RNA modifications

Learn different applications and techniques for determining the presence and distribution of RNA modifications in mRNA, tRNA and more.

​​The field of epigenetics is branching down many new and exciting avenues. One of these avenues is the area of RNA modification research. Recent advancements in the development of RNA modification detection and sequencing methods eg m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) has meant that it is becoming easier and faster to discover new modifications and map them to different species of RNA within any cell type or model organism. The advancements in this technology lead to a boom in the number of known RNA modifications. Currently, there are over 100 known RNA chemical modifications (Roundtree et al., 2017). You find these on mRNA, tRNAs, rRNAs and other non-coding RNAs including miRNAs. Each of these modifications also has its own function, including RNA structure, export, stability, and mRNA splicing. The future is bright for this field of research; there is still much to uncover regarding the function of some of these new modifications.​​

Figure 1. The distribution of RNA modifications on mRNA and tRNAs. To find out more take a look at our RNA modifications poster 

Of all the RNA species, tRNAs contain the most RNA modifications: almost one in five nucleotides within tRNAs are thought to contain RNA modifications (Kirchner et al., 2015). The modifications on tRNA are incredibly diverse and require step-by-step formation by multiple enzymes7. You can commonly find modifications in the anticodon loop of the tRNA, which help promote translation efficiency by aiding codon-anticodon interactions and preventing frameshifting (Stuart et al., 2003).

The field of RNA modifications is relatively new but growing more and more every day. In this section, we will go through some of these protocols and offer a few tips and advice for working with RNA modifications.

Getting antibodies that are specific to your RNA modification of choice can be difficult. We have many well-cited and validated RNA modification antibodies available including our m6A antibody, cited in several great publications including a Nature Methods paper which uses it for single-base resolution sequencing of m6A (Linder et al., 2015). This same m6A antibody also features in a Nature paper, looking at the role of m6A in mRNA stability (Mauer et al., 2017). Click here for a full list of our RNA modification antibodies.

RNA modification antibodies: technical considerations

Antibody specificity and consistency. It is important to be sure that the antibody you are using is specific for the modification you are interested in using the right controls and testing the antibody in your model system. Once you have a working antibody you should ensure that between batches you are still getting specificity. Recombinant RabMAb® technology eliminates batch-to-batch variation by using the same immunogen for each round of antibody production. This gives you a consistent antibody and reproducible results throughout your project. Many of our RNA modification antibodies are RabMAb® monoclonal antibodies including key targets such as m6A, m1A, Mettl3, m2,2G, and many more.

RNA immunoprecipitation (RIP)

RIP is an antibody-based technique used to map in vivo RNA-protein interactions. The RNA binding protein (RBP) of interest is immunoprecipitated together with its associated RNA for identification of bound transcripts (mRNAs, non-coding RNAs or viral RNAs). Transcripts are detected by real-time PCR, microarrays or sequencing.

Beyond transcription and subsequent translation, there is still is much more to the function of RNA. For example, RNA-protein interactions can modulate mRNA and noncoding RNA function. This new appreciation for the potential of RNA has lead to the development of novel methods allowing researchers to map RNA-protein interactions. RIP is one such protocol for the study of the physical association between individual proteins and RNA molecules.

Take a look at our full RIP protocol here.

Adapted from Khalila et al. (2009), Hendrickson et al. (2009), Hendrickson et al. (2008) and Rinn et al. (2007).

​​

Figure 2. Schematic of RIP protocol workflow. Top uses a native approach without cross-linking. Bottom method uses formaldehyde cross-linking. 

RIP: technical considerations

RNase contamination. Avoid contamination using RNase-free reagents such as RNase-free tips, tubes, and reagent bottles. Use ultrapure distilled, DNase-free, RNase-free water to prepare buffers and solutions.

Plan your controls carefully. One or more negative controls should be maintained throughout the experiment, eg no antibody sample or immunoprecipitation from knockout cells or tissue. Knockdown cells are not recommended for negative control experiments.

Downstream analysis. The RNA isolated from your pull-down can be analyzed using several techniques. Choose the best method based on the questions you want to answer and use multiple methods to confirm your results. For example, any novel results you obtain using RIP-seq should then be confirmed using RIP-qPCR.

