一种测量方法RNA<em>ñ</em⁶ -methyladenosine细胞和组织修改

Summary

中描述了RNA测定N ⁶ -methyladenosine（M ⁶ A）修改的改进RNA印迹法。当前的方法可以检测在各种实验设计在不同的RNA修改和控制。

Abstract

N⁶-Methyladenosine (m⁶A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m⁶A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m⁶A modifications. Herein, we present a northeastern blotting technique for measuring m⁶A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m⁶A modifications in various sources of RNA. While m⁶A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m⁶A modifications have immense possibility to provide key insights into molecular physiology.

Introduction

Dynamic and reversible RNA modifications have important roles in RNA homeostasis. Four decades ago, N⁶-methyladenosine (m⁶A) modifications were found to be abundant in eukaryotic transcriptomes¹. They have diverse functions in messenger RNA (mRNA), ribosomal RNA (rRNA), small nucleolar RNA, transfer RNA, and microRNA². The m⁶A modifications of mRNA influence their splicing³, translation⁴, degradation⁵, and localization². Moreover, they affect ribosome biogenesis and microRNA function⁶. The evolutionary conservation of m⁶A modifications of RNA is noted in unicellular bacteria to multi-cellular humans⁷. Delineation of the roles of m⁶A modifications is currently under extensive exploration. The efforts are expected to provide new insights into transcription control. Recent studies reveal that other chemical modifications of mRNA⁸ also play critical roles in RNA metabolism.

Circadian rhythms⁹, stem cell pluripotency¹⁰, triglyceride metabolism⁴, fibrosis¹¹, obesity¹², and major depression¹³ are a few examples of processes where m⁶A modifications are known to control outcomes. Many circadian clock gene transcripts have m⁶A sites¹⁴. Modulation of m⁶A methylase or demethylase elicits circadian period changes¹⁵. Mettl3, an m⁶A transferase, is a regulator for stem cell pluripotency. A deficiency of Mettl3 leads to early embryonic lethality and aberrant lineage priming at the post-implantation stage¹⁰. A deficiency of fat mass- and obesity-associated (FTO), an m⁶A demethylase, in adipocytes affects fatty acid mobilization and body weight through posttranscriptional regulation of Angptl4⁴. These studies reveal that m⁶A not only controls mRNA processing, but also plays critical roles in embryological development and patho-physiology. The function of m⁶A modifications holds implications for therapeutic considerations in the future.

Several methods are available to measure m⁶A modifications of RNA^16-18. Traditionally, thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are used to study the distribution of m⁶A in several RNAs^19,20. Mass spectrometry is a sensitive tool for the detection of m⁶A modifications in RNA. However, the RNA needs to be excised by RNase into short fragments before analysis by mass spectrometry²¹. Methyl-RNA immunoprecipitation and sequencing (m⁶A-Seq)²² immunoprecipitates fragmented RNAs with m⁶A-specific antibodies and performs parallel RNA sequencing. This method generates transcriptome-wide m⁶A landscapes. High-resolution mapping of m⁶A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) further maps m⁶A modifications at a single-nucleotide resolution²³. Both methods provide details of m⁶A modification across the whole transcriptome, with specific genes’ information. However, quantifications and standardization in both methods are difficult if experiments require the comparison of multiple conditions. Moreover, fragmentations of RNA for m⁶A-Seq alter the original RNA structure, which may affect native m⁶A levels. To detect global m⁶A modifications of RNA and their changes under different experimental conditions, we report a method that employs a modified northern blotting protocol. This method resolves RNA by molecular weight, using gel electrophoresis¹⁸. This procedure provides better standardization and quantifications for experiments that involve multiple conditions or samples. It also provides specific m⁶A modification information for different RNAs, whether rRNA, mRNA, or microRNA.

