There are at least 170 known chemical modifications on RNA molecules in all organisms. Most of these modifications are on tRNAs, rRNAs and small RNAs. But over the past two decades, researchers found that eukaryotic mRNAs also carry some modifications, the most abundant being N6-methyladenosine (m6A). Most methods to date used various RNA sequencing technologies to detect such modifications or selectively sequence modified RNAs. But these lack spatial information on where these mRNAs reside. In 2021, a m6A-Specific In Situ Hybridization Mediated Proximity Ligation Assay (m6AISH-PLA) was published. But it only recognizes modified mRNAs. ARPLA is a novel method that detects glycosylated RNAs, but not at single-molecule resolution. DART-FISH is a new in situ method that combines detection of m6A-modified and unmodified mRNAs at single-molecule resolution.
mRNA modifications are another level of regulation on mRNA localization, translation and stability. Almost all of the work so far was done either by mutating/eliminating the proteins involved in writing, reading or erasing these modifications or by looking at global RNA modifications outside of cell context (i.e. by sequencing). But where are these mRNAs in the cells? How are they distributed? What can we learn from single-molecule analysis?
In the intro I mentioned m6AISH-PLA, developed by the group of Jinghong Li from Tsinghua University, Beijing. This method is based on the ability to ligate two proximal DNA strands, then amplifying it. I DNA strand comes from a FISH probe with a long “tail”. The probe hybridizes to the mRNA in close proximity to the m6A site. The second DNA strand is brought by a complicated complex: first there’s a primary antibody for m6A-RNA. Then a secondary antibody which recognize it. The DNA strand is conjugated to biotin, and is connected to the secondary antibody via a biotin-streptavidin-biotin bridge. Once both ding the mRNA, a proximity ligation followed by rolling circle amplification (PLA-RCA) is performed in situ, and fluorescent probes recognize the amplified DNA (see figure 1).
Figure 1: m6AISH-PLA. A) Scheme depicting the method. B) Example of m6AISH-PLA detection of m6A-HSP70 mRNA before and after heat-shock (HS). (Source: Ren et al. Angew. Chem. Int. Ed. 2021)
This method allowed for single-molecule m6A-mRNA detection and the authors used to show a (mild) change in nuclear-cytoplasmic ration of m6A-HSP70 mRNA before and after heat-shock, but they didn’t look any further.
A similar approach, developed by the group of Yi Lu from University of Texas, was recently used to image glycosylated RNAs. The method, called “sialic acid aptamer and RNA in situ hybridization-mediated proximity ligation assay” (ARPLA), uses a complicated set of probes, including an aptamer that specifically binds the sialic acid group – which is a very cool tool to have (Figure 2). But the basic idea is to have PLA-RCA followed by fluorescent probes. Using this method, they managed to visualize glycoRNA on the cell surface of various cell types and conditions. But unlike m6AISH-PLA, ARPLA did not provide single-molecule resolution.
Figure 2: ARPLA. A) Scheme depicting the method. B) Example of ARPLA detection of glycoRNA on the cell surface. (Source: Ma et al. Nature Biotechnology 2023)
DART-FISH uses a different approach. It is based on DART-seq (deamination adjacent to RNA modification targets), an RNA sequencing method developed by Kate Meyer lab from Duke University, North-Carolina to identify m6A-labeled mRNAs. While most sequencing methods pull-down m6A-mRNA using anti-m6A antibody, DART-seq uses APO1-YTH, a fusion between the C-to-U deaminase APOBEC1 and the m6A-binding YTH domain. When APO1-YTH is expressed in cells, it directs C-to-U deamination of cytidine residues which follow nearly all m6A sites. The C-to-U change can be detected by RNA-seq.
So now, instead of sequencing, Meyer’s lab developed a set of padlock probes with unique identifiers that can discriminate between the C and U nucleotides. After they bind, there’s an RCA step followed by hybridization with fluorescent probes that bind the unique identifiers. The result, again, is single-molecule detection, only this time it can simultaneously detect both m6A and non-m6A mRNAs (Figure 3).
Figure 3: DART-FISH. A) Scheme depicting the method. B) Example of DART-FISH detection of m6A or non-m6A β-actin mRNA at position A1222. C) Example of cell heterogeneity of m6A on HNRNPA1 position A1225. (Source: Sheehan et al. Nucleic Acids Research, 2023)
They verified the method by looking at m6A1222 in the 3UTR of β-actin mRNA and could detect labeling of m6A and non-m6A mRNAs. The m6A, as expected, was dependent on the m6A writing machinery. She later looked at other mRNAs, in order to study cell-cell heterogeneity. Through these experiments, they found that single-cell DART-seq which was used in the past missed a lot of m6A mRNAs due to the arbitrary threshold they used. DART-FISH was also used to detect isoform specificity of m6A, and look at stress-granule localization of the m6A vs non-m6A Actin mRNA. Frankly, I would rather Meyer looked at actin mRNA localization to lamelipodia or focal adhesions – something more meaningful than SG.
Both m6AISH-PLA and DART-FISH look like cool methods to get single-cell single-molecule data on modified mRNAs, with DART-FISH having the advantage of also detecting the non-modified mRNA. However, DART-FISH relies on the deamination activity of the APO1-YTH – which might not access certain mRNAs or m6A sites, or become inactive under certain conditions. In addition, Meyer mentions that the padlock method identifies only about 30% of the transcripts that are detected by regular smFISH, so this method could miss a lot of data. Yet this is a great first step and I’m sure future work will improve either of these methods (or come up with new better ones).
Meyer wrote a detailed protocol for DART-FISH which should be published in a new book on FISH which I am editing in the Methods in Molecular Biology series. I will add a link once its published.
I am looking forward to more innovative methods to in situ detect other mRNA/RNA modifications.
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A nice review on mRNA modifications by Wendy Gilbert & Sigrid Nachtergaele
“mRNA Regulation by RNA Modifications” (2023) Ann Rev Biochem 92:175-198
A nice review by Kate Meyer on “The Proteins of mRNA Modification: Writers, Readers, and Erasers” (2023) Ann Rev Biochem 92:145-173
Single-Cell Imaging of m6A Modified RNA Using m6A-Specific In Situ Hybridization Mediated Proximity Ligation Assay (m6AISH-PLA) Xiaojun Ren, Ruijie Deng, Kaixiang Zhang, Yupeng Sun, Yue Li, Jinghong Li Angew. Chem. Int. Ed. 2021, 60, 22646
Spatial imaging of glycoRNA in single cells with ARPLA. Ma, Y., Guo, W., Mou, Q. et al. Nat Biotechnol (2023)
In situ visualization of m6A sites in cellular mRNAs, Charles J Sheehan, Bahjat Fadi Marayati, Janvi Bhatia, Kate D Meyer Nucleic Acids Research, 2023, gkad787
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