因素。然而，虽然所有这些机制似乎发挥作用，但它们不能完全解释后面疟疾镰状细胞抗性的机制。
一个潜在的一套红细胞因素仍然知之甚少是成熟红细胞中的miRNA存在的大池。微RNA是小的非编码RNA，19-25个核苷酸的大小，其中由3'UTR内的碱基配对介导的翻译和/或靶mRNA的稳定性。他们有牵连的哺乳动物的免疫反应的控制，包括抑制Ò˚F病毒复制10，并分别显示出赋予对病毒在植物中它们也已显示出调节一些红细胞过程，包括红细胞生成11,12和铁代谢13。以前的研究确定了红细胞的miRNA，其表达显着改变的HBSS红细胞14,15的丰富和多样化的人口。由于成熟的红细胞缺乏积极的转录和翻译，这些红细胞miRNA的功能作用尚不清楚。由于显著物质交换的宿主细胞和P之间发生的红细胞内发育周期（IDC）16中疟原虫，有人推测的HbS红细胞内改变的miRNA谱可以直接有助于细胞内在疟疾电阻。
这些研究最终导致管道的发展分离，鉴定和功能上的米内研究人类miRNA的作用alaria寄生虫P.疟原虫，这表明这些宿主/人miRNA的最终共价保险丝然后平移地抑制寄生虫mRNA转录物17。这提供了通过转拼形成的第一跨物种的嵌合转录物的示例，并且暗示，这种miRNA的体mRNA融合物可以在其它物种中，包括其他寄生虫可以发生。所有锥虫的mRNA为反式剪接与拼接领袖（SL）来调节多顺反子转录物18的分离。由于P.疟原虫缺乏直系同源对的Dicer /阿戈19,20，它是可能的红细胞的miRNA劫持在巴斯德类似SL机械恶性融入靶基因。最近在体育研究疟原虫有实际上表示的5'剪接前导序列21的存在。这项研究详细介绍了导致人类寄生虫的miRNA基因融合基因的发现，这些方法包括转录和translational调节技术。这些方法的总体目标是研究的小RNA在P的基因调控，表型和翻译潜在的影响疟原虫成绩单。
人类寄生虫嵌合成绩单初步查明依赖的RNA分析技术，如实时PCR，转录组测序和EST库的采集，其中包括总额和小RNA，而不是用技术只孤立的小RNA使用。分离所有的RNA一起在一个大的池，而不是分开，允许两个易位人类小RNA在寄生虫的标识以及这些小RNA序列的存在作为较大序列的一部分。然后，这需要这些融合mRNA的翻译状态的分析，以确定这些融合的功能性后果。
而在寄生虫的基因组的表征广泛的努力ð转录纷纷加入到寄生虫的生物学22-25的了解，远不如所了解的mRNA的转录组跨越P的生命周期中的翻译调控疟原虫 26。寄生虫的蛋白质组的这种有限的理解已经阻碍了双方的了解寄生虫的生物学，并确定为下一代抗疟疾治疗的新目标的能力。寄生虫的细胞生物学的理解，这仍存在差距主要是由于缺乏足够的技术来研究翻译调控的P.疟原虫 。最近的一篇论文描述了使用P的核糖体足迹的恶性确定全球翻译状态21。一个公认的转录的平移电位测定是由多核糖仿形测定相关的核糖体的数目。然而，当这种技术应用到P.疟原虫</ em>的，它是无法收回大部分多核糖体和捕获主要monosomes。近日，几组27,28优化了P.疟原虫多核糖体的技术，通过同时裂解红细胞和寄生虫保存多核糖体和它们在宿主红细胞28无性发育过程中表征这些疟原虫的核糖体占用和翻译潜能。
总的来说，这些方法证明人类miRNA与寄生虫mRNAs的所观察到的融合调制那些融合的mRNA，其使用先前报道的方法27表明的寄生虫蛋白质翻译，并且是在疟疾的HBA阻力的主要决定因素和HBSS红细胞17。这些方法将在任何系统，查找以确定和功能上探索RNA剪接事件是有用的，这些融合的RNA是否在第疟原虫或其他真核系统。
Protocol
Representative Results
Discussion
哈佛商学院在疟疾流行地区最常见的异常血红蛋白之一，主要是因为它提供保护，防止造成P.重症疟疾疟原虫 。该技术需要在P的基因调控的人类微小RNA的作用表征恶性详述整个手稿。通过提取总RNA以这样的方式，以包括所有的小RNA，并通过执行一个相对简单的寄生虫溶胞步骤，这些融合的RNA能够通过各种独立技术来鉴定。
这些步骤都比较简单，但?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This research was funded by Doris Duke Charitable Foundation, Burroughs Wellcome Fund, NIH R21DK080994, Duke Chancellor’s pilot project fund, the Roche Foundation for Anemia Research and The Bill and Melinda Gates Foundation. G.L. is supported by Duke’s UPGG and the NIH (Grant # 5R01AI090141-03) and K.A.W. by the NSF Graduate Research Fellowship Program.
Materials
| REAGENTS-All reagents must be RNAse-free. |
| Diethyl pyrocarbonate (DEPC; Sigma, cat. no. D5758) |
| DEPC-treated water (see REAGENT SETUP) |
| Yoyo-1 DNA fluorescent dye (Thermo Fisher) |
