Poster abstracts
Poster number 111 submitted by Hina Zain
Multiple endonucleases for digestion of tRNA anticodon regions to conduct modification mapping by LC-MS/MS
Hina Zain (University of Cincinnati, Cincinnati, OH), Yu Sun (Humboldt-Universitt zu Berlin, Berlin, Germany), Bibek Hamal (University of Cincinnati, Cincinnati, OH), Asif Rayhan (University of Cincinnati, Cincinnati, OH), Ann Ehrenhofer-Murray (Humboldt-Universitt zu Berlin, Berlin, Germany), Patrick Limbach (University of Cincinnati, Cincinnati, OH)
Abstract:
Transfer RNAs are the most diversified modified species among all the RNAs. These modifications play multitude roles in protein translation and its fidelity depending upon their position within the tRNA. Modifications at the wobble position are crucial for the efficiency of codon-anticodon pairing and it also regulates the modification at other positions within the anticodon loop. This crosstalk can be well studied by using the bottom-up approach which involves the base-specific ribonucleases to produce smaller oligonucleotides. Liquid chromatography coupled with mass spectroscopy (LC-MS/MS) is one of the state-of-the-art techniques for mapping the modification. For the full-range profiling of tRNA modifications, multiple endonucleases for digestion of tRNA anticodon region can be used to generate overlapping digestion products.
RNase T1(Gp specific) is the most common ribonuclease used for this purpose. Oligonucleotides produced by T1 digestion were studied in LC-MS/MS. RNase T1 was not enough for the analysis as it often generates redundant products. This was avoided by using various ribonucleases with different cleavage specificity to generate overlapping oligonucleotides. Most of the ribonucleases are not adequately available commercially or it has been focused on the digestion of messenger RNA (mRNA). However, transfer RNAs (tRNAs) contain far more modifications than are found in mRNAs. In the present study, different incubation periods and enzyme concentrations were optimized to obtain overlapping oligomers that can be well studied in LS-MS/MS.
In the present study, first RNase T1 was used to digest the total tRNA of yeast, and oligomers were analyzed on LC-MS/MS. Modification at position 34/37 within the anticodon loop of four tRNA (His, Tyr, Asn, and Asp) was observed. this crosstalk modification regulates the translation and fidelity of the codons. For the verification of the crosstalk modification, the other ribonuclease, Human RNase 4 was used. Different incubation periods and enzyme concentrations were optimized for the appropriate tRNA digestion. Once the conditions were optimized, the reaction was applied to yeast total tRNA.
In this study, we have focused on the crosstalk modification within the anticodon loop of tRNA using multiple endonucleases.
References:
1. Richard P et al. (2021) PloS one, 16(8), e0253216.
2. Sun, Y., et al. (2023). Nucleic acids research, 51(20), 11197–11212.
3. Thakur, P., Atway, J., Limbach, P. A., & Addepalli, B. (2022). International journal of molecular sciences, 23(13), 7021
4. Wolf, E. J. et.al. (2022). Nucleic acids research, 50(18), e106
Keywords: tRNA modifications, LC-MSMS, RNase 4