CLIP

CLIP is an antibody-based technique used to study RNA-protein interactions related to RNA immunoprecipitation (RIP) but differs from RIP in that it uses UV radiation to cross-link RNA binding proteins to the RNA. This covalent bond is irreversible, allowing stringent purification conditions. Unlike RIP, CLIP provides information about the actual protein binding site on the RNA.

Different types of CLIP exist, including high-throughput sequencing-CLIP (HITS-CLIP), photoactivatable-ribonucleoside enhanced CLIP (PAR-CLIP), and individual CLIP (iCLIP).

You can find our full CLIP protocol here adapted from Konig et al. J. Vis. Exp. 2011. “iCLIP -Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution.” 

Figure 3. Schematic of CLIP protocol workflow. 

CLIP: technical considerations 

4-thiouridine pre-incubation. Optional 4-thiouridine pre-incubation and UV-A crosslinking may be necessary for certain proteins. 4-thiouridine enhances crosslinking of some proteins. Details for this can be found in the complete protocol.
Optimize antibody concentration. The amount of antibody required should be optimized before you start your experiment (Huppertz et al., 2014). A no-antibody sample is a good negative control. If your target of interest has not been studied using CLIP before, you could start by using an antibody that already works in IP, which is a good indication that it will work in CLIP.

miCLIP

Although m6A is the most abundant modified base in eukaryotic mRNA, current methods to accurately study it has limitations. New approaches to high-resolution mapping of m6A will be essential for understanding this epigenetic RNA modification.

miCLIP allows for high-resolution detection of single m6A residues and m6A clustering across the entire RNA (figure 14). Using miCLIP, it is possible to map m6A and the related dimethylated version m6Am (N6,2′-O-dimethyladenosine), at single-nucleotide resolution in human and mouse mRNA (Linder et al., 2015).

miCLIP is applicable to smaller RNAs. The authors of this protocol (Linder et al., 2015) discovered that m6A is present in small nucleolar RNAs (snoRNAs), a class of small non-coding RNAs. This was impossible to establish with previous applications due to lack of specificity and bioinformatic challenges.

You can find our full miCLIP protocol here.

miCLIP: technical considerations

Not 100% accurate. The method is unable to identify the specific location of modified residues and only determines the general location of m6A sites.

Bias in bioinformatic m6A calling. Data analysis uses assumptions based upon known consensus sequences that harbor m6A residues, and so it misses modifications outside these motifs.


Figure 4: Schematic of miCLIP protocol workflow

Liquid chromatography tandem-mass spectrometry (LC/MS-MS)

Similar to DNA modifications, if you have access to LC-MS/MS, then this is the best way quantify the amount of RNA modification within total genomic DNA. Also similar to measuring DNA modifications you can use absolute quantification methods limited only by which isotopic standards you have available to use as a standard to measure your sample against.

Using this technique combined with RIP (RIP-MS) will allow you to determine if your RNA modification antibody is binding to your modification of interest and see if it binds any other non-specific modifications. If you generate LC-MS/MS data of your RIP input and pull-down samples, you should see an enrichment of your modification of interest in the pull-down sample compared to the input. You can also then check other modifications with these same data to see if anything else came out as enriched in your samples to test for non-specific antibody binding.

LC/MS-MS: technical considerations

Technically challenging. Mass spec equipment is costly and very specialized. The machine itself will require an enormous amount of maintenance and often requires its own technician to keep on top of things. Operating the machine is very complicated and requires specialized training so it may be difficult to obtain this type of mass spec data on your own. Consider obtaining this data through collaborations or paid services if it is not feasible for you to purchase your own LC/MS-MS equipment.

RNA modification control experiments

Due to the nature of RNA modifications their chemical structures are often very similar. To make sure you are getting the most accurate results from your antibodies, you need to test them in your model system thoroughly. Controls for RNA modification antibodies can be done using a range of applications. See below for some of our advanced controls and tips to make your RNA modification research easy.