Protocol

注:总RNA米⁶ A级包括rRNA基因，基因，和其他小RNA。由于核糖体RNA具有丰富的米⁶一修改，m的测量⁶ A水平将需要考虑这一事实。

1. RNA分离

1. 使用核糖核酸酶净化解决方案（喷在纸巾），擦拭移液器和实验室工作台表面。无核酸酶水的湿纸巾，再次擦拭移液器和实验室工作台表面。
2. RNA提取
   1. 总RNA分离
      1. 用顶置式搅拌器，均化50-100毫克组织或5-10×10 ^6个细胞在1mL的RNA分离溶液保持在4℃。
         注:更多组织（100-150毫克）可能需要从因为较低的RNA浓度在其中的小鼠脂肪组织样品。样品从小鼠心脏，肝脏，骨骼肌，肺，脑来源，和巨噬细胞先前已经采用^4。
      2. 在cubate样品匀浆在室温下5分钟。
      3. 添加100μL的每1毫升样品匀浆1-溴-3-氯丙烷（BCP）的。摇晃试管剧烈15秒，然后继续根据制造商的说明²⁴总RNA分离。
   2. 从分离的总RNA多腺苷酸mRNA纯化
      1. 净化用可获得的试剂盒或协议多腺苷酸的mRNA。
         1. 摇彻底重新暂停各通过以下方法解决:2倍的结合溶液，寡（dT）的聚苯乙烯珠和洗涤溶液。确保寡（dT）在使用前珠温至室温。
         2. 传输的每制备洗脱溶液120微升成管并加热至70℃的加热块中。
         3. 吸取到的总RNA的500微克到离心管中。无核酸酶的水，将音量调节到250微升。
         4. 添加的2倍结合液250微升总RNA所以lution和涡简单地混合内容。
         5. 添加低聚15μL（DT）珠和涡彻底混合。
         6. 孵育在70℃下该混合物3分钟。
         7. 除去来自加热块的样品，并让它在室温下放置10分钟。
         8. 离心在15,000rpm xg离心2分钟。
         9. 小心取出上清液，留下约50微升。
         10. 加的洗涤液500微升重新悬浮，通过移液沉淀。
         11. 移液器悬挂到自旋过滤/收集管套。
         12. 离心在15,000rpm xg离心2分钟。除去从收集管色谱柱和丢弃流过。返回列至收集管。
         13. 吸取500微升洗涤液到旋转过滤器。
         14. 离心15,000× 克2分钟。
         15. 转移自旋过滤到一个新的收集管。
         16. 移液器50的洗脱液μL加热至70℃到旋转过滤器的中心。
         17. 在70℃孵育2-5分钟。离心在15,000rpm xg离心1分钟。
         18. 吸管至70℃洗脱溶液加热额外的50微升到旋转过滤器的中心。
         19. 重复步骤1.2.2.1.18。
      2. 加入100微克/毫升糖原，0.1体积的3M乙酸钠缓冲液（pH 5.2）和2.5体积的无水乙醇中。在-80℃沉淀过夜。
      3. 离心机以15,000 xg离心在4℃下25分钟。弃去上清液。
      4. 用1mL的75％乙醇洗涤沉淀。离心机以15,000 xg离心在4℃下15分钟。小心地取出乙醇。
      5. 干燥空气中为3-5分钟。
      6. 加的不含核酸酶的水10μL的以溶解沉淀。
      7. 储存于-70℃。
3. RNA定性和定量
   1. 使用分光光度计，确定由NotI位RNA浓度纳克在260nm和280nm的吸光度。在A ₂₆₀ / A ₂₈₀的比例应为1.8-2.2。
   2. 检查用0.8％琼脂糖凝胶电泳²⁵的RNA样品的质量。
      注:完整的真核细胞的总RNA应该表现出强烈的28S和18S rRNA的乐队。 28S的18S与rRNA基因带强度有一个良好的RNA制备的比率是〜2。

