Quantification and localization of pseudoUridine in RNA
PseudoUridine (Ψ) is the most abundant post-transcriptionally modified ribonucleoside. It is widely used for modification of RNA, including ribosomal RNA, small nuclear RNA and selective messenger RNAs, which has important biological significance. And pseudouridylation at different site of varies RNAs may play different and important roles. Therefore, quantification and localization of pseudoUridine in RNA are indispensable tools for the study of pseudouridylation of certain RNA.
Quantification of pseudoUridine
- Two dimensional cellulose thin-layer chromatography (2D-TLC)
- High-performance liquid chromatography (HPLC) and Mass spectrometry (MS)
The 2D-TLC system contains two different chromatography systems. The first dimension can use the mixture of isobutyric acid, ammonia and water, the second dimension can use a mixture of isopropanol, hydrochloric acid and water or a mixture of sodium phosphate, ammonium sulfate and isopropanol. The RNA is cleaved into oligonucleotides using RNaseA or RNaseT1 and RNaseT2, subsequently the 5' end is labeled with 32P, and finally 5'-32P-labeled nucletide is obtained using nuclease P1. Due to the different polarity of different bases with different position on 2D-TLC and the high sensitivity of 32P, the presence of pseudoUridine in RNA can be determined by analyzing the 2D-TLC pattern for the presence of pseudoUridine. Also, the relative content of pseudoUridine can be determined by examining the ratio of the exposure intensity at the pseudoUridine position to that of other common bases (A, G, C, U).
After enzymatic decomposition of RNA into single nucleosides, HPLC is used to separate different nucleosides, ultraviolet (UV) detectors are the most commonly detectors used to measure the absorbance of the analytes. These have been applied to determine pseudoUridine content in RNA. However, the chromatographic conditions required up to 85 minutes to analyze a sample, making this method ineffective as a screening method. MS is also used to detect pseudoUridine content. The molecules are fragmented in the presence of an electric field, giving individual molecules with special base-to-charge ratios. Triple quadrupole mass spectrometry was used for the quantification of modified nucleosides by HPLC-MS coupling, which can reduce the detection time to 40 min. Replacing HPLC with ultra performance liquid chromatography (UPLC) has also improved the efficiency of nucleoside analysis, with single sample being analyzed in less than 15 minutes. The sensitivity of nucleoside quantification has been greatly improved by using these methods of coupled LC/MS. However, given the high cost of MS analysis, such complete RNA modification analysis may not be necessary for many simple research questions.
Localization of pseudoUridine
- Localization of pseudoUridine via RNase H cleavage specific sites
- Localization of pseudoUridine via CMC
- Localization of pseudoUridine via SCARLET
RNase H cleaves RNA only at the unmodified 2'-OH position of the ribose ring. If the 2’-OH on ribose ring is methylated, the cleavage at that site will be hindered. Notably, this method can also be quantitative, as the degree of resistance to cleavage quantitatively reflects the level of 2’-O-methylation. This site-specific RNase H cleavage can be extended to the detection and quantitation of base modifications including pseudoUridines. The approach to detect and quantify modified bases in RNA can be divided into three steps. Firstly, the test RNA is specifically cleaved at the 5’ side of the modified nucleotide of interest. This is readily achieved by using RNase H site-specific cleavage directed by 2’-O-methyl RNA–DNA chimeras, consisting of a DNA sequence complementary to the region containing the pseudoUridine and the 2’-O-methylated RNA at both ends of the DNA sequence. This design ensures that the cleavage site of RNase H is just in front of the pseudoUridine. To produce a 5’-half RNA with a 3’-OH end and a 3’-half RNA with a 5’-phosphate end, and the pseudoUridine is located at the 5' end of the 3'-half RNA. Secondly, the 3’-half RNA is dephosphorylated at its 5’ ends by using alkaline phosphatase (CIP) and subsequently labeled with [γ-32P]ATP by polynucleotide kinase (PNK). Thirdly, the 5’-labeled RNA is completely digested with nuclease P1, generating a mixture of nucleotides which can be resolved by TLC. Because only the pseudoUridine and the Uridine are labeled with 32P, this method allows the determination of not only whether the site contains pseudoUridine in RNA, but also the percentage of pseudoUridine at the site.
The most popular method for studying pseudoUridine in RNA is based on chemical derivatization of RNA by N-cyclohexyl-N’-β-(4-methylmorpholinium)ethylcarbodiimide (CMC) p-tosylate. The CMC p-tosylate reacts with all guanosine, Uridine, guanosine-like, and Uridine-like residues to form adducts. However, under weak base conditions, the adducts on Uridine and guanine are removed and only the adduct on 3’- position of pseudoUridine is retained. Thus, the adduct of pseudoUridine with CMC can be obtained specifically. CMCT reacts with the N3 of pseudoUridine, which is located on the Watson-Crick side of pseudoUridine, affecting base complementary pairing and prevents the reverse transcription due to the larger steric hindrance of CMC. Therefore, the primer extension assay with reverse transcriptase will be explored to sequence-specifically determine the location of each pseudoUridine modification site. The reverse transcription process is terminated when the primer labeled with the 32P encounters the adduct of pseudoUridine with CMC. The products via reverse transcription process that stops at the position of pseudoUridine can be distinguished by SDS-PAGE.
RNase H specific cleavage can be used to localize modifications in RNAs, but this method is not suitable for studying modifications on low abundance mRNAs or lncRNAs because it requires the isolation of a particular RNA first. Thus, the site-specific cleavage and radioactive labeling followed by ligation-assisted extraction and TLC (SCARLET) have been used to locate the pseudoUridine in RNA. This method does not require the initial purification of a specific RNA. The 5' end of the modification site is cleaved with RNase H and labeled with 32P, and then splint ligation to a 116 nt DNA oligonucleotide. Only the target RNA can be ligated to DNA in the presence of splint DNA. The DNA-linked RNA strand is cleaved using RNase A/T1, and the 32P-labeled modification site will then form a DNA-32P-Xp or DNA-32P-XCp structure with the DNA strand. The band is then purified by SDS-PAGE, cut into individual nucleotides using nuclease P1, and separated using 2D-TLC to determine whether the site contains a modification and the proportion of the site that is modified.
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