RNase treatment

Whether you are carrying out ICC/IHC or RIP-qPCR, it is essential to have an RNAse-treated control alongside your experimental samples (Delatte et al., 2016). For example, if you see a clear bright signal in your experimental IHC samples, but you get no signal in your RNAse treated control samples, you can be confident that the signal you are getting is within the RNA and is not background signal from a non-specific source. This suggests that the antibody recognizes the modification within RNA and not the DNA.

  • It is crucial to ensure that you are not detecting high levels of non-specific background signal from DNA when using RNA modification antibodies. You can quickly add an RNase treatment step to your normal RNA modification IHC or RIP protocol. It is important not to leave the samples in the RNase solution for too long, this can lead to degradation of the DNA, and this then makes it difficult to carry out counterstains such as DAPI.
  • For each different sample type, you should test different concentrations of RNase and try leaving on your samples for varying lengths of time. For example, an IHC may need an RNAse time of up to an hour depending on the tissue type whereas an ICC will require much less time – try 10–30 mins as a starting point.

Whether carrying out ICC/IHC or dot blot, it is essential to have an RNAse-treated control alongside your experimental samples to ensure that you are not picking up non-specific background signal from DNA when using RNA modification antibodies. For example, if you see a clear, bright signal in your experimental IHC samples, but you get no signal in your RNAse-treated control samples, you can be confident that the signal you are getting is from the RNA and not background signal from a non-specific source. This suggests that the antibody is recognizing the modification within RNA and not the DNA. 

You can quickly add an RNase treatment step to your normal RNA modification IHC, RIP, or dot blot protocol.

  • It is important not to leave the samples in the RNase solution for too long, as this can lead to degradation of the DNA, which makes it difficult to carry out counterstains such as DAPI.
  • For each different sample type, you should test different concentrations of RNase and try leaving on your samples for varying lengths of time. For example, an IHC may need an RNAse time of up to an hour depending on the tissue type whereas an ICC will require much less time – try 10–30 mins as a starting point.

DNase treatment

In addition to RNase-treated controls, you should carry out DNAse-treated controls. If you are concerned that your RNA modification antibody is recognizing a similar modification within DNA, the best way to test for this is to treat your samples with DNAse. Many modifications are within both RNA and DNA, so this is a common problem. For example, 5mC within DNA has the same chemical modification as m5C within RNA.

  • ​If you carry out IHC using an RNA modification antibody, it is a good idea to have a DNAse-treated control alongside your experimental samples. If you get a clear, strong signal from your experimental samples, but your DNAse-treated control has no signal, it suggests that your antibody is binding to a modification within DNA.
  • For this type of control, it is also important to optimize the conditions. Leaving your samples in DNAse treatment for too long can lead to degradation of RNA, so be sure to test different DNAse concentrations and the duration of the treatment.

Competition assays

Another way to ensure the specificity of your RNA modification antibody is to use a competition assay. This assay uses a synthetic modification-containing oligonucleotide which can be pre-incubated with your antibody (Meyer et al., 2012). When you then use this pre-incubated antibody for your applications, eg ICC/IHC or dot blot, you should see a reduction in the signal obtained when compared a sample stained with the antibody alone. You can try adding the competitor oligonucleotide to your antibody solution at increasing concentrations; a decreasing gradient of the signal reflects the amount of competitor you add to the antibody. For example, try a gradient of 0 ng, 10 ng, 100 ng, and 1µg of the competitor oligonucleotide.

Dot blot

Carrying out a dot blot using RNA modification antibodies can be a quick and simple way to test for their specificity. A dot blot works like a simplified version of a western blot. For this technique, the sample is spotted directly on to the membrane, cross-linked, and then undergoes blotting. For more details take a look at our dot blot protocol here. If you have access to synthetic RNA molecules containing your modification of interest, this can act as the perfect positive control. Similarly, loading an unmodified molecule or a molecule containing a different modification can serve as a negative control and help you to gauge any non-specific binding or cross-reactivity.