2.凝胶电泳和转让

注:缓冲液配制协议在表1中给出。

1. 甲醛凝胶制备（1％）
   1. 冲洗所有电泳设备酸二乙酯（DEPC）水。
   2. 熔融2.5克在215毫升DEPC水的琼脂糖完全在微波炉。
   3. 加12.5毫升10×3-（N-吗啉代）丙磺酸（MOPS）缓冲液和22.5毫升37％的甲醛。
   4. 倒入用1.5毫米厚的梳子的电泳装置。
   5. 消除泡沫或推Ť下摆凝胶用干净的梳子的边缘。
   6. 使凝胶在室温下固化。
2. 样品制备
   1. 混合样品缓冲液中的1-10微克总RNA和11.3微升。
   2. 混合RNA标记（1微克/微升）的2微克的样品缓冲液11.3微升。
   3. 不含核酸酶的水从2.2.1和2.2.2添加到样本以16微升的总体积。
   4. 热，在60℃进行5分钟，然后在冰上冷却。
   5. 混合来自2.2.3样品16微升与4微升跟踪染料，其含有0.1微克/微升的溴化乙锭在冰上。
      注意:如果吸入溴化乙锭可能是致畸和毒性。阅读溴化乙锭安全技术说明书。
3. 电泳
   1. 加入200毫升的10倍MOPS缓冲至1800毫升的DDH ₂ O，使一个1X MOPS运行缓冲液。
   2. 使用1×MOPS运行缓冲液冲洗凝胶的孔中。
   3. 预RUN T给他凝胶在20 V 5分钟。
   4. 加载RNA样品到凝胶的孔中。
   5. 用铝箔纸覆盖，避免光线照射。在35 V大约17小时运行（过夜）
   6. 检查和UV光下拍摄的凝胶。
4. 转让
   1. 切胶删除未使用的部分。
   2. 制备500毫升的10×SSC缓冲液（250毫升20×SSC缓冲液和250mL DEPC处理的水）。
   3. 用10倍的SSC缓冲液中以50rpm摇动洗凝胶两次20分钟。
   4. 切1滤纸片大到足以用作灯芯纸，它从传送盘（ 图1）吸收转移缓冲液。
   5. 切4张该滤纸的尺寸相同的凝胶。
   6. 切成1片带正电荷的尼龙膜，以相同的尺寸的凝胶。
   7. 标记在凝胶上的凹口和膜作为标记，以确保正确的方向。
   8. 在2×SSC缓冲液浸泡的膜15分钟。
   9. 倒入500毫升邻˚F10X SSC缓冲到传输托盘。
   10. 铺设横跨玻璃板的大滤纸片并用转移缓冲液浸湿的滤纸。
   11. 推出用吸管任何气泡。
   12. 浸泡2片预先切割的滤纸在转移缓冲液，并将其放置在滤纸上的从步骤2.4.5的中心。删除任何气泡。
   13. 莱凝胶顶面朝下放在滤纸。
   14. 躺在凝胶的顶端膜上。对齐凝胶和膜的槽口。
   15. 放置在膜的顶部2个预切割滤纸和湿用转移缓冲液的滤纸上。
   16. 取出层之间的任何气泡。
   17. 应用保鲜膜凝胶周围，以确保毛巾只吸收缓冲器通过凝胶和膜。
   18. 堆叠在滤纸的顶部的纸毛巾。
   19. 将一个超大的玻璃板上的毛巾上。
   20. 放置一个重量上堆叠的顶部。
   21. 保持过夜以允许所述RNA从凝胶转移到膜上。

3. N ⁶ -methyladenosine检测

1. 交联膜
   1. 保持该膜在2×SSC缓冲液潮湿。
   2. 将浸有10X SSC缓冲到UV交联剂滤纸2张。
   3. 放置在滤纸上的顶部的膜，与所述RNA吸附面朝上。
   4. 选择”autocrosslink模式”（120,000μJ），并按下”开始”按钮启动照射。
   5. 检查在膜和凝胶用UV凝胶图像捕获器，以确认从该凝胶中的RNA转移到膜上。
2. 免疫印迹
   1. 1小时阻断与吐温20（TBST）缓冲的Tris缓冲盐水在室温下，在5％非脂肪牛奶的膜。
   2. 用TBST缓冲液三次洗15分钟。
   3. 孵育膜M ⁶ A（隔夜<青霉> N ⁶ -methyladenosine）抗体溶液（1:4℃在5 1,000％非脂肪牛奶的TBST缓冲液）。
   4. 用TBST缓冲液三次洗膜15分钟。
   5. 孵育在HRP标记的驴抗兔抗体溶液（1:2,000，在5％非脂肪牛奶的TBST缓冲液）的膜1小时在室温下。
   6. 用TBST缓冲液三次洗膜15分钟。
   7. 应用增强的化学发光底物（每膜厘米² 0.125毫升）。
   8. 捕捉与数字成像器的最佳设置的化学发光，根据制造商的说明^26。
3. 米⁶的量化
   1. 测量使用ImageJ的软件相对米⁶化学发光强度。
      1. 根据ImageJ的软件菜单中，选择”文件”选项打开相关文件。
      2. 从ImageJ的”矩形”工具和周围画一个框的信号。
      3. 如果核糖体RNA不是实验的重点，避免用于计算的18S和28S核糖体RNA米⁶ A频带。
      4. 点击”命令”和”1″来确认所选择的车道。
      5. 按”命令”和”3″以显示所选的情节。
      6. 点击”直线”工具，画线分割区分开。
      7. 点击”棒”工具来记录测量。
      8. 导出数据。
      9. 规范化M ⁶ A的水平与来自溴化乙锭染色的18S核糖体RNA带。

Representative Results

Discussion

Modifications of RNA have important roles in cellular function and physiology. The current understanding of the regulation, function, and homeostasis of these modifications is still being explored and expanded⁸. Therefore, a precise and gold-standard method to evaluate the modifications of RNA is needed. The modified northern blotting method provides precise quantification of RNA modifications and clear delineation of the modifications in diverse RNAs. Although the method requires at least 3 days, it can be standardized and can be used in various experimental designs. Moreover, with different antibodies, it can detect different RNA modifications²⁷.

