طريقة لقياس الحمض النووي الريبي<em> N</em<sup> 6</sup> -methyladenosine تعديلات في الخلايا والأنسجة
Summary
ووصف طريقة النشاف شمال المعدلة لقياس N 6 -methyladenosine (م 6 أ) التعديلات في الحمض النووي الريبي. الطريقة الحالية يمكن الكشف عن تعديلات في الرنا متنوعة والضوابط في إطار مختلف التصاميم التجريبية.
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 RNA16-18. Traditionally, thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are used to study the distribution of m6A in several RNAs19,20. 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
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 RNA28,29. 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.
Divulgations
The authors have nothing to disclose.
Acknowledgements
C.Y.W. received support from the National Health Research Institute (NHRI-EX101-9925SC), the National Science Council (101-2314-B-182-100-MY3, 101-2314-B-182A-009), and Chang Gung Memorial Hospital (CMRPG3B1643, CMRPG3D1002, CMRPG3D0581, CMRPG380091, and CMRPG3C1763).
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 400 | GE Healthcare | 17-0300-10 | Protocol 2.2.5 tracking dye |
| Bromophenol blue | Sigma | 114391 | Protocol 2.2.5 tracking dye |
| Xylene Cyanol | Sigma | X4126 | Protocol 2.2.5 tracking dye |
| Sodium chloride | JT Baker | 3624 | Protocol 2.4.2 20X SSC buffer |
| Trisodium citrate | Sigma | S1804 | Protocol 2.4.2 20X SSC buffer |
| filter 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-m6A (N6-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 |
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