  • For your experimental samples, it is possible to test whether your RNA modification antibody is specific by carrying out a dot blot with the right controls. For a negative control, use samples that contain a KO for the enzyme responsible for producing your specific RNA modification (Jia et al., 2011).
  • If you load RNA from your wild-type and KO samples onto a membrane for dot blot, you should see a clear difference between the two samples. The wild-type sample will display a clear signal and the KO should appear blank when the membrane is stained using an antibody against your RNA modification of interest. 

RIP-MS

If you have access to LC-MS/MS, then this is really the best way to test for RNA modification antibody specificity (Kellner et al., 2014). Using this technique combined with RIP (RIP-MS) allows you to determine if your antibody is binding to your modification of interest exclusively.

  • Using either absolute or relative quantification methods, LC-MS/MS gives you parallel quantification of all the RNA modifications found in total RNA from any organism and cell type. If you generate LC-MS/MS data of your RIP input and pull-down samples you should see an enrichment of your modification of interest in the pulldown sample compared to the input.
  • You can also then check other modifications with these same data to see if anything else came out as enriched in your samples to test for non-specific antibody binding. There is software being developed now that can even help you with this type of analysis (Yu et al., 2017). 


References

Delatte B, Wang F, Ngoc LV, Collignon E, Bonvin E, Deplus R, Calonne E, Hassabi B, Putmans P, Awe S, Wetzel C, Kreher J, Soin R, Creppe C, Limbach PA, Gueydan C, Kruys V, Brehm A, Minakhina S, Defrance M, Steward R, Fuks F.RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine. (2016) Science. 2016 15:282-5

Hendrickson DG, Hogan DJ, Herschlag D, Ferrell JE, and Brown PO (2008). Systematic Identification of mRNAs Recruited to Argonaute 2 by Specific microRNAs and Corresponding Changes in Transcript Abundance. PLoS One 3 (5), 2126.

Hendrickson DG, Hogan DJ, McCullough HL, Myers JW, Herschlag D, Ferrell JE, and Brown PO (2009). Concordant Regulation of Translation and mRNA Abundance for Hundreds of Targets of a Human microRNA. PLoS Biology 7 (11), 2643.

Huppertz et al. iCLIP: Protein–RNA interactions at nucleotide resolution. Methods. (2014).

Jia G, Fu Y, Zhao X, Dai Q, Zheng G, et al. (2011). N6-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat. Chem. Biol. 7:885–87

Kellner S, Ochel A, Thüring A, Spenkuch F, Neumann J, Sharma S, Entian KD, Schneider D, and Helm M. (2014) Absolute and relative quantification of RNA modifications via biosynthetic isotopomers. Nucleic Acids Res. 42(18): e142.

Khalila AM, Guttman M, Huarte M, Garbera M, Rajd A, Morales DR, Thomas K, Pressera A, Bernstein BE, Oudenaarden AV, Regeva A, Lander ES, and Rinn JL (2009). Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression. PNAS 106, 11667–72.

Kirchner, S., and Ignatova, Z. (2015). Emerging roles of tRNA in adaptive translation, signalling dynamics and disease. Nat. Rev. Genet. 16, 98–112.

Konig et al. J. iCLIP -Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution. Vis. Exp. (2011).

Linder B  et al. Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat Methods 12:767-72 (2015)

Mauer J  et al. Reversible methylation of m(6)Am in the 5' cap controls mRNA stability. Nature 541:371-375 (2017).

Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR.(2012) Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons. Cell 1635-46

Rinn JL, Kertesz M, Wang JK, Squazzo SL, Xu X, Brugmann SA, Goodnough LH, Helms JA, Farnham PJ, Segal E, and Chang HY (2007). Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs. Cell 129, 1311–1323.

Roundtree, I., Evans, M., Pan, T., & He, C. (2017) Dynamic RNA Modifications in Gene Expression Regulation. Cell, 1187-1200

Stuart, J.W., Koshlap, K.M., Guenther, R., and Agris, P.F. (2003). Naturally occurring modification restricts the anticodon domain conformational space of tRNA (Phe). J. Mol. Biol. 334, 901–918.

Yu N, Lobue PA, Cao X, Limbach PA. (2017) RNAModMapper: RNA Modification Mapping Software for Analysis of Liquid Chromatography Tandem Mass Spectrometry Data. Anal Chem 10744-10752




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