It is important to separate different RNAs when analyzing RNA modifications. Ribosomal RNA comprises a large portion of the total amount of RNA^28,29. The results from analyzing RNA modifications only in total RNA will represent mostly the changes of rRNA. Methylation and other such modifications of rRNA could potentially mask the changes in other RNAs. With the procedure of gel separation, the modifications of mRNA and other small RNAs can be more accurately analyzed.

Transcriptome-wide mapping with m⁶A immunoprecipitation and sequencing provides detailed insight into the modification of each type of RNA²². It provides information on the specific RNAs and a resolution of around 80-120 bp. Although m⁶A-Seq can compare the modifications between different experimental conditions, the selection of proper standards and controls for such experiments is difficult¹⁸. Immunoprecipitation is difficult to reproduce, often giving significant variations amongst repeats. Moreover, m⁶A-Seq requires the fragmentation of RNA samples before immunoprecipitation and sequencing³⁰. The fragmentation process could potentially induce undue influences on the original RNA modifications. If the experiment does not need the specific gene’s information but requires different conditions for comparison, the current method provides better visualization and control for diverse experimental setups.

Modification of RNA is an important step in regulating transcriptional control³¹. However, the homeostasis and the regulatory mechanisms of various RNA modifications under diverse physiological realms are still unclear. Using the present modified northern blotting method, different RNA modifications can be quantified and compared. Furthermore, the changes and regulations of RNA modifications can be investigated in greater detail. In the future, it could also be possible to combine the experimental data from both the classical northern blotting and the modified northern blotting protocols, providing greater insights into RNA biology.

The most important factor determining the success of the modified northern blotting protocol is the integrity of the RNA sample. RNAs with some amount of degradation may yield good classical northern blotting results, but this could potentially have significant impact on the modified northern blotting results. The modifications of RNA in different tissues or cell lines could also vary significantly. It is important to test the suitable RNA sample loads for different tissues before performing the final experiments.

As blotting procedures have been traditionally named after Dr. Southern and different geographical directions, we propose the name “northeastern” blotting for the current technique.

Materials

RNaseZap solution	Ambion	AM9782	Protocol 1.1
TRI Reagent solution	Ambion	AM9738	Protocol 1.2.1.1
1-Bromo-3-chloropropane (BCP)	Sigma	B9673	Protocol 1.2.1.3
Ethanol	JTbaker	8006	Protocol 1.2.1.3
Isopropanol	Sigma	I9516	Protocol 1.2.1.3
GenElute mRNA Miniprep Kit	Sigma	MRN70	Protocol 1.2.2.1
Glycogen	Ambion	AM9510	Protocol 1.2.2.2
Sodium acetate	Fluka	71183	Protocol 1.2.2.2
Nuclease-free water	Ambion	AM9930	Protocol 1.2.2.6
DEPC	Sigma	D5758	Protocol 2.1.1
Agarose	JT Baker	A426	Protocol 2.1.2
MOPS	Sigma	M1254	Protocol 2.1.3
37% formaldehyde Solution	Sigma	F8775	Protocol 2.1.3
EDTA, Disodium Salt	JT Baker	8993	Protocol 2.1.3 10x MOPS buffer
formamide	Sigma	F7503	Protocol 2.2.1
RNA Millennium Marker	Ambion	AM7150	Protocol 2.2.2
Ethidium Bromide	Amresco	X328	Protocol 2.2.5
Ficoll PM400	GE Healthcare	17-0300-10	Protocol 2.2.5 tracking dye
Bromophenol blue	Sigma	114391	Protocol 2.2.5 tracking dye
Xylene Cyanol FF	Sigma	X4126	Protocol 2.2.5 tracking dye
Sodium chloride dihydrate	JT Baker	3624	Protocol 2.4.2 20x SSC buffer
Trisodium citrate	Sigma	S1804	Protocol 2.4.2 20x SSC buffer
Hybond Blotting Paper	GE Healthcare	RPN6101M	Protocol 2.4.4
GE Hybond-N+ membrane	GE Healthcare	RPN303B	Protocol 2.4.6
Stratalinker UV Crosslinker 2400	Stratagene	400075	Protocol 3.1.2
Gel Catcher 1500	ANT Technology	Gel Catcher 1500	Protocol 3.1.5
Anti-m<sup>6</sup>A (N<sup>6</sup>-methyladenosine)	Synaptic Systems	202003	Protocol 3.2.3
Amersham ECL Anti-Rabbit IgG, HRP-linkd whole Ab	GE Healthcare	NA934	Protocol 3.2.5
ChemiDoc MP System	BIO-RAD	1708280	Protocol 3.2.8