| Gene Pulser II (or comparible) electroporator (Bio-Rad) |
| 0.2 cm Electroporation cuvettes |
| 4 M potassium acetate (Mallinckrodt, cat. no. 6700) |
| 2 M potassium HEPES (pH 7.2; Sigma, cat. no. H0527) |
| 1 M magnesium acetate (Sigma, cat. no. M-2545) |
| 1 M dithiothreitol (DTT; Research Products International, cat. no. D11000) |
| 100 mM phenylmethylsulfonate fluoride (PMSF; dissolved in isopropanol; Sigma, cat. no. P7626) |
| 10% (v/v) Igepal CA-630 (Sigma-Aldrich, cat. no. I3021) |
| 10% (w/v) sodium deoxycholate (DOC; Sigma, cat. no. D-6750) |
| Cycloheximide (CHX; Sigma, cat. no. C-7698) |
| Sucrose (Mallinckrodt, cat. no. 8360-04) |
| RNAseOUT (Invitrogen, cat. no. 100000840) |
| RPMI-1640 w/ L-glutamine (Cellgro, cat. no. 10-040-CV) |
| 10% AlbuMAX I (w/v in sterile water) (Invitrogen, cat. no. 11020-039) |
| Gentamicin (Gibco, cat. no. 15750-060)) |
| HT supplement (100x) (Invitrogen, cat. no. 11067030) |
| 45% (w/v) sucrose (Sigma, cat no. G8769) |
| 1 M HEPES buffer (Gibco, cat. no. 15630-080) |
| 1x phosphate buffered saline (PBS; Cellgro, cat. no. 2109310CV) |
| Corning 500 ml vacuum filter flask (VWR, cat. no. 430769) |
| Glass slides (VWR, cat. no. 48311-702) |
| Giemsa stain (50X) (VWR, cat no. m708-01) |
| mirVana miRNA isolation kit (Ambion, ThermoFisher Scientific). |
| 5' Biotin conjugated mature miRNA (sequence varies by miRNA of interest) (Dharmacon) |
| Biotin powder (Sigma-Aldrich) |
| 1M Potassium Chloride |
| Streptavidin Sepharose High Performance Beads (GE Healthcare) |
| Ribosome resuspension buffer (see REAGENT SETUP) |
| Lysis buffer (see REAGENT SETUP) |
| 15% (w/v) sucrose gradient solution (see REAGENT SETUP) |
| 50% (w/v) sucrose gradient solution (see REAGENT SETUP) |
| 0.5 M sucrose cushion (see REAGENT SETUP) |
| 60% (w/v) sucrose chase solution (see REAGENT SETUP) |
| Plasmodium cell culture media (see REAGENT SETUP) |
| RNA capture Wash Buffer (see REAGENT SETUP) |
| RNA Elution Buffer (see REAGENT SETUP) |
| REAGENT SETUP |
| Solutions |
| Plasmodium cell culture media |
| 500 ml RPMI-1640 w/ L-glutamine |
| 0.5 ml gentamicin |
| 5 ml HT supplement |
| 2.5 ml 45% glucose |
| 25 ml 10% AlbuMAX I |
| 12.5 ml 1 M HEPES |
| Sterile filter with 0.2 mm vacuum filter flask. |
| Plasmodium cell culture media containing 10x CHX |
