2 Quantitative Analysis of RNA Modifications

Creative BioLabs is a contract research organization based in New York that specializes in antibody discovery and engineering. RNA modifications are important for the accurate expression of genetic information. Pre-messenger RNA undergoes processing to become mature messenger RNA, which is then exported to the cytoplasm for protein synthesis. Ribosomal RNA and transfer RNA also undergo modifications. Detection and quantification of RNA modifications can be challenging, but new technologies such as mass spectrometry and ligation-based methods show promise. A method combining primer extension and ribonuclease cleavage has been successfully applied to detect and quantify base modifications in RNA. Welcome to Creative BioLabs, 100% of the effort, 100% of the service. As a dynamic contract research organization, we are based in New York and serve the whole world. Our seasoned scientists are skilled in antibody discovery, antibody engineering, and biomanufacturing solutions. Good evening, dear friends. As we all know, it is crucial for every organism that the genetic information is accurately passed down from DNA to protein to be expressed. The intrinsic process of higher eukaryotic gene expression is the processing of pre-messenger RNA and the synthesis of protein. Pre-messenger RNA needs to be processed in many ways to become mature messenger RNA. The process involves RNA splicing and modification. The mature messenger RNA can then be exported to the cytoplasm to direct protein synthesis. Our researcher David is an expert on the topic of RNA modifications. Thank you for your time joining us today, David. So first, what are some key points when we think about RNA modifications? Thanks for the invitation. It is great to be here and let more people know about RNA modifications. To answer your question, the key points about RNA modifications are both prokaryotic and eukaryotic RNAs are synthesized in the form of more complex primary transcripts and then processed into mature RNA molecules. The transcription and translation of eukaryotes are separated in time and space. The newly transcribed messenger RNA is a large molecular precursor, namely heterogeneous nucleus RNA. Only about 10% of heterogeneous nucleus RNA molecules are transformed into mature messenger RNA. And the rest will be degraded during post-transcriptional processing. Can you tell us more about the modification of messenger RNA? Sure. The processing modification of eukaryotic messenger RNA mainly includes the modification of 5' and 3' ends and the splicing of the middle part. Most eukaryotic messenger RNAs have a 3' end polyadenylate tail. Polyadenylate tails are not encoded by DNA, but are added to the nucleus after transcription. Eukaryotic genes are often broken genes. What it means is that the nucleotide sequence encoding a protein molecule is separated by multiple inserts. After a complex process, the introns are cut off and the coding nucleotides, also known as exons, are connected. First, the introns were cut off by endonuclease. Then, under the action of ligase, each part of the exon is connected to become mature messenger RNA, which is called splicing. As far as I know, there are three common types of RNA. Namely the messenger RNA, ribosomal RNA, and transport RNA. What about the other two RNAs? Do they undergo post-transcriptional modifications, like messenger RNA does? Right. Start from ribosomal RNA, without exception, it belongs to the group that can be modified. Ribosomal RNA is a component of the ribosome, which has an evolutionarily conserved structure, and is very important for translation. There are some sequence differences among different species. The presence of modified nucleotides is a common feature of ribosomal RNA. Pseudouracil and methylation are the most common modifications in ribosomal RNA. If messenger RNA modification is to ensure the correct protein synthesis, what is the significance of ribosomal RNA modification? Several pieces of evidence suggest that ribosomal RNA modifications are essential for the function of ribosome. Although there is no significant difference in size or quantity of ribosomal RNA, from prokaryotes to lower and higher eukaryotes, the number of modifications increases with the complexity of biological evolution. Now we have talked about messenger RNA and ribosomal RNA, both can be post-modified for special purposes. I think it is needless to say that, transport RNA undergoes modification as well. Right, newly transcribed transport RNA precursors, in prokaryotes and eukaryotes, generally have no biological activity and need to be modified. They are cut into small molecules, under the action of transport RNA cleavage enzymes. There are many rare bases in mature transport RNA molecules, generally pseudoridine nucleotides and methylated nucleotides. In the end, three nucleotides, CCA are added to the three prime end of the transfer RNA, under the action of nucleotides transferase. RNA modification definitely can affect many cellular processes. How do we elucidate the mechanism of these modifications? It is necessary to have the ability to detect and quantify the presence of these modifications in RNA molecules. You see, transcriptional modified ribonucleotides were found, in the hydrolysates of RNA half a century ago. We now know that, almost all kinds of RNA contain post-transcriptional modifications. However, although more than 50 years have passed, since the discovery of unconventional RNA for the first time, the functions of many RNAs are still unclear. The premise of elucidating the function, of post-transcriptional modified RNAs is to know, their exact location and quantity, or the quantitative analysis of their existence. Before the early 1990s, the detection of RNA base modification was time-consuming and laborious. A variety of techniques need to be combined, including in vivo radiolabeling, nuclease digestion, and chromatography, or fingerprinting. Then after the 1990s, with the maturity of primer extension technology, the emergence of reverse transcription-based methods, greatly promoted the research on post-transcription modification. The basic principle of these methods is mainly based on one fact. That is to say, some modified nucleotides will stop early, during primer extension reactions. Can you give us a specific example? For instance, the chemical derivatization of pseudoridine with a molecule abbreviated CMC, blocks reverse transcription of one nucleotide prior, to the CMC-modified pseudoridine. Similarly, the presence of two oxymethylated nucleotides, leads to premature termination, when the primer is extended, at low concentrations of deoxin. This method eases the detection of RNA-modified base. In a sense yes, but they still have shortcomings. For example, most base modifications require chemical derivatization, to induce early termination of primer elongation, except for sugar ring modification. Why is it so difficult to recognize the base modification of RNA? Because there are many base modifications in RNA, it is a difficult task to identify the specific chemical modifiers, and reaction conditions of all known modifications. In addition, although these methods based on primer extension, are suitable for detection, they cannot quantitatively analyze the presence of modified bases. Are there other disadvantages of the detection method, based on the principle of reverse transcription? Yeah, another one I can think of, is the visual observation of early stop, by gel electrophoresis. It's not an accurate method, not at all, which may lead to errors, in the recognition of post-transcriptional modifications. So, there must be more efficient, and more sensitive detection technology developed. Right, the ones based on mass spectrometry. So is it widely used in scientific research? So far, not on a large scale. These technologies are impractical in general laboratories, because they require expensive specialized equipment, mass spectrometer and high-pressure liquid chromatography. Another big drawback is that, they can't detect all kinds of base modifications. But there got to be some ways, that we can analyze RNA modifications comprehensively. No more efficient and economical technology. Oh, don't worry. New technology always arrives. Recently, a promising ligation-based method has been described. Which utilizes the ability of T4 DNA ligase, to distinguish modified nucleotides. It has the potential, for high-throughput screening of modified nucleotides. Although it has not reached the ideal state, for the detection of all modifications. In other words, it is not currently optimized for all modifications. We just need to give it a bit more time. I heard that researchers are also developing a technology, that can combine the advantages of the above technologies. Yes, a method that can combine primer extension, which is a very effective method, to detect and quantify the modified nucleotides, in various RNAs. It has a very simple basic principle. Based on the fact that, ribonuclease H cleavage only occurs, at the 2-hydroxyon modified site of RNA. The cleavage can be directed to the specific nucleotide of interest, by using two oxymethyl RNA-DNA chimeric oligonucleotides. RNA can then be digested into single nucleotides, by ribonuclease after radiolabeling. The digested nucleotides can be separated, by thin-layer chromatography. And the interested nucleotides can be shown by autoradiography. Has it been successfully applied? Yes, I'm happy to report that, we have successfully applied this method, to the detection and quantification of various base modifications, including pseudouracil and 5-fluorouracil. As an example, we used it to quantify the pseudouracil, at position 34 of an RNA in the mouse brain. Our analysis shows that, almost 100% of uridine at this location, is converted to pseudouridine. That's great to hear. Thank you for educating us, on mRNA modification and its analysis today. It sounds like a process that involves many techniques, like the affinity selection of oligonucleotides, site-specific cleavage, and radioactive labeling, nuclease digestion, and thin-layer chromatography. But let's save these for the next program. Thank you for listening. See you next time.