| Same recipe as Plasmodium cell culture media above, with the addition of: |
| 2 mM cycloheximide |
| Media is prepared as normal, then add CHX to a final concentration of 2 mM and filter. |
| 1x PBS containing cycloheximide |
| Cycloheximide is added fresh to 1x PBS to a final concentration of 200 mM, and kept at 4 OC until use. |
| Ribosome resuspension buffer |
| 400 mM potassium acetate |
| 25 mM potassium HEPES, pH 7.2 |
| 15 mM magnesium acetate |
| 200 mM cycloheximide (add fresh) |
| 1 mM DTT (add fresh) |
| 1 mM PMSF (add fresh) |
| 40 U/ml RNaseOUT (add fresh) |
| Lysis buffer |
| Same recipe as ribosome resuspension buffer above, with the addition of: |
| 1% (v/v) Igepal CA-630 |
| 0.5% (w/v) DOC |
| Sucrose solutions |
| Same recipe as ribosome resuspension buffer above, with the addition of varying amounts of sucrose: |
| 15% (w/v) sucrose to make 15% sucrose gradient solution |
| 50% (w/v) sucrose to make 50% sucrose gradient solution |
| 0.5 M sucrose to make 0.5 M sucrose cushion |
| As above, cycloheximide, DTT, PMSF, and RNaseOUT must be added fresh. |
| The 60% sucrose chase solution requires only sucrose and water, no other components |
| 60% (w/v) sucrose in DEPC-treated water to make 60% sucrose chase solution |
| RNA Capture Wash Buffer |
| 20mM KCl |
| 5unit/ml Rnase Out (Invitrogen) |
| RNA Capture Elution Buffer |
| Same recipe as RNA Capture Wash Buffer, with the addition of: |
| 2mM Biotin |
| EQUIPMENT |
| SW55 Ti ultracentrifuge rotor (Beckman, cat. no. 342194) |
| SW41 Ti ultracentrifuge rotor (Beckman, cat. no. 331362) |
| Ultra-ClearTM ½ x 2 in (13 x 51 mm) ultracentrifuge tubes for the SW55 (Beckman, cat. no. 344057) |
| Polyallomer 9/16 x 3 ½ in (14 x 89 mm) ultracentrifuge tubes for the SW41 (Beckman, cat. no. 331372) |
| L8-80M ultracentrifuge (Beckman) |
| Steel blunt syringe needle, 4 inch, 16G (Aldrich, cat. no. Z261378) |
| 1 ml syringe with 27G½ needle (Becton Dickinson, cat no. 309623) |
| 5 ml syringe (Becton Dickinson, cat. no. 309646) |
| Parafilm |
| Tube rotator, end-over-end |
| Microcentrifuge, refrigerated |
| Density gradient fractionation system (Teledyne Isco, cat. no. 69-3873-179) |
| TracerDAQ (Measurement Computing) |
| Eppendorf 5810R centrifuge |
| Eppendorf A-4-81 rotor (Eppendorf, cat. no. 022